Keratins are cytoskeletal proteins that hierarchically arrange into filaments, starting with the dimer sub-unit. They are integral to the structural support of cells, in skin, hair and nails. In skin, keratin is thought to play a critical role in conferring the barrier properties and elasticity of skin. In general, the keratin dimer is broadly described by a tri-domain structure: a head, a central rod and a tail. As yet, no atomistic-scale picture of the entire dimer structure exists; this information is pivotal for establishing molecular-level connections between structure and function in intermediate filament proteins. The roles of the head and tail domains in facilitating keratin filament assembly and function remain as open questions. To address these, we report results of molecular dynamics simulations of the entire epithelial human K1/K10 keratin dimer. Our findings comprise: (1) the first three-dimensional structural models of the complete dimer unit, comprising of the head, rod and tail domains; (2) new insights into the chirality of the rod-domain twist gained from analysis of the full domain structure; (3) evidence for tri-subdomain partitioning in the head and tail domains; and, (4) identification of the residue characteristics that mediate non-covalent contact between the chains in the dimer. Our findings are immediately applicable to other epithelial keratins, such as K8/K18 and K5/K14, and to intermediate filament proteins in general. Intermediate filaments (IF) are one of only three types of fibrous proteins in the human body. Keratins are the most numerous IF and are found in skin , hair  and nails . MoleculExperimental evidence confirms that all IF protein dimers have the same tri-domain structure: a head, a central rod, and a tail domain ,10 Fig . Howