Saccharomyces cerevisiae, which is involved in the epigenetic repression and plays a crucial role in transcriptional regulation and developmental events. Though the N-terminal catalytic HDAC domain of Hda1 is well characterized, the function of the C-terminal ARB2 domain remains unknown. In this study, we determine the crystal structure of the ARB2 domain from S. cerevisiae Hda1 at a resolution of 2.7 Å. The ARB2 domain displays an α/β sandwich architecture with an arm protruding outside. Two ARB2 domain molecules form a compact homo-dimer via the arm elements, and assemble as an inverse “V” shape. The pull-down and ITC results reveal that the ARB2 domain possesses the histone binding ability, recognizing both the H2A-H2B dimer and H3-H4 tetramer. Perturbation of the dimer interface abolishes the histone binding ability of the ARB2 domain, indicating that the unique dimer architecture of the ARB2 domain coincides with the function for anchoring to histone. Collectively, our data report the first structure of the ARB2 domain and disclose its histone binding ability, which is of benefit for understanding the deacetylation reaction catalyzed by the class II Hda1 HDAC complex.Hda1 is the catalytic core component of the H2B- and H3- specific histone deacetylase (HDAC) complex from  In eukaryotes, the structural unit of chromatin is the nucleosome that contains 147 bp of DNA wrapping around an octamer composed of two molecules each of the four histones-H2A, H2B, H3, and H4. The tails of these histones are unstructured and protruding from the core component3456in vivo, and contains an abundant source of epigenetic information11+-dependentAcetylation is one of the first discovered histone modificationSaccharomyces cerevisiae. Two related proteins Hda2 and Hda3 are essential for the activity of Hda1 both in vivo and in vitro16Hda1 is a typical class II histone deacetylase from S. cerevisiae Hda1. The ARB2 domain indicates structural resemblance to the α/β fold hydrolases. Largely distinct from these enzymes, the insertion region of the ARB2 domain protrudes from the core α/β fold and mediates the homodimer formation, which disrupts the corresponding substrate binding pocket. However, the unique homodimer architecture enables the ARB2 domain to bind to the histones. The ITC experiment shows that the ARB2 domain binds to the H2A-H2B dimer and H3-H4 tetramer with a Kd of 5.46 ± 0.69 μM and 3.24 ± 0.49 μM, respectively. Given the sequence homology, our results also provide some hints for understanding the function of the Arb2 protein, an essential subunit of the yeast Argonaute complex.Here, we report the first structure of the ARB2 domain from Hda1 consists of an N-terminal catalytic domain and a C-terminal non-catalytic domain (ARB2). The catalytic domain of Hda1 shows high sequence homology to the HDACs of which the structures were reported and the catalytic mechanism has been well elucidated. However, the non-catalytic ARB2 domain is poorly defined. To gain insight into the role of the C-terminal non-catalytic domain of Hda1 functioning in the deacetylation process, we determined the crystal structure of ARB2 domain of Hda1. The structure was solved by the SAD method using the Se anomalous signal. The final model has been refined to 2.7 Å resolution and the details of the data collection and refinement statistics are summarized in 512, Ile523 and Leu525 located in the protruded arm region of each molecules form a hydrophobic core, which dominates the domain-domain interaction  server and scores 1.0 in Complex Formation Significance Score 512, Ile523 and Leu525 located in the arm region of each molecules mediates the homodimer interaction, we designed a ARB2 mutant, ARB2-M0  to disrupt the homodimer interface. The size-exclusion chromatography assay shows that the mutant ARB2-M0 eluted with a molecular weight of approximately 29 kDa, which is coincident with the theoretical value of a monomer. Taken together, these results strongly suggest that the ARB2 domain of Hda1 exists as a homodimer, and this is consistent with the fact that the Hda1 protein functions as a dimer during the deacetylation reaction512, Ile523 and Leu525 that play key role in the domain-domain interaction are relatively conserved (Further examination of symmetry-related molecules indicates that two ARB2 domain molecules form a homodimer. As shown in eraction . The homonserved . We specLactobacillus johnsonii (PDB code 3S2Z)Anabaena variabilis ATCC 29413 (PDB code 2O2G), with a Dali Z-score of 12.9 and an RMSD of 3.4 Å for 166 Cα atoms (Pseudomonas putida IFO 12996 (PDB code 1ZOI)Considering that no structures exhibiting sequence homology to the ARB2 domain of Hda1 were deposited into the PDB database, the Dali server was used to search for the structures that are similar to the ARB2 domain. The result shows that the ARB2 domain is structural homologous to some hydrolases, such as the cinnamoyl esterase LJ0536 from Cα atoms , and thein vitro, and performed the GST pull-down assay to investigate the interaction between the histone octamer and the ARB2 domain. As shown in in vitro. The ITC experiment shows that the ARB2 domain could bind to both the H2A-H2B dimer and H3-H4 tetramer, with a Kd of 5.46 ± 0.69 μM and 3.24 ± 0.49 μM, respectively , ARB2-M2 (residues Tyr468 and Glu472 mutated to Ala), ARB2-M3  and ARB2-M4   . Next, wSaccharomyces cerevisiae, the Hda1 HDAC complex is recruited to specific promoters through the transcriptional corepressor Tup1. The N-terminal repressor domain of Tup1 could make direct interaction with the catalytic subdomain of the Hda1 HDAC complexSaccharomyces cerevisiaeTUP1 knock-out mutationin vivo and in vitroThe yeast class II Hda1 HDAC complex shows similarity to the mammalian class II HDACs, such as HDAC4, HDAC5, HDAC6, HDAC7, HDAC9 and HDAC10, which play an essential role in nucleosomal repression of transcription. In in vivo. That may come from the multi-component of the yeast Hda1 HDAC complex and unique features to fulfill the function. As reported, the histone deacetylase Hda1 could not exert the histone deacetylase activity alone, and two related proteins Hda2 and Hda3 are essential for the activity of Hda1. Besides, posttranslational modifications of core histone proteins are usually accompanied with chromosome remodeling. Generally, acetylated histones are correlated with more open chromatin and active gene expression, whereas deacetylated histones are correlated with closed chromatin and repressed gene expression. The Hda1 homologue in fission yeast (S. pombe), Clr3, forms a multi-protein complex in vivo, which recruits helicase Mit1 to perform both deacetylation and chromatin-remodeling functionsAs we known, histone octamer is wrapped by DNA to form nucleosome in chromosome and the physical character of the histone octamer is totally different from that of the nucleosome. Although our results indicate that the ARB2 domain of Hda1 can bind to histone octamer, it is important to investigate how the ARB2 domain functions The ARB2 domain of Hda1 is the first structural and functional characterized member of the Arb2 family. Given the sequence homology similarity, we speculate that the Arb2 protein, to a large extent, possesses the histone binding ability. This will greatly facilitate us to understand how the Argonaute complex works in the progress of histone methylation, heterochromatin assembly, and siRNA generation. In addition, whether the ARB2 domain existed as a conserved histone binding module in other members of the Arb2 family is an interesting question that deserves to address. Further experiments are in progress to confirm this hypothesis.457–698) was amplified from Saccharomyces cerevisiae genome, and cloned into modified pET-28a vector (Novagon) with six-His tag at the N-terminus and pGEX-4T-2 vector (Novagon). The mutations of the ARB2 domain were generated using the MutanBEST Kit (Takara). Overexpression of all recombinant proteins was induced in Escherichia coli BL21 (DE3) cells (Novagen) with 0.5 mM IPTG when the cell density reached an OD600 nm of 0.6–0.8. After growth for approximately 20 h at 16 °C, the cells were collected and lysed. The recombinant proteins were purified using Ni2+ -nitrilotriacetate affinity resin  in buffer  and eluted with 500 mM imidazole. The proteins were further purified using HiTrap Q FF (5 ml) and HiLoad 16/60 Superdex 200 . The final proteins were concentrated to 15 mg/ml for crystallization trials. Since there were no homologous structures published, a selenomethionine derivative Hda1457–698 was obtained to solve the phase problem. The selenomethionine derivative Hda1457–698 was overexpressed in the same competent cells as native Hda1457–698 but using M9 medium based on a methionine-biosynthesis inhibition method. The purification of selenomethionine derivative Hda1457–698 followed the same protocol as used for the native Hda1457–698.The gene encoding the C-terminal ARB2 domain of Hda1  vector with six-His tag at the C terminus. The recombinant plasmids were transformed into the E. coli Rosseta (DE3) strain for expression. The yeast reconstituted histones were prepared as described previously27The ORF of yeast histones H2A, H2B, H3 and H4 were amplified from 457–698 and selenomethionine derivative Hda1457–698 were grown using the hanging-drop vapour diffusion method at 285 K and grew to maximum size in approximately 1 day in the buffer containing 0.1 M Sodium acetate trihydrate pH 4.6, 2.0 M Sodium formate. For data collection, crystals were transferred to cryoprotectant solution consisting of the respective reservoir solution supplemented with 25% (v/v) glycerol and then flash-cooled in liquid nitrogen. Data sets for all crystals were collected at 100 K on beamline BL17U of synchrotron-radiation at Shanghai Synchrotron Radiation Facility (SSRF). The data sets were processed and scaled with HKL-2000 and programs from the CCP4 package457–698 was determined by the single-wavelength anomalous dispersion (SAD) phasing technique using AutoSol as implemented in PHENIXAutoBuild in PHENIX. Using the Se- Hda1457–698 structure as the search model, the structure of native Hda1457–698 was determined by the molecular-replacement method using MOLREPCCP4i. All of the initial models were refined using the maximum-likelihood method implemented in REFMAC5CCP4 program suite and rebuilt interactively using CootMolProbityPROCHECKPyMOL.Crystals of both the native Hda1The apparent molecular masses of the ARB2 domain of Hda1 and the ARB2-M0 mutant were estimated with a Superdex 200 column . Briefly, protein samples or molecular-mass standards were applied onto the Superdex 200 column at a flow rate of 0.5 ml/min with 25 mM Tris-HCl pH 7.5, 200 mM NaCl. The standard proteins  used in this assay were β-amylase (200.0 kDa), alcohol dehydrogenase (150.0 kDa), albumin (66.0 kDa), carbonic anhydrase (29.0 kDa) and cytochromec(12.4 kDa). The void volume was determined with blue dextran .The ITC binding studies were performed using an ITC200 (GE) at 20 °C with 0.04 ml of 0.5 mM ARB2 or ARB2 mutants  in the injector cell and 0.3 ml of 0.2 mM H2A/B or 0.25 mM H3/4 in the sample cell, respectively. All proteins were kept in a buffer consisting of 25 mM Tris-HCl pH 7.5 and 200 mM NaCl. Twenty microliters injection volumes were used for all experiments. Two consecutive injections were separated by 2 min to reset the baseline. The control experiment, consisting of titration of ARB2 against buffer, was performed and subtracted from each experiment. ITC data was analyzed with a single-site fitting model, using Origin 8.6 (OriginLab Corp).A similar amount of purified recombinant GST-ARB2 and GST were immobilized on 100 μl of GST resin. After washing twice (1 ml each time) with buffer C, the beads were incubated with histone octamer for 1 h. Then, the beads were washed thoroughly with buffer C. Finally the beads were mixed with 100 μl of 2 × SDS-PAGE loading buffer and boiled for 10 min at 90 °C. The samples were stained with Coomassie Brilliant Blue.How to cite this article: Shen, H. et al. Structural and histone binding ability characterization of the ARB2 domain of a histone deacetylase Hda1 from Saccharomyces cerevisiae. Sci. Rep.6, 33905; doi: 10.1038/srep33905 (2016).


Peptidyl-prolyl isomerases (PPIases) catalyze cis/trans isomerization of peptidyl-prolyl bonds, which is often rate-limiting for protein folding. SlyD is a two-domain enzyme containing both a PPIase FK506-binding protein (FKBP) domain and an insert-in-flap (IF) chaperone domain. To date, the interactions of these domains with unfolded proteins have remained rather obscure, with structural information on binding to the FKBP domain being limited to complexes involving various inhibitor compounds or a chemically modified tetrapeptide.Thermus thermophilus (TtSlyD) in terms of binding thermodynamics and enzyme kinetics through the use of isothermal titration calorimetry, nuclear magnetic resonance spectroscopy, and site-directed mutagenesis. We show that the affinities and enzymatic activity of TtSlyD towards these peptides are much higher than for the chemically modified tetrapeptides that are typically used for activity measurements on FKBPs. In addition, we present a series of crystal structures of TtSlyD with the inhibitor FK506 bound to the FKBP domain, and with 15-residue-long peptides bound to either one or both domains, which reveals that substrates bind in a highly adaptable fashion to the IF domain through β-strand augmentation, and can bind to the FKBP domain as both types VIa1 and VIb-like cis-proline β-turns. Our results furthermore provide important clues to the catalytic mechanism and support the notion of inter-domain cross talk.We have characterized the binding of 15-residue-long unmodified peptides to SlyD from We found that 15-residue-long unmodified peptides can serve as better substrate mimics for the IF and FKBP domains than chemically modified tetrapeptides. We furthermore show how such peptides are recognized by each of these domains in TtSlyD, and propose a novel general model for the catalytic mechanism of FKBPs that involves C-terminal rotation around the peptidyl-prolyl bond mediated by stabilization of the twisted transition state in the hydrophobic binding site.The online version of this article (doi:10.1186/s12915-016-0300-3) contains supplementary material, which is available to authorized users. Due to unfavorable steric and electronic effects, the cis form is by far the least favored, except for peptidyl-prolyl bonds where the unique N-alkylation of proline markedly reduces the energy difference between the two conformations  and one with the modified tetrapeptide suc-ALPF-pNA bound to the FKBP domain [PDB: 3LUO] /[cis].In order to study binding of the S2-P25A peptide to TtSlyD1H spectra of 500 μM suc-ALPF-pNA tetrapeptide containing 2 μM, 4 μM, or 6 μM of TtSlyD were recorded for the different mutants [cis and the trans states were derived by lineshape analysis of one methyl resonance of the leucine residue, which displays different chemical shifts for the cis and trans states. kcat/KM values were subsequently determined by linear regression of the exchange rate versus enzyme concentration. Because the kcat/KM values are determined by lineshape analysis in an equilibrium experiment, they are not expected to be directly comparable to the kcat/KM values obtained from traditional initial-rate stopped-flow experiments. Nevertheless, our validation experiments (see below) give the same results within the range of errors. Furthermore, the kcat/KM values obtained for a series of mutant enzymes can be directly compared as a measure of relative efficiency, even if the values are not directly comparable to the ones obtained in a traditional experiment.For tetrapeptide activity studies, one-dimensional  mutants . Apparen13C-labeled proline residue. 1H–13C HSQC experiments were used to separate the peptide signals in two dimensions, such that the cis and trans forms could both be detected without overlap. Further analysis focused on the signals from the β and γ position, because they displayed the largest chemical shift differences and appear in isolated regions of the 1H–13C HSQC spectrum. One-dimensional 1H slices of the β and γ protons for the cis and trans forms were extracted from the two-dimensional spectrum, and lineshape analysis was applied. Michaelis-Menten-like studies were performed using a fixed concentration of TtSlyDFL of 0.2 μM and peptide concentrations ranging from 10 to 1000 μM.Activity studies using the 15-residue-long S2-P25A peptide utilized a sample with a uniformly FL and TtSlyDΔIF were concentrated to 30–60 mg/ml. For complex formation, the protein was typically incubated with a threefold excess of peptide or the compound FK506 for at least 1 hour prior to crystallization. Crystals were grown by the sitting-drop vapor diffusion method at 4 °C in 96-well crystallization plates using total drop volumes of 0.2–0.3 μl. Crystallization conditions varied depending on which ligand was used, but in all cases the pH was in the range of 5.0–8.0 and polyethylene glycol (PEG) was used as precipitant , as well as a metal ion coordinated by six histidines, which we have interpreted as a co-purified Ni2+ ion . For all structures, refinement was carried out using iterative cycles of manual rebuilding in Coot [FL:T1 structure where we used anisotropic B-factor refinement without TLS, and the 1.75 Å TtSlyD∆IF:S2-W23A structure, where we used a combination of isotropic B-factor refinement with TLS (protein and peptide chains) and anisotropic B-factor refinement without TLS (ions).We used XDS and XSCALE  for proc program  of the P program . General AutoSol  instead  in Coot  and maxi in Coot . Refinem in Coot . We geneAnalysis of hinge regions was performed using DynDom version 2.0 ; protein


Soft phononic crystals have the advantages over their stiff counterparts of being flexible and reconfigurable. Normally, the band gaps of soft phononic crystals will be modified after deformation due to both geometric and constitutive nonlinearity. Indeed these are important properties that can be exploited to tune the dynamic properties of the material. However, in some instances, it may be that one wishes to deform the medium while retaining the band gap structure. A special class of soft phononic crystals is described here with band gaps that are independent or almost-independent of the imposed mechanical deformation, which enables the design of phononic crystals with robust performance. This remarkable behaviour originates from transformation elasticity theory, which leaves the wave equation and the eigenfrequencies invariant after deformation. The necessary condition to achieve such a property is that the Lagrangian elasticity tensor of the hyperelastic material should be constant, i.e. independent of deformation. It is demonstrated that incompressible neo-Hookean materials exhibit such a unique property. Semilinear materials also possess this property under special loading conditions. Phononic crystals composed of these two materials are studied theoretically and the predictions of invariance, or the manner in which the response deviates from invariance, are confirmed via numerical simulation. Recently, however, attention has switched to the study of compliant or soft phononic crystals (SPCs) due to their potential for flexibility, tunability and multifunctionality. To date, almost all work on SPCs is related to their tunability, i.e. band gaps are tuned via mechanical deformation or some other mechanism such as an imposed electrical or magnetic field.Phononic crystals (PCs) are periodic structures that can control the propagation of acoustic or elastic waves via wave filtering in specific frequency ranges –10. Poteet al. ,,Wi+Wj=0 3=1). In this situation, equation (0+μ0 in equation (ν0 because λ0+μ0=μ0/(1−2ν0). If Poisson's ratio A straightforward case to consider, but which will inevitably lead to stretch-dependence of equation  reduces In addition to the effect of stretch, as described above, the transformed ET 5.homogeneous neo-Hookean and semilinear materials is studied in this section. A homogeneous sample is stretched uniformly along the X1 direction with stretch ratio λ1=Λ. The medium is constrained along the X3 direction, i.e. λ3=1 is imposed. The condition associated with stretch in the X2 direction will be chosen for each example to be considered. For neo-Hookean materials, the focus shall be on the horizontally polarized  and vertically polarized  shear waves, while for the semilinear material the additional longitudinal (P mode) wave is considered.In §X1 direction in an incompressible neo-Hookean material under uniform pre-stretch. The stretch ratios are taken to be λ1=Λ,λ2=Λ−1,λ3=1 so the deformation is isochoric. The Lagrangian wave speed for plane waves can be derived from equation  propagating in the equation  as5.1vLequation , as A212X2 direction, i.e. imposing P22=0, so that the pre-stretch ratios can be shown to be 5.2λ1=Λquations –4.14), , X2 direequation . The Lag1=Λ, λ2=2−Λ and λ3=1. The Lagrangian wave speeds of the in-plane P-SV waves are not affected by the pre-deformation. However, the components of the ET corresponding to SH wave propagation will be affected by the pre-stress. Suppose that an SH wave propagates in a direction defined by an angle θ subtended from the X1 axis. The corresponding Lagrangian wave speed is thenΛ. This effect of induced anisotropy for the SH wave is illustrated in Now consider a third case; the special scenario associated with the imposition of equation , which gTo summarize, the analysis above indicates that the Lagrangian wave speed of shear waves in an incompressible neo-Hookean material is independent of the deformation. Hence, the neo-Hookean material has potential in designing PCs with deformation-independent band gaps for shear wave modes. On the other hand, the semilinear material cannot exhibit such total invariant behaviour. Having said this, the Lagrangian wave speed of plane waves in a deformed semilinear material are unaffected (for P waves) or slightly affected (for SV and SH waves) by pre-deformation. Hence, one should expect that band gaps in semilinear PCs can be almost-independent of the pre-deformation in general. For a specific case, the semilinear material may exhibit a constant in-plane ET for the special pre-deformation mode as in equation . This gi6.(a)a) is considered with cylinders organized on a square lattice. For the medium of type (iii), the inclusion phase is stiff enough so that its ET is independent of deformation since the induced strain is infinitesimal. For the type (ii) material the inclusion is a void. The matrix phase is neo-Hookean in the first example, and semilinear in subsequent examples. The phononic band structures are calculated by using the finite-element package ABAQUS (v. 6.13-1) and adopting the method proposed by Åberg & Gudmundson [Based on the above analysis, a PC comprising an incompressible neo-Hookean material will have deformation-independent band gaps for shear waves, while one composed of a semilinear material will have band gaps that are almost-invariant for all wave modes. Full in-plane invariance of the semilinear material is achievable for the special deformation case given in equation , which lfigure 2dmundson ,13. One (b)I1= U2, J= det F, the shear modulus μm=25.9 MPa, bulk modulus κm=10μm and density ρm=1000 kg m−3. The cylindrical inclusions are taken to be aluminium with shear modulus μc=25.9 GPa, Poisson's ratio νc=0.33, and density ρc=2700 kg m−3. Additionally in this example, uniaxial stretching  is applied to the PC, as illustrated in figure 5b. For the stretch deformation, the lattice deformation gradient components are FL11=1+ϵ, FL12=FL21=0 and FL22 is determined by the stress-free condition on the lateral sides. The band structures for the undeformed and stretched PCs are shown in figure 5a,b, respectively, with dispersion curves ωn(K) calculated along the path Γ-G1-M-Γ-G2-M in the Brillouin zone . The dimensionless frequency is defined asω is the angular frequency and a=0.1 m is the characteristic length of the unit cell. Additionally, the radius of the cylindrical inclusions is 0.03 m. By comparing the band gaps in x1−x2 mode) disappears after stretching. This is because in this case the ET components associated with in-plane waves are deformation-dependent. The stretch deformation induces symmetry breaking of the Brillouin zone so that the G1 and G2 points are no longer equivalent. This is seen by considering the third band ω3 associated with in-plane wave modes in figure 5b. By contrast, the position of the two band-gaps associated with antiplane waves (the x3 mode) remain unchanged after stretching since the neo-Hookean material with strain energy function defined in m=50.4 MPa, shear modulus μm=25.9 MPa and density ρm=1000 kg m−3. The material configuration is the same as for the neo-Hookean material example considered in the previous section , except that now the compliant matrix is semilinear. Because the semilinear medium is not completely invariant to deformation, two different deformation modes, stretch and shear, are considered for this example. The resulting band structures of the semilinear SPC are shown in FL12=γ, FL21=0, and FL11 and FL22 are determined from the free normal stress conditions on the four sides.Consider an SPC with semilinear hyperelastic matrix medium with Lamé constant λa, only one band gap is observed for in-plane waves while two band gaps are determined for antiplane waves in the undeformed state. In the deformed state, the band gaps are slightly tuned, as shown in figure 6b for the stretching case (ϵ=0.15) and figure 6c for the shearing case (γ=0.3), respectively. The dependence of the pre-deformation on the band gaps is more clearly shown in figure 6d,e for the stretch and shear deformation cases, respectively. It is observed that the stretch deformation will affect the band gaps slightly, while the influence of the shear deformation on the band gaps is negligible. One explanation for this difference is that the maximum material stretch in the case of shearing is less than that in the case of uniaxial stretching. It is thus concluded that the band gaps in semilinear SPCs are slightly dependent on the pre-deformation and the influence also depends on the deformation mode considered. The advantage of using materials of semilinear type in SPCs then is that they exhibit relatively robust band gaps for both P-SV and SH waves. This more general case has broader significance and potential applications than using a neo-Hookean material, for which SPCs exhibit invariance only for the antiplane wave mode.The ET x3 modes. It is found that the two dispersion curve branches, which originally coincide at point M in figure 6a, become separated in the corresponding band structures in figure 6b,c. This is actually induced by the symmetry breaking of the PC once the square lattice is distorted due to the deformation [x3 modes. However, these two branches are slightly separated at point M in figure 5b due to the existence of the stiff elastic cylinders. Detailed analysis of the symmetry breaking is quite complicated and will be discussed in future work.Another interesting phenomenon observed in ormation ,40, e.g.X3 direction  while the other two directions are left to be load-free. In this case, the in-plane deformation is homogeneous everywhere (λ1=λ2=1+νm−νmλ3) and it can be verified that equation , i.e. solid–void, with semilinear matrix. Fully invariant P-SV band gaps are possible when the medium is stretched in the equation  is satisequation , while tIn summary, a material of type (iii)  has been shown to exhibit invariant band gaps for SH waves (for arbitrary deformations), whereas a medium of type (ii)  exhibits invariant band gaps for P-SV waves .7.It has been demonstrated here that it is theoretically possible to design SPCs with deformation-independent band gaps or band gaps that are close to being deformation-independent. This was achieved by appealing to hyperelastic transformation theory. This apparently unusual phenomenon has particular utility in engineering applications requiring both robust frequency response and structural flexibility. In order to achieve this non-trivial property, the key issue is to find materials with deformation-independent ETs so that the Lagrangian-formulated incremental equations possess the property of being invariant to deformation, which is the key notion of transformation elasticity theory. It has been proved that only incompressible neo-Hookean materials satisfy such a constraint exactly . Semilinear hyperelastic materials exhibit almost-constant ETs for the deformation modes considered here and in-plane invariance for specific materials under axial stretch. It was indicated that the invariance of the band gaps is associated with the invariance of the Lagrangian wave speeds in the medium, which are derived from transformation elasticity. Numerical simulations illustrate that a specified SPC with semilinear hyperelastic matrix and aluminium stiff cylindrical inclusions does indeed have band gaps that are almost-deformation-independent for wave modes in the frequency ranges considered. An example that considered the same cylindrical inclusions in an almost-incompressible neo-Hookean medium on the other hand illustrated that only SH wave band gaps are deformation independent, in accordance with the theoretical predictions. An example involving a solid–void PC of the semilinear type illustrated that deformation-independent P-SV band gaps are possible, but only when the structure is stretched axially. This can be thought of as the counterpart to the invariant SH band gap case for neo-Hookean materials, although while the neo-Hookean case holds for arbitrary deformation the semilinear SPC is only fully invariant under axial stretch. Although the deformation-independence is verified for the Bragg-type band gaps in this work, other types of band gaps, e.g. the one caused by local resonance, are expected to be deformation-independent as long as the Lagrangian-formulated wave equations are invariant.Future work could be directed towards exploring SPCs of other types, as well as the more practical issue of the design and fabrication of polymeric materials that behave as per the suggested strain energy functions. For example, many soft rubbers such as silicone and neoprene  behave a


Trifunctional PutAs also have an N-terminal ribbon–helix–helix (RHH) DNA-binding domain and moonlight as autogenous transcriptional repressors of the put regulon. A unique property of trifunctional PutA is the ability to switch functions from DNA-bound repressor to membrane-associated enzyme in response to cellular nutritional needs and proline availability. In the present study, we attempt to construct a trifunctional PutA by fusing the RHH domain of Escherichia coli PutA (EcRHH) to the bifunctional Rhodobacter capsulatus PutA (RcPutA) in order to explore the modular design of functional switching in trifunctional PutAs. The EcRHH–RcPutA chimaera retains the catalytic properties of RcPutA while acquiring the oligomeric state, quaternary structure and DNA-binding properties of EcPutA. Furthermore, the EcRHH–RcPutA chimaera exhibits proline-induced lipid association, which is a fundamental characteristic of functional switching. Unexpectedly, RcPutA lipid binding is also activated by proline, which shows for the first time that bifunctional PutAs exhibit a limited form of functional switching. Altogether, these results suggest that the C-terminal domain (CTD), which is conserved by trifunctional PutAs and certain bifunctional PutAs, is essential for functional switching in trifunctional PutAs.Proline utilization A (PutA) is a bifunctional flavoenzyme with proline dehydrogenase (PRODH) and Δ In the rganisms  and haveCDH . In rganisms  and schirganisms –9. Prolinfection ,11 and i in mice .Escherichia coli and Salmonella typhimurium encode PutAs with a DNA-binding domain, which allows PutA to repress transcription of putA and putP (proline transporter) genes  is total concentration of protein [DNA-binding activity was determined by gel mobility shift assays using fluorescently labelled escribed . A disso protein .1FractiE. coli polar lipids (Avanti Polar Lipids) [Lipid pull-down assays were performed anaerobically under a nitrogen atmosphere in an anaerobic chamber (Belle Technology Glovebox) as described previously using  Lipids) . In thesE. coli and has a molecular size on SDS/PAGE consistent with the predicted M of 126 kDa. The UV–visible spectrum of EcRHH–RcPutA has absorbance maxima at 380 nm and 451 nm showing incorporation of flavin into the protein fold. The flavin absorbance spectrum indicates 92% flavin content. The incorporation of one FAD per protein monomer is consistent with previous and recent studies of RcPutA [EcRHH–RcPutA was expressed as a soluble protein in f RcPutA  and othef RcPutA ,30,31.Km and kcat values for proline as the variable substrate are 5.3 mM and 0.89 s−1, which are close to those of RcPutA (5.6 mM and 1.0 s−1). The catalytic efficiencies of the chimaera and RcPutA differ by just 6%. Using CoQ1 as the variable substrate, the parameters are Km of 161 μM and kcat of 1.1 s−1. These values are within a factor of two of those of RcPutA. These results show that fusing the EcPutA RHH domain on to RcPutA does not significantly affect the PRODH kinetic parameters.The PRODH steady-state kinetic parameters of EcRHH–RcPutA were determined for comparison to those previously reported for RcPutA . The Km Coupled PRODH–P5CDH assays were performed to further explore the kinetic properties of EcRHH–RcPutA. These assays report on the activity of both the PRODH and P5CDH active sites by monitoring NADH formation, which is used as a read out of the overall conversion of proline to glutamate. The domain organization of RcPutA provides an excellent opportunity to explore the impact of the RHH and CTDs on the oligomeric organization of PutAs. Previous studies have shown that the RHH domain mediates dimerization of EcPutA . In contM of approximately 315 kDa whereas RcPutA elutes at an apparent M of approximately 122 kDa consistent with that reported recently for RcPutA [EcRHH–RcPutA and RcPutA were first analysed by gel filtration chromatography. r RcPutA . These rM value of 256 kDa (M of 251 kDa). Thus, fusion of the EcPutA RHH domain induces dimerization of RcPutA.EcRHH–RcPutA was also analysed by analytical ultracentrifugation. Sedimentation equilibrium experiments were performed at three protein concentrations. The equilibrium data for EcRHH–RcPutA B were fi 256 kDa . These rRg, Dmax, M and molecular shape envelope [Rg values of 49.5–51.9 with an average of Rg=51±1 Å (r)) suggest Rg of 53–56 Å for assumed Dmax of 170–210 Å (Rg and Dmax of RcPutA are only 32 Å and 107 Å respectively [r) curve for EcRHH–RcPutA exhibits a major peak at r=42 Å with a shoulder on the high r side of the major peak. This distribution of interatomic vectors resembles that of EcPutA (r) curve for RcPutA is monomodal with maximum at r=37 Å  as described above. The estimated Porod volume is 383000 Å3. The assumption of a dimeric protein leads to a protein density of 1.1 g/ml, which is well within the characteristic range for proteins of 0.9–1.5 g/ml [The c method  and Poroc method   for EcRHH–RcPutA with put control DNA was determined by varying EcRHH–RcPutA concentration in the binding assays as shown in n=1.2), from which a Kd value of 58±20 nM was estimated. This value is nearly the same as that determined previously for EcPutA (Kd=45 nM) [From the analysis above, it appears EcRHH–RcPutA adopts a similar oligomeric state and spatial arrangement of the DNA-binding and catalytic domains as that of EcPutA. We next sought to determine if EcRHH–RcPutA exhibited the functional switching properties of EcPutA. The DNA-binding activities of EcPutA, RcPutA and EcRHH–RcPutA were compared using gel mobility shift DNA-binding assays. d=45 nM) .E. coli polar lipid vesicles in the absence and presence of 50 mM proline. After incubation for 1 h, the soluble and lipid fractions were separated by centrifugation and analysed by SDS/PAGE to monitor PutA partitioning in the soluble and lipid fractions. Lipid pull-down assays were used to test whether the membrane binding properties of RcPutA and EcRHH–RcPutA are regulated by proline reduction of the flavin cofactor as observed for EcPutA ,26. RcPuFusing the EcRHH domain to RcPutA generated a stable EcRHH–RcPutA dimer providing evidence that dimerization in trifunctional PutAs is mediated through the DNA-binding domain ,46. KineB. japonicum was shown to be insensitive to proline [R. capsulatus, putA expression is activated in response to proline by the PRODH activator, PutR [Our findings suggest the trifunctionality of type C PutA derives from a dimeric structure anchored by a RHH domain that is sandwiched between two catalytic lobes. The spacing of the catalytic lobes is likely critical to enable the RHH domain to access DNA. Although the oligomeric state and catalytic properties of RcPutA were previously determined, the membrane binding properties RcPutA were unstudied . In the  proline . The phyor, PutR . PutR coor, PutR . NeverthThe CTD may be an element of the functional switching apparatus, since this domain is absent in type A PutAs and present in types B and C PutAs. Recent studies of RcPutA suggested that the CTD is a structural domain that couples the PRODH and P5CDH domains, and facilitates P5CDH activity and substrate channelling . The dat


Teneurins are ancient cell–cell adhesion receptors that are vital for brain development and synapse organisation. They originated in early metazoan evolution through a horizontal gene transfer event when a bacterial YD-repeat toxin fused to a eukaryotic receptor. We present X-ray crystallography and cryo-EM structures of two Teneurins, revealing a ~200 kDa extracellular super-fold in which eight sub-domains form an intricate structure centred on a spiralling YD-repeat shell. An alternatively spliced loop, which is implicated in homophilic Teneurin interaction and specificity, is exposed and thus poised for interaction. The N-terminal side of the shell is ‘plugged’ via a fibronectin-plug domain combination, which defines a new class of YD proteins. Unexpectedly, we find that these proteins are widespread amongst modern bacteria, suggesting early metazoan receptor evolution from a distinct class of proteins, which today includes both bacterial proteins and eukaryotic Teneurins. Teneurins are cell-cell adhesion receptors that evolved through horizontal gene transfer in which a bacterial YD-repeat protein fused to a eukaryotic receptor. Here the authors present crystallographic and cryo-EM structures of two Teneurins, revealing an ancient YD-repeat protein super-fold. The intracellular domain of Teneurin is proteolytically cleaved and acts as a transcriptional repressor8. Homologue-specific Teneurin-mediated cell adhesion is crucial for neural wiring, e.g., in axon targeting and circuit formation of the visual system9 and in synaptic organisation7. Elegant recent experiments further reveal the importance of Teneurin in the wiring of the hippocampus, highlighting the importance of alternatively spliced loops that regulate homophilic Teneurin binding10. Teneurins have been linked with cancers11 and specific neurological disorders14. For example, a missense mutation in Ten1, P1610L, is associated with congenital general anosmia (the inability to smell), and mice carrying this mutation display an olfactory deficit15.Teneurins  are functionally important type II single-pass transmembrane proteinstrans interactions across the synapse. It contains eight membrane-proximal epidermal growth factor-like repeats (EGF1–8), which bear similarity to the vertebrate extracellular matrix protein tenascin18 and harbour intermolecular disulphide bonds that covalently dimerise Teneurins20. The EGF-like repeats are followed by a large 1850-residue segment of unknown structure, in which few parts have been annotated. A part of this segment has been derived from a bacterial tyrosine-aspartate (YD) repeat toxin by a horizontal gene transfer event during early metazoan evolution, an event that may have contributed to the evolution of multicellularity21. The YD-repeat motif has been structurally characterised in the context of heterodimeric bacterial toxin complexes (TcB-TcC), revealing that it forms a protective shell-like structure, encapsulating the proteolytically cleaved toxic C termini of these proteins23. In addition, the 1850-residue segment contains an ‘NCL-1, HT2A and Lin-41’ (NHL) domain that defines specificity for homophilic Teneurin trans interactions24.The Teneurin extracellular region defines its adhesive specificity and controls both homophilic and heterophilic 15. Here we present X-ray and electron microscopy (EM) structures for two out of four Teneurin (Ten2 and 3) ectodomains, revealing a ~200 kDa super-fold with a 170 kDa rigid core. Eight sub-domains, of which five were previously unreported, form an intricate structure centred on a spiraling YD-repeat shell domain. Unlike in known bacterial toxin structures of the YD-repeat family23, the ancient, now inactive C-terminal cytotoxic domain of Teneurin is included in our structure and, unexpectedly, lies outside of the shell. The shell is ‘plugged’ at the N-terminal side via a fibronectin (FN) and plug domain combination, which we also observed in current, structurally uncharacterized bacterial YD-repeat proteins. Taken together, our data provide structural insights into early receptor evolution, YD-repeat protein architecture and Teneurin function.Lack of detailed structural information on the Teneurin extracellular region has hampered progress with understanding Teneurin functions and related neurological diseasesCT, residues 955–2802, Fig. CT, residues 846–2715, Fig. CT, Supplementary Fig. CT) to 2.4 and 3.8 Å resolution, respectively. This combination of structural techniques allowed us to assess the flexibility of TenCT. Comparison of the two structures reveals a structurally conserved core fold with two flexibly attached domains . Indeed, the human Teneurin paralogues share 58–70% sequence identity, suggesting that the large extracellular Ten structure is similar for all four mammalian paralogues.Here we reveal a conserved and surprisingly compact domain arrangement for the ~200 kDa C terminus of chicken Ten2 -like domains and attaches peripherally onto the super-fold. TTR-like domains are widely distributed in species of bacteria, plants and animals, and gain their names from their structural similarity to TTR, a transport protein for thyroid hormones and retinol25. Other TTR-like domains have roles in protein aggregation26 and lipid recognition27.Previously unpredicted, the structures reveal three β-stranded domains upstream of the Teneurin NHL domain. The most N-terminal domain, resolved in Ten2CT. The FN-plug domain forms numerous hydrogen-bonding and hydrophobic interactions with the shell interior 28. The interface is formed by multiple hydrogen bonds and hydrophobic interactions . This domain is the most structurally conserved region of Teneurins and the TcB-TcC toxin complexes  for the protein with highest sequence identity to this region of chicken Ten2 (a protein from Bacillus subtilis (B. subtilis), NCBI accession WP_08111228.1, 27% identity; Supplementary Fig. Downstream of the NHL lies a series of YD or rearrangement hotspot (Rhs) repeats, forming a striking ~800 residue YD-shell domain Fig. . It formain Fig. . Given tA linker seals the YD shell and threads through it. The Ten2CT FN-plug and YD-shell combination encloses a volume of ~130 nm3. The residues downstream of the Rhs-associated core (residues 2467–2592 in Ten2CT and 2382–2508 in Ten3CT) are located within the shell itself. This ‘internal linker domain’ is well resolved and has no detectable structural homologues  maps to a highly conserved area in the Ten2CT, Fig. 32. In our Ten2CT structure, the Tox-GHH domain reveals highest structural similarity to the catalytic domains of the colicin DNAse family of bacteriocins  domains and a TTR-like domain upstream of its Teneurin-like core domain , an additional domain of unknown structure and function (~180 residues) separates the FN-plug and TTR in the B. subtilis protein. This domain of unknown function persists in the ancient Teneurins encoded by Monosiga brevicollis and Branchiostoma sp.21, but is absent in Teneurin proteins encoded by arthropods and higher chordates. Future analysis of this domain should provide further insights into the early evolution of Teneurins from bacterial proteins.Teneurins have recently emerged as vital specificity-determining cell–cell adhesion receptors41. The Rhs-associated core domain of the bacterial Teneurin-like proteins shares higher sequence identity with that of TcC proteins than with the Teneurins, including sequence conservation of the auto-proteolytic cleavage site itself (which is not conserved in the Teneurins).Amongst the bacterial Teneurin-like proteins, diverse uncharacterised C-terminal regions lie downstream of the YD repeats and Rhs-associated core domain, suggesting that the Teneurin-like core fold evolved as a platform or delivery machine to which diverse variable domains have been added. This is in agreement with previous reports suggesting that YD repeats and their associated C-terminal regions are evolutionarily decoupled in bacteriaHomology modelling of the region downstream of the auto-proteolytic site reveals that some bacterial Teneurin-like proteins show similarity to lipases in this region, including conservation of catalytically important histidine residues . Together these observations suggest that the bacterial Teneurin-like proteins could be cleaved in their Rhs-associated core domain and could function as toxins.23), but has it merely been retained in Teneurins as a vast surface for protein–protein interaction? Suggestively, the gap in the YD-shell that forms the exit site for the C-terminal linker region in mammalian Teneurins coincides with the interface between the two separate subunits (B and C), of the TcB-TcC toxins. This prompts us to speculate that folding-intermediates, or indeed alternate functional states of Teneurin may exist, in which the shell is opened up at that position. Moreover, the Ten2CT YD shell + FN-plug encloses a space of ~130 nm3. The C termini of the bacterial Teneurin-like proteins typically range from 100–150 amino acids in length, and are therefore not dissimilar to the length of the internal linker we report for Teneurin. This suggests that the C termini of the bacterial Teneurin-like proteins, some of which encode putative toxic activity, could be shielded within the YD shell, similar to what is observed for TcB-TcC toxins.The intricate nature of the fold begs the question, which functions are encoded in the conserved core of Teneurin and Teneurin-like proteins. The YD-shell of bacterial Teneurin-like proteins may act to hide a toxin that can be revealed upon a trigger  or array-like, reminiscent of assemblies postulated for other neuronal homophilic cell adhesion receptors, such as the protocadherins42. How heterophilic ligand interaction impacts on Teneurin structure and assembly-formation will need to be established.In multicellular animals, the addition of EGF domains to the Teneurin fold has added covalent 24 (Q9DER5)  and mouse Lphn2Lec  into the AgeI-KpnI cloning sites of vectors from the pHLSec family43. Ten2CT for crystallisation was fused to a C-terminal hexahistidine tag and Ten2ecto (both wild type and P1680L) for SPR was fused to a C-terminal AviTag and hexahistidine tag. The plasmid encoding full-length mouse Ten3 lacks amino acids 741–749 (AHYLDKIVK) and amino acids 1219–1225 (RNKDFRH) . Ten3CT (residues 846–2715) was subcloned into the BamHI/NotI restriction sites of the pUPE107.03 vector . Primers used for cloning and mutagenesis are shown in Supplementary Table We cloned constructs of chicken Ten243. Glycan maturation was inhibited during protein expression as specified below, to allow the structural analysis of homogenously glycosylated protein samples. Ten2 proteins were produced in kifunensine-treated HEK293T cells (American Type Culture Collection (ATCC) CRL-3216). Four days post transfection the cell culture medium was clarified by centrifugation and filtration and diafiltrated into 20 mM Tris (pH 7.5) and 500 mM NaCl. Ten3CT was expressed in acetylglucosaminyltransferase I-deficient Epstein–Barr virus nuclear antigen I-expressing HEK293 cells  and 6 days post transfection Ten3CT-containing medium was diafiltrated into 25 mM HEPES (pH 7.8) and 500 mM NaCl. All proteins were purified by Ni-NTA affinity chromatography and size-exclusion chromatography using an S200 16/60 column (Ten2 constructs) or a Superose6 column . For Ten2 constructs the gel filtration (GF) buffer was 20 mM Tris (pH 7.5) and 200 mM NaCl. The GF buffer for Ten3CT was 20 mM CHES (pH 9) and 150 mM NaCl. Lphn2Lec for SPR was produced in HEK293T cells without kifunensine. The medium was harvested 2 days post transfection and dialysed against phosphate-buffered saline (PBS) at 4 °C for 24 h.We expressed all proteins in a secreted form using transiently transfected HEK293 cellsCT was purified as described above and concentrated to 5 mg ml−1 in 10 mM Tris (pH 7.5), 200 mM NaCl and 200 mM non-detergent sulfobetaine (NDSB-256). Initial, weakly diffracting crystals were grown using the vapour-diffusion method in sitting drops at 18 °C by mixing protein solution and crystallisation solution in a 1:1 ratio. The crystallisation solution was 0.1 M Tris (base)/Bicine (pH 8.5), 20% v/v glycerol, 10% w/v PEG4K, 30 mM di-ethyleneglycol, 30 mM tri-ethyleneglycol, 30 mM tetraethyleneglycol and 30 mM pentaethyleneglycol. Crystal quality was optimised by a combination of varying the drop ratio, linearly reducing the concentration of all components of the crystallisation solution and microseeding. Pt-derivative crystals were produced by adding K2PtCl4 in powder form to the drop containing Ten2CT crystals and incubating for 21 h. Derivative crystals were back-soaked in crystallisation solution prior to flash-cooling. All crystals were cryo-cooled in the crystallisation solution described above.Ten2λ = 0.97626 Å). Wedges of native data having suffered the least radiation damage were selected for merging to produce the final native data set 45.Due to the radiation sensitivity of the crystals, 90° wedges of native diffraction data from three isomorphous crystals were collected up to 2.4 Å resolution on Diamond Light Source at 100 K . Data were processed as above, but Bijvoet pairs were unmerged  accession 3HRP). The resulting model was subsequently used for iterative phase improvement using the Phaser SAD pipeline49 and manual re-building in Coot48. Partial sequence assignment was facilitated by homology models of the Ten2 Rhs-associated core domain and YD-shell region based on the structure of YenC2 (PDB 4IGL)22 and the knowledge that Pt atoms typically bind to either methionine or histidine residues.For anomalous data collection, data sets from two isomorphous crystals were collected at the European Synchrotron Radiation Facility (beamline ID29) at 100 K. To maximise anomalous signal, data were collected at the L-III Pt edge  restraints54. Thermal motion was parameterized by treating each macromolecular chain as a single translation libration screw group. The quality of the final model was assessed using MolProbity55.The resulting structure was used to phase the higher-resolution native data by molecular replacement with PhaserCT in GF buffer was diluted in MilliQ water to a final salt concentration of 75 mM NaCl and a final protein concentration of 100 μg ml−1. Three-microlitre aliquots of Ten3CT were applied onto glow-discharged holey carbon Quantifoil R2/2 grids. Grids were blotted for 2 s at 95% humidity with a blot force of −2 using a Vitrobot Mark IV (FEI). Grids were stored in liquid nitrogen until further use.Purified Ten3CT data set was collected on a Talos Artica (FEI) operating at 200 kV, equipped with a GIF quantum energy filter (Gatan) and K2 Summit camera (Gatan). Movies were collected in super-resolution counting mode at a nominal magnification of 130 000, corresponding to a physical pixel size of 1.03 Å at the specimen level. The exposure time was 7.2 s, every 0.4 s a frame, corresponding with an electron dose of 56 e− Å−2 for the integrated image and 3.1 e− Å−2 per frame. A total of 2775 micrographs were collected using EPU software (FEI), with a defocus range of −1.2 to −3.0 µm.The Ten356. After CTF modelling with Gctf57, micrographs with resolution estimates to 5 Å or better at CC = 50% were selected . The particle coordinates were further improved by particle polishing in Relion2.0 and local CTF determination in gCTF and imported back into cryoSPARC for a final refinement. The two resulting half maps were imported to Relion2.0 to calculate the Fourier Shell Correlation (FSC) by generating a mask that was extended by 2 Å and by adding a cosine-shaped soft edge of 4 Å. This resulted in a resolution of 3.8 Å at the FSC = 0.143 resolution criterion. Local resolution estimates were determined by the local resolution algorithm in Relion2.0 that samples the map with a soft-spherical mask.All micrographs were corrected for beam-induced motion and drift using MotionCor2CT as a template a model for Ten3CT was generated in Modeller v9.1660. The TTR domain (residues 872–964), the ABD and Tox-GHH domains (residues 2513–2715) and residues 1219–1228, 1401–1411, 2413–2424 and 2487–2496 were omitted from the model due to lack of density in the Ten3CT density map. The structure was refined by manual re-building in Coot48 and by real-space refinement in Phenix61 , where C is analyte concentration calculated as monomer).Equilibrium binding experiments were performed at 25 °C using a Biacore T200 instrument  using PBS + 0.005% (v/v) polysorbate 20 (pH 7.5) as running buffer. The regeneration buffer was 2 M MgClCT) and 6FAY (Ten3CT). The cryo-EM density map for Ten3CT has been deposited in the Electron Microscopy Data Bank under the accession number EMD-4219.Data supporting the findings of this manuscript are available from the corresponding authors upon reasonable request. Atomic coordinates have been deposited in the PDB with accession numbers 6FB3 (Ten2Supplementary Information


The role of Cdc45 in genomic duplication has remained unclear since its initial identification as an essential replication factor. Recent structural studies of Cdc45 and the evolutionarily-related archaeal GAN and bacterial RecJ nucleases have provided fresh insight into its function as co-activator of the MCM helicase. The CMG helicase of the last archaeal/eukaryotic ancestor might have harboured a single-stranded DNA nuclease activity, conserved in some modern archaea.  •The structure of human Cdc45 provides insight into its function in DNA replication as co-activator of the MCM helicase.•Structural analysis of human Cdc45 and bacterial RecJ illustrates the structural elements of their shared nuclease fold.•The structure of the archaeal GAN nuclease shows that it is an orthologue of eukaryotic Cdc45.•The Cdc45 subunit of the ancestral predecessor of archaeal and eukaryotic CMG helicases was a single-stranded DNA nuclease. In the case of Cdc45, the DHH residues coordinating the catalytic metal ions are found to be consistently mutated, and therefore Cdc45 has no catalytic activity. An unexpected feature of the structure of human Cdc45 is the presence of an intra-molecular tether between DHH and DHHA1 domains, formed by the insertion of invariant DHHA1 residue F542 into a hydrophobic pocket on the DHH surface c. As a rA second striking aspect of the Cdc45 structure is the unusual conformation of a large insertion present in the C-terminal of the DHH domain c. The inCMG-interacting domain or CID, located after the N-terminal DHH domain and before the three-helix bundle of the RecJ/Cdc45 fold. Conversely, the interaction with GINS affects a distinct site on the Cdc45 surface, mainly comprising its N-terminal DHH domain. Neither of the two interfaces appears sufficiently large to confer stability to a binary Cdc45-MCM or Cdc45-GINS interactions, in support of the current step-wise model of CMG assembly requiring the regulated intervention of conserved replication factors such as Sld3/Treslin to chaperone Cdc45 to the helicase ring.Recent cryoEM analyses of yeast and fly CMG have elucidated the relative position of Cdc45 within the helicase assembly and, together with crystallographic data, helped define its interaction interfaces with MCM and GINS 3Thermococcus kodakaraensis, which bound to and was stimulated by the Gins51 subunit of the GINS heterodimer An important piece of the Cdc45 puzzle was solved recently with the determination of the crystal structure of the archaeal GAN, alone and in complex with the C-terminal domain of the Gins51 subunit (Gins51C) Thermococcus kodakaraensis, as well as the CMG of other archaeal organisms possessing GAN orthologues These biochemical and structural observations suggest that the CMG helicase assembly of 4Deinococcus radiodurans RecJ bound to a oligodeoxynucleotide substrate Possible insights into the mechanism of GAN interaction with ssDNA can be gleaned from the recently determined co-crystal structure of Given the remarkable degree of GAN's structural similarity with RecJ and same 5′-to-3′ polarity of digestion, it is reasonable to assume that the RecJ-ssDNA provides a reliable template to predict the geometry of the GAN-ssDNA interaction. One important difference concerns the path of ssDNA as its 3′-end emerges from the back of the enzyme. Whereas in RecJ the 3′-DNA end engages in further interactions with an OB domain, the same trajectory in GAN would lead to a steric clash with its CID b. IntrigFurther extrapolation of these considerations to Cdc45 and the eukaryotic CMG would require in the first place a conformational change leading to the physical separation of the DHH and DHH1 domains, to allow Cdc45 to engage with ssDNA. No evidence for such rearrangement is available at the moment, although it should be noted that Cdc45 is poised to interact with other components of the replisome, beyond MCM and GINS, that might conceivably drive such change, under specific yet-unknown circumstances. Of note, Cdc45’s tertiary structure contains conserved elements of secondary structure, such as the long helical insertion in its DHH domain c, that a5bona fide component of the archaeal CMG, and consequently a role for its nuclease activity is likely to exists in DNA replication. The recent reconstitution and biochemical characterization of an archaeal CMG In summary, these recent findings define at atomic level the Cdc45/RecJ fold, explain how Cdc45 interacts within the eukaryotic CMG and highlight some unexpected features of the Cdc45 structure that are likely to be relevant for replisome function. They further show that the GAN nuclease must be considered a


Collagen plays a fundamental role in all known metazoans. In collagens three polypeptides form a unique triple-helical structure with a one-residue stagger to fit every third glycine residue in the inner core without disturbing the poly-proline type II helical conformation of each chain. There are homo- and hetero-trimeric types of collagen consisting of one, two or three distinct chains. Thus there must be mechanisms that control composition and stagger during collagen folding. Here, we uncover the structural basis for both chain selection and stagger formation of a collagen molecule. Three distinct chains  of the non-collagenous domain 2 (NC2) of type IX collagen are assembled to guide triple-helical sequences in the leading, middle and trailing positions. This unique domain opens the door for generating any fragment of collagen in its native composition and stagger. Collagen is the most abundant protein in the human body and is the major building block for bone, cartilage, tendon, ligament and skin. Collagen molecules are crucial to tissue organization and physiology with their functions ranging from bulk mechanical strength to delicate instructions to cell receptors.Every molecule of the 28 types of collagen in humans or other collagen-like proteins contains a collagenous fragment with repeated G-X-Y sequences, where X and Y are any amino acid but often proline and hydroxyproline residues, respectively. Three chains associate with a one-residue shift  to fit glycine residues in the inner core. This type of packing was confirmed by multiple crystal structures of collagen and collagen-like fragments2345910The collagen repeating sequence has an intrinsic ability to form irregular alignments, which under certain conditions leads to a formation of a gel, also known as gelatin. To avoid such complications every type of collagen has a unique trimerization domain that selects and aligns three specific chains1314161812There are other collagens that use different types of trimerization domains. Such domains greatly vary in size, position and structure with the smallest known domain discovered in type IX collagen of only ~35 residues4-(GXY)4-(GPP)3, where (GXY)4 sequences are from the α1 and α2 chains of human type I collagen, were recombinantly linked to chains A, B and C  of the NC2 domain in the following combinations designated as α1Aα1Bα1C (for short 111), α1Aα1Bα2C (112), α1Aα2Bα1C (121), α2Aα1Bα1C (211) and α2Aα2Bα2C (222) All five constructs were screened for crystallization, but only three of them yielded crystals of sufficient quality, crystal structures for 111, 211 and 121 were independently solved using the MAD phasing from selenomethionine derivatives  to 2.25 Å, 2.10 Å and 1.6 Å, respectively , Table 1In accord with the secondary structure prediction2222e.g. in lung surfactant protein DIt was suggested that many collagens contain α-helical coiled coil domains that might help in trimerization and stagger formationAα2Bα1 C) translates into staggering order of α2α1α1, whereas 211 translates into α1α2α1.The overall structure of the triple helical sequences is typical for the structure of a triple helix. Despite the variations of composition (homo-trimeric for 111) and stagger (121 or 211) that might not represent a native stagger, the regions of sequences derived from type I collagen are well structured. A set of unique side chain interactions is observed for each composition . The mos(G)…O = C(X) hydrogen bonds (characteristic collagenous bonds between glycine in one chain and an amino acid in X position of an adjacent chain) that form within the triple helix and the beginning of the NC2 domain (4-(GXY)4-(GPP)3 sequences linked to the NC2 domain. Moreover, first glycine residues that were originally assigned to the beginning of the NC2 domain are still part of the triple helix without any sign of disturbance. Even an alanine residue  in the leading chain (chain B) does still form a reliable hydrogen bond with lysine 37 in the trailing chain (chain C) (The right-handed bundle of α helices (not a very common structure) of the NC2 domain congruently continues into the right-handed superhelix of the collagenous part. Visual analysis of the interface region suggests a broadening of the triple helical end before the NC2 domain starts. To analyze and quantify the opening of the triple helix and transition into the NC2 domain we identified and plotted the ladder of recurrent N–H2 domain . Remarkachain C) .The actual opening of the “triple helix” happens only at already the non-collagenous sequence of the NC2 domain , where r10-foldon constructCollagen is the most plentiful protein in our body fulfilling structural and biologically active roles in multiple physiological processes as well as in pathology. Numerous heritable and acquired diseases are associated with collagen. Atherosclerosis, fibrosis, osteoarthritis, rheumatoid arthritis, diabetes, cancer are just few diseases where collagen function is adversely affected. 28 collagen types are formed from polypeptides encoded by 42 distinct genes, frequently in several isoforms. In addition, more than 20 additional proteins adopt collagen-like structures such as collectins, ficolins, and scavenger receptorsin vitro make this system easily adoptable in any laboratory with basic molecular biology techniques. If needed the expression system can be transferred to eukaryotic cells to obtain certain post-translational modifications of proline and lysine residues. Moreover a peptide synthesis with specifically modified residues is still possible for these sizes.As revealed by the structural analysis of the host-guest system reported here, such a method is now available. A collagenous sequence connected to the α2 chain of the NC2 domain will have the leading position, whereas collagenous sequences linked to α1 and α3 chains will be in the middle and trailing positions, respectively. To avoid confusion we suggest to label α1, α2 and α3 chains of the NC2 domain as chains A, B and C. Connecting collagenous sequences to respectively B, A and C chains will place them in the leading, middle and trailing positions (the BAC translation rule). The small size of the NC2 domain and the ability to recombinantly express individual chains in bacteria and later assemble them Crystal structures of 111, 121 and 211 constructs demonstrated that a staggering order can be manipulated at least for short native collagenous sequences, meaning that such sequences are rather adaptive to various abnormal conformations. Nevertheless, these alternative conformations demonstrated differences in thermal stability and affinity to a ligandAt least for type IX collagen we can conclude now that the stagger of the central collagenous domain (COL2) is α2α1α3 and it is determined by the NC2 domain. Staggers and its staggering mechanisms of other collagenous domains in type IX and other collagens remain to be elucidated. The most intriguing structural studies would include a junctional region between a triple-helical and trimerization domain in such hetero-trimeric collagens as type I and IV, as well as in hetero-trimeric collagen-related complement protein C1q.In summary, these data detail the structural organization of the triple-helix- to- trimerization domain interface of type IX collagen and the mechanism of staggering. The current constructs provide a straightforward tool to produce any collagen fragments of interest with a controlled composition and stagger.E. coli strain B834 (DE3) and a medium composed of SelenoMet Medium Base and SelenoMet Nutrient Mix (Athena Enzyme Systems).All constructs were expressed, accordingly assembled and purified as describedThe complexes were crystallized by vapor diffusion with the following crystallization conditions:111sm: 0.1 M BisTris pH 6.4, 14% PEG MME 5,000 + 20% glycerol (cryo)121nat and 121sm: 0.1 M HEPES pH 7.5, 50 mM Na-Acetate, 17% PEG 3,350 + 20% glycerol (cryo)211sm: 0.1 M BisTris pH 6.0, 16% PEG MME 5,000 + 20% glycerol (cryo).Diffraction data were collected at the Advanced Light Source beamline 4.2.2. The diffraction images were indexed, integrated, and scaled using HKL2000Crystal diffraction data, phasing and refinement statistics are presented in Accession codes: Atomic coordinates and structure factor amplitudes have been deposited in the Protein Data Bank under accession numbers 5CTD, 5CTI, 5CVA and 5CVB.How to cite this article: Boudko, S. P. and Bächinger, H. P. Structural insight for chain selection and stagger control in collagen. Sci. Rep.6, 37831; doi: 10.1038/srep37831 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Here we show that GROGER, found in the N-terminal domain of collagens I and III, is only weakly recognised by α10β1, an important collagen receptor on chondrocytes, contrasting with the other collagen-binding integrins. Alignment of I domain sequence and molecular modelling revealed a clash between a unique arginine residue (R215) in α10β1 and the positively-charged GROGER. Replacement of R215 with glutamine restored binding. Substituting arginine at the equivalent locus (Q214) in integrins α1 and α2 I domains impaired their binding to GROGER. Collagen II, abundant in cartilage, lacks GROGER. GRSGET is uniquely expressed in the C-terminus of collagen II, but this motif is similarly not recognised by α10β1. These data suggest an evolutionary imperative to maintain accessibility of the terminal domains of collagen II in tissues such as cartilage, perhaps during endochondral ossification, where α10β1 is the main collagen-binding integrin.The collagen-binding integrins recognise collagen through their inserted (I) domain, where co-ordination of a Mg •Integrin α10β1 binding to collagen is mapped onto Collagen Toolkits.•Charged residue in α10 I domain clashes with some binding sites that are unique to collagen II.•Mutant constructs of other integrin I domains mimic this charge effect.•Implications for evolution of collagens and cartilage with reference to bone formation Upon ligation, the octahedral co-ordination shell surrounding the metal ion is perturbed and reorganised by an incoming ligand, a negatively-charged glutamate residue that is crucial to the tight ligation of the receptor The collagen-binding integrin family is complete, with four established members, α1β1, α2β1, α10β1 and α11β1. These are highly-conserved, heterodimeric receptors with an important structural feature in common, the presence of an I (inserted) domain which contains the metal ion-dependent adhesion site (MIDAS). The MIDAS is a constellation of electronegative residues at the distal surface of this 200-residue domain that, in the resting, non-ligated state, can co-ordinate a Mg5 extensions (host sequence) that adopt triple-helical conformation, and so ensure that the quite diverse guest sequences are presented in their native conformation. The Toolkit approach revealed the presence in collagens II and III of several Gxx′GEx″ motifs with varying affinity for the collagen-binding integrins, depending also on the activation state of the integrin Understanding of integrin activation advanced with the elucidation of a co-crystal structure comprising the α2 I domain and a synthetic triple-helical peptide (THP) containing the  motif, GFOGER Much less is known about the ligand propensity of α10β1, and it is with this topic in view that the present study was designed. In vivo, α10β1 appears to be expressed primarily on the chondrocyte surface but also in some junctional fibroblasts, in chondrogenic mesenchymal stem cells, and in cells lining endosteum and periosteum α10β1 was identified in cartilage, but, as mentioned above, it is not exclusive to that tissue although its tissue distribution has not been exhaustively examined 2 +, and was detected using anti-GST, as described in Experimental: data are shown in 2 +-dependent manner, in rank order, to Toolkit peptides II-8, II-7, II-28, II-22, II-27, II-31, II-44 and II-32  that allows label-free, real-time monitoring of cell adhesion to intact collagens and to selected positive Toolkit peptides. C2C12-α10β1 bound quite well to collagens I, II and III, better to collagen IV, with Peptide III-7 (containing GLOGEN) as a strongly positive control. For simplicity, endpoint data only of xCELLigence data are shown in Using a set of short peptides, α10 I domain bound strongly to a THP containing GLOGEN and less well to GLOGER and GFOGER, shown in In LDH-based SPBA , transfeIn real time xCELLigence assays testing the short peptide motifs, C2C12-α10β1 showed poor binding to the THP containing the GROGER sequence, of comparable level to the GPP10 negative control peptide , whereasWe re-examined the PDB structure, 1DZI, a co-crystal of the collagen hexapeptide motif, GFOGER, with the α2 I domain, to establish the contacts between the peptide x residue and the I domain surface. In that context, the peptide F residue makes some contact with Q215 of the α2 I domain . ReplaciTo test this hypothesis, we replaced the corresponding Q214 and Q215 in α1 and α2 I domains, respectively, with R, as in α10, and we replaced the R215 in the α10 I domain with Q, as in α1 and α2. The expressed mutants of α1-Q214R and α2-Q215R I domains failed to bind to GROGER, whereas the α10 I domain R215Q mutant showed better binding to GROGER C and D.2 +, detected using anti-GST as before, and data are shown in 2 +-dependent, reproducing the wild type Toolkit II data shown in Ligand binding activity of the human recombinant GST-α10-R215Q I-domain expressed in bacteria was examined using conventional colorimetric 96-well SPBA. The I domain was first applied to the collagen II and III Toolkits in the presence or absence of MgD on the order of 10 μg/ml  as a selective ligand in preference to GFOGER. No other peptide (or shorter derivatives) from Toolkit III approached the binding activity of III-7/GLOGEN. In the setting of cartilage, collagen III, although enriched in the pericellular environment, is much less abundant than collagen II The only other peptide from Toolkit II that shows good affinity for α10β1 and α10 I domain is II-22, that contains no obvious integrin motif, but is a good ligand for the discoidin domain receptors, DDR1 and DDR2, and for the ECM components SPARC and von Willebrand factor. It may well be that some of these proteins augment the regulation of chondrocytes, by co-localising with α10β1, although this is beyond the scope of the present study. A potential role for other collagen receptors was revealed by the use of a blocking α10 antibody By sequence alignment and molecular modelling, we establish that the α10 I domain differs from other members of the family by the presence of a positively-charged residue, R215, at the site where the long hydrophobic residue x (of Gxx′GEx″) would interact with the I domain surface. Other I domains that lack such a positive residue at this site bind GROGER well, and presumably the long aliphatic stem of the collagen arginine sidechain can support a hydrophobic interaction with the I domain surface. This is not the case with native α10β1 where the electropositive R215 will repel GROGER. This α10 residue is conserved in many other species; rat or mouse Why would collagen II differ in this respect? Nine conserved integrin-binding sites are distributed across the fibrillar collagens see ; Site 1 The collagen fibre adopts its canonical quarter-stagger assembly, with 300 nm triple-helical tropocollagen monomers laid end-to-end with an axial gap of ~ 35 nm between. The 67 nm axial offset between adjacent monomers defines the D-period observed in transmission electron micrographs of native collagen fibres. This simple model allows side-by-side location of binding sites in adjacent collagen molecules to be considered, and reveals that Site 1 in collagen II (GROGER) and Site 9 (GRSGET), both unable to bind α10β1, are almost perfectly co-located in the D-period overlap region. Thus, this specific axial locus in the collagen fibre cannot be ligated by the principal chondrocyte integrin, suggesting that occlusion of this specific site by α10β1 ligation is not tolerated and there is a need to preserve access to this region of the collagen II fibre for other activities. Similarly, this locus in collagens I and III is protected against α10β1 binding, implying that in α10β1-expressing tissues, i.e. in cartilage, it is also preserved from integrin ligation.Callorhyncus milii) is a cartilaginous fish whose evolution diverged prior to the emergence of the bony fish and other vertebrates Since a collagen II orthologue is considered to precede the other fibrillar collagens in evolutionary history C. milii genome. These shark integrins all contain the critical, permissive, Glu residue, so that they are predicted to bind Site 1 in collagens I and III. In contrast, bony fish  resemble most tetrapods in terms of the distribution and identity of collagen-binding integrins and integrin-binding sites in the collagens.However, further inspection of the cartilaginous fish genome sheds little direct light: the ghost shark contains orthologues of α1β1, α2β1 and α11β1, but appears to lack α10 This suggests that co-evolution of collagens I and III with the calcified skeleton may have occurred, and required the prohibition of chondrocyte binding to Site 1 in collagens I and III, along with the absence of any cellular binding to Site 1 in collagen II. One might speculate that these properties would maintain access of osteoclasts and their precursors to the nearby OSCAR motifs in collagen II, GAOGPQGFQ in D1 and the collagen II-specific GAOGASGDR motif The increased expression of collagen III has been proposed as a repair response after damage or disease in cartilage The three α10-repellent motifs identified here, GROGER, GKOGER and GRSGET, summarised in Unless stated otherwise, reagents were from Sigma .5GPC host peptides, which confers triple-helical form on the shorter peptides.Toolkit peptides, as C-terminal amides, were synthesised on TentaGel R-Ram resin using either an Applied Biosystems Pioneer peptide synthesiser as described previously Mouse anti-glutathione S-transferase antibody (Horseradish peroxidase-conjugated) was from GE Healthcare (cat. no. RPN1236).Recombinant human α10 I domain-encoding plasmid (pGEX-2T-α10 I domain) was cloned using standard procedures pGEX-2T-α1, α2 and α10 I were used to generate the Q to R or R to Q mutations using QuikChange PCR kit from Stratagene (200518).d-thiogalactopyranoside . Cells were harvested by centrifugation at 4500g for 20 min, and pellets for GST-fusion were resuspended in 10 ml Dulbecco's phosphate-buffered saline, containing 1 tablet of protease inhibitor cocktail (Roche) and 5 mg of lysozyme (Fluka). Suspensions were sonicated and Triton X-100 was added to 1% (v/v). Suspensions were mixed on a roller for 15 min at room temperature, then centrifuged at 18,000 × g for 20 min, and supernatants were pooled. The lysate was passed down a glutathione-agarose column equilibrated in Tris-buffered saline ; the column was washed with 10 volumes of Tris-buffered saline containing 1 M NaCl and 1% (v/v) Triton X-100, and the glutathione S-transferase–I domain fusion proteins were eluted with 10 mM glutathione reduced in 50 mM Tris–HCl (pH 8.0). The proteins were then dialyzed against Tris-buffered saline and concentrated using a Microcon-3 . I domain purity and degradation were checked using 10% SDS-PAGE and Western blotting. Nitrocellulose blots were probed with horseradish peroxidase-conjugated anti-glutathione S-transferase polyclonal antibody .To express α2 wild type and mutant I domains, a 100-ml overnight culture of transformants (Origami strain) was used to inoculate 1 L of Luria broth containing 100 μg/ml ampicillin, 15 μg/ml Kanamycin and 12.5 μg/ml Tetracyclin. The culture was grown for 2 h at 37 °C and then induced at room temperature for 4 h with isopropyl β-2 or EDTA for 1 h at room temperature. Wells were washed five times with 200 μl of adhesion buffer containing MgCl2 or EDTA, before adding 100 μl of adhesion buffer containing the anti-GST-HRP conjugate at 1:10,000 dilution for 1 h at room temperature. After washing, colour was developed using an ImmunoPure TMB Substrate Kit (Pierce) according to the manufacturer's instructions.I domain adhesion was determined colorimetrically, as described 6/ml in Tris-buffered saline supplemented with 5.5 mM glucose and either 2 mM EDTA or 2 mM MgCl2. SPBA was performed as follows: 100 μl of the cell suspension was added to peptide-coated wells The mouse myoblast cell line, C2C12,  was maintained in RPMI medium containing 10% fetal bovine serum, 2 mM glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 2.5 μg/ml amphotericin. Cells were harvested by centrifugation, washed, and suspended at 0.5 × 10n − Rb) / 15; where Rn is the electrical impedance of each cell-containing well and Rb is the background impedance of the well with medium aloneTo quantitate C2C12-α10β1 binding to collagen peptides, we used the impedance-based xCELLigence system (Acea) that allows label-free, dynamic monitoring of cell adhesion in real-time 4 cells/well in an E-plate (Roche Applied Science) pre-coated with peptides as for SPBA, and CI was measured for up to 2 h.Briefly, C2C12 cells were seeded at 2.5 × 10Values shown in all figures are mean ± s.d.British Heart Foundation programme grants, RG/15/4/31268 and RG/09/003/27122, and by a Wellcome Trust Biomedical Resource grant, 094470/Z/10/Z.This work was supported by


Escherichia coli outer membrane. Here we investigate the conformational behavior of the T-domain of colicin N (ColN-T) to understand why such domains are widespread in toxins that target Gram-negative bacteria. Like some other intrinsically disordered proteins in the solution state of the protein, ColN-T shows dual recognition, initially interacting with other domains of the same colicin N molecule and later, during cell killing, binding to two different receptors, OmpF and TolA, in the target bacterium. ColN-T is invisible in the high-resolution x-ray model and yet accounts for 90 of the toxin’s 387 amino acid residues. To reveal its solution structure that underlies such a dynamic and complex system, we carried out mutagenic, biochemical, hydrodynamic and structural studies using analytical ultracentrifugation, NMR, and small-angle x-ray scattering on full-length ColN and its fragments. The structure was accurately modeled from small-angle x-ray scattering data by treating ColN as a flexible system, namely by the ensemble optimization method, which enables a distribution of conformations to be included in the final model. The results reveal, to our knowledge, for the first time the dynamic structure of a colicin T-domain. ColN-T is in dynamic equilibrium between a compact form, showing specific self-recognition and resistance to proteolysis, and an extended form, which most likely allows for effective receptor binding.Intrinsically disordered regions within proteins are critical elements in many biomolecular interactions and signaling pathways. Antibacterial toxins of the colicin family, which could provide new antibiotic functions against resistant bacteria, contain disordered N-terminal translocation domains (T-domains) that are essential for receptor binding and the penetration of the  Rg  have emerged in the last decade as a challenging and exciting area of structural biology . GeneralRg , originaRg , 7. MoreRg , 9, 10. Rg , these sIn this article, we investigate the native conformation of the antibiotic protein colicin N, which has an N-terminal, 90-residue, intrinsically disordered region (IDR) , 12. ThiEscherichia coli colicins  domain that usually contains an IDR, a central receptor binding (R-) domain, and a C-terminal toxic domain  Fig. 1)Fig. 1). E. coli (1A87 (We study colicin N (ColN), which is the smallest pore-forming colicin at 42 kDa, and which kills the target cell by inserting its C-terminal pore-forming (P)-domain into the cytoplasmic membrane. The receptor binding R-domain of colicin N binds to lipopolysaccharide (LPS), the main lipid component of the outer surface of E. coli . All 90 li , ColN40–387 (ColN-Δ1-39), ColN90–387 (ColN-RP), ColN1–90 (ColN-T), and mutants ColN-Y62A and ColN-K145A each had a –SSHHHHHH at their C-terminus and were purified as previously described  was performed as previously described  or Avance III spectrometer  equipped with triple resonance and pulsed field gradient probes, operating at 1H frequencies of 499.865, and 800.229 MHz, respectively, and 15N frequencies of 50.66 and 81.09 MHz, respectively, at 15°C. Resonance assignments of 13C/15N-labeled R domain were obtained from standard triple-resonance experiments, and assignments of ColN and ColN-T were taken from the literature  using both absorbance at 280 nm and interference optics. All AUC runs were carried out at a rotation speed of 48,000 RPM at 4°C, using an eight-hole An-Ti50 rotor and double-sector aluminum-Epon centerpieces . The sample volume was 400 μL and the concentrations were ranged between 0.3 and 1.5 mg/mL and usually up to seven sample dilutions were used. The partial specific volumes c(s) model implemented in the program SEDFIT (c(M) distribution. Each peak on the distribution plot was integrated to obtain the weight-averaged values for the sedimentation coefficient and molecular mass. The integrated values of sedimentation coefficient s obtained at experimental conditions were converted to the standard conditions s20,w, which is the value of the sedimentation coefficient in water at 20°C and extrapolated to zero concentrations of protein.SEDNTERP  and extrSEDNTERP . The denm SEDFIT . To deteHydrodynamic parameters and folding state. The theoretical Stokes radii Rs for native state N, molten globule MG, premolten globule PMG, intrinsically disordered coil, and denatured urea/guanidinium chloride proteins were calculated as described in  data for colicin N samples were collected on the Beamline BM29  as entries SASDC23 (ColN WT), SASDC33 (ColN Δ1-39), SASDC43 (ColN K145), and SASDC53 (ColN-T).The experimental SAXS data were submitted to the SASBDB database (Flexible domain modeling. The flexible translocation domain was modeled using both the program BUNCH (1A87). The model was fitted simultaneously against two experimental data sets of ColN, the full-length protein, and its truncation mutant, ColN-Δ1-39.am BUNCH  and the am BUNCH . BUNCH wRg and Dmax for plausible protein conformations were modeled using the EOM package (1A87). To model ColN K145A, the atomic coordinates of lysine 145 in PDB: 1A87 were changed to alanine using the program COOT (Iexp(q) and calculated I(q) curves,K is the number of experimental points, σq values are standard deviations, and μ is a scaling factor.The distributions of  package , employiram COOT , 40. In Hydrodynamic modeling. The program HYDROPRO (Rg)2de chain . The b vterature . For theβ-sheet region of the ColN-R domain displays a concave face, and we thought this could be a binding site for LPS. We created the mutation K145A to remove a centrally exposed cationic residue in an attempt to inhibit both receptor binding and toxicity. This failed, as its activity against E. coli was unchanged. However, it was observed that, upon storage, the T-domain degraded more quickly than usual. Limited proteolysis assays confirmed this by showing that the T-domain of ColN-K145A was more sensitive than the WT to proteolysis by trypsin (A). Far UV-CD difference spectra compared with WT of the two mutants showed a larger change, with more disorder for ColN-Y62A than ColN-K145A (B). Thus, three independent measurements indicate that the T-domain is more disordered in the mutants, and two indicate that ColN-Y62A is more unfolded than ColN-K145A.The  trypsin . The deg trypsin . The int trypsin  in the i trypsin , which s trypsin A. Far UlN-K145A B. Thus,15N-1H HSQC NMR spectrum in which the majority of backbone amide proton resonance peaks can be assigned to specific residues. In full-length colicin N, self-recognition and thus decreased mobility of ColN-T residues leads to the disappearance of a number of ColN-T-specific peaks belonging to residues 40–67 within the TABS , but this is structurally more revealing because the changes in the ColN-T spectrum result from a mutation outside of ColN-T. The seven mobilized residues in ColN-K145A correspond to the start of the TABS between W44 and D59 (B). Thus, the region of ColN-T that is mobilized by the ColN-K145A mutation 44–59 is N-terminal to that mobilized by ColN-Y62A 51–75. Also, the effect of K145A appears spatially more limited than that of Y62A. In a previously published experiment, addition of free unlabeled ColN-T to 15N-labeled ColN-RP altered the chemical shifts of several amide protons corresponding to a limited number of ColN-RP residues (C), which are on the helix and loop next to the first resolved residue S91 in the high-resolution x-ray crystallographic model. These combined results further define the extent of the self-recognition site on the R-domain, and show that K145 may interact with the region that includes residues 40–60.In aqueous solution, free ColN-T (residues 1–90) has a well-resolved the TABS , 24. In ognition . The resognition A, but t and D59 B. Thus,residues C, whichA; The ColN translocation domain ColN-T, monomeric in solution, has been shown experimentally to be unstructured , 48, in q2Iq versus q, where q is the scattering angle and I(q) is the scattering intensity, provide information about the globularity of a particle in solution . The dimensionless Kratky plot qRg for the isolated T-domain derived from SAXS data resembles a saturation-like curve, suggesting an unstructured molten globule conformation without any residual secondary structure (A). The distance distribution function gives the maximal dimension Dmax of the particle at ∼115 Å (B).SAXS curves represented in the form of Kratky plot solution . The dimears ago , gives aears ago . The scaears ago  and httptructure A. The dt ∼115 Å B.FigureA (inset). SAXS data were treated as a flexible system using the EOM (B). The fit to the data is shown in A and yields a good agreement between experimental data and curve fit χ-value = 1.22 without any systematic deviations. The size-distribution Dmax of structures fitting the ColN-T scattering curve revealed two major peaks centered around 65 and 93 Å, respectively, with the selected population of conformations shifted toward more disordered particles versus the general pool of structures. The distribution of radii of gyration Rg displays a similar shape (B), with the main peaks at 23 and 29 Å. According to Rg values centered around 29 Å is characterized by a statistical length of 15.5 Å, which is 7.8 Å in terms of persistence length, thus corresponding to 2.8 residues per length. The latter population represents an extended disordered polypeptide segment with virtually no compact regions. Thus, the quasi-native ColN-T population relates to compact structures and agrees well with sedimentation velocity results, whereas the second one relates to an extended form, which, surprisingly, was not detected by AUC (C). The occurrence of extended and compact conformations of solution-forms of the T-domain obtained from EOM are shown in C, superimposed upon the c(s) distribution derived from sedimentation velocity experiments. It should be noted, however, that this comparison could be done only in the constrained pool of EOM-selected structures considered to be instantaneous conformations, which provides defined atomic coordinates from which to calculate sedimentation coefficients. This fact results in significantly lower numbers for population frequency compared with Rg or Dmax histograms obtained directly from scattering data. Two illustrative examples of modeled ColN-T extended and compact conformations are shown in C. Based on the values of hydrodynamic radius and sedimentation coefficient , these her SoMo , respectRS), for the isolated T-domain to be 19.3 Å (Rs for a molten globule state (19.2 Å)    and AUC S ≅ 1.4) . ReferriFitting the SAXS data using ab initio dummy atom models resulted in a reliable fit for ColN-RP residues 91–384, thus confirming the quality of the data collected. However, as the amount of T-domain increased, and especially when ColN-K145A was modeled, the fits became increasingly poor . From thqRg2Iq/I0 versus qRg for all the T-domain-containing ColN constructs WT, Δ1-39, and K145A (A) show the results typical for a combination of a folded, compact fragment with a disordered segment tail (P(r) plots, both ColN-Δ1-39 and ColN-RP are more compact than the much smaller, isolated T-domain, confirming the importance of the self-recognition behavior in constraining ColN-Δ1-39 (B). The data for the purely globular ColA pore-forming domain (friction ratio 1.14), calculated by SoMo, are shown for comparison with ColN-RP  demonstrating in a semiquantitative way the increased asymmetry for the ColN-RP molecule and all other ColN constructs (B). The full-length ColN, which includes residues 1–39, shows a more extended particle in solution that is even more pronounced in the ColN-K145A mutant. Aggregation of the Y62A samples, observed by AUC, precluded their analysis by SAXS. The P(r) plots of ColN and ColN-K145A describe elongated particles  by CRYSOL with a rigid single structure generated by I-TASSER, suggesting a relatively well-defined conformation , whereas the full-length ColN molecule cannot be fitted in this way (χ = 2.09) . This supports the NMR data that show residues 1–39 as disordered (χ = 0.673) (A and B). This approach was extended to the WT ColN, where each stipulated flexible region 1–39 and 72–90 could be modeled separately (C) and the contribution of the flexible regions can be seen in total conformation space. It is worth noting, however, that in the more general case, when the T-domain region is modeled as being totally flexible, the Rg (Dmax (A) distributions occupy the same conformational space as when modeled as a compact TABS region surrounded by flexible fragments . The unrestricted fit of Rg to the data is shown in χ-values of 0.675 and 1.08, respectively, for ColN-Δ1-39 and ColN. The equivalent Dmax distribution generated by EOM for ColN shows a bimodal form with peaks at ∼100 and ∼117 Å (A). We previously estimated from KD values that the extended form of ColN-WT would represent ∼5% of the population (A) agrees with this by showing a minor second peak for the WT protein. However, the EOM fit for ColN-K145A  that clearly defines two distinct, separated peaks—one similar to the compact peak of ColN-WT (Dmax ∼ 100 Å), and a second one representing ∼25% of the total population at ∼145 Å, which represents a significantly more extended and mobile structure. Sedimentation coefficients for selected conformations generated by EOM (considered to be instantaneous) were calculated and are presented in B superimposed with independently obtained c(s) distribution from sedimentation velocity AUC experiments. Because the TABS is shown by NMR to have reduced mobility, it is of particular interest to compare the configuration of the TABS in the EOM models with the observed hydrodynamic behavior of ColN (B). The calculated sedimentation coefficient of selected ColN-T domain structures taken from the EOM ensemble increases as the TABS becomes more compact by forming a tight loop (highlighted) and consequently approaches the experimentally observed AUC value. Similarly, one observes for the full-length construct a compaction of the T-domain taking place when it comes within reach of the R-domain (B). Conversely, in the case of the extended fraction for the K145A mutant, the T-domain is more extended and the TABS is often situated at a considerable distance from the compact R- and P-domains.The ColN-Δ1-39 scattering curve was fitted satisfactorily (sordered . Further= 0.673) , A and Bparately C and th, the Rg  and DmaxRg  how does the self-recognition affect the spatial mobility of the ColN-T, and 2) can we define the inherent flexibility of the colN-T and understand how it accesses its twin targets OmpF and TolA?2His6-tag follows directly after residue 90, so may induce compaction but, due to ColN-T’s protease sensitivity, we cannot remove it proteolytically. When the hydrodynamic radius RS is calculated, including the effect of the His-tag, according to , which indicate additional disorder for all constructs that include regions of the T-domain. A combination of rigid body modeling combined with an additional N-terminal flexible polypeptide segment using BUNCH (B) not only led to better fits to the experimental SAXS curves but also predicted sedimentation behavior similar to the observed AUC data  at the cell surface and TolA within the periplasm. The ColN-T/OmpF interaction  employs The results from NMR, AUC, SAXS, and EOM have brought the study of colicin dynamics to a new level. SAXS can obtain reliable data from such proteins in solution, but attempts to resolve the unfolded state clearly indicate the need for a distribution of conformers. However, instead of a poorly defined data set, the clear identification of a two-state equilibrium, assisted by the discovery of K145A, helps us resolve the self-recognition state and understand how it works in vivo. The EOM-modeled structures are independently refined by the genetic algorithm and these consistently represent the TABS as a compact conformation whereas the other T-domain sections show various degrees of disorder. This independently supports the TABS ordering previously identified by mutagenesis, NMR, and PONDR analysis , 24. ThiC.L.J. and H.W. carried out molecular biology and protein biochemistry experiments and protein purification. O.H., C.M., and G.R.M. performed and analyzed NMR experiments. A.S.S. performed and analyzed SAXS and AUC experiments. C.L.J., J.H.L., and J.G.G. contributed to SAXS experiments and data analysis. A.S.S. and J.H.L. wrote the article.


Here we report that a cluster of arginine residues in the first domain required for selective voltage-gating of TPC1 map not to the voltage-sensing fourth transmembrane region (S4) but to a cytosolic downstream region (S4-S5 linker). These residues are conserved between TPC isoforms suggesting a generic role in TPC activation. Accordingly, mutation of residues in TPC1 but not the analogous region in the second domain prevents Ca2+ release by NAADP in intact cells. Our data affirm the role of TPCs in NAADP-mediated Ca2+ signalling and unite differing models of channel activation through identification of common domain-specific residues.Two-pore channels (TPCs) are two-domain members of the voltage-gated ion channel superfamily that localize to acidic organelles. Their mechanism of activation P TPC1 constructs in which either R219 or R223 had been mutated individually to glutamine expressed and colocalized with wild type TPC1 (2+ responses in cells expressing wild type TPC1 (To examine the functional role of the S4–S5 linker, we examined the consequences of mutating conserved arginine residues on Caype TPC1 A–C. Micrype TPC1 A. The reype TPC1 A.Fig. 2TWe also mutated R554 in the S4–S5 linker of domain II. This residue together with R552 is conserved in TPC1 but not TPC2 across species, and like the conserved residues in domain I, is placed within a helix . Again, 2+ fluxes in intact cells.Collectively, these data summarised in 3.32, which regulates both TPC isoforms In sum, prompted by new structural data, we identify a domain-specific requirement for the first S4-S5 linker in activation of TPCs by NAADP. Together with previous work implicating this same region in voltage-sensing 2+ imaging. DC performed the confocal microscopy. SP designed the study, performed the bioinformatics and molecular biology, and wrote the paper.EB performed the CaThe authors declare that they have no competing interests.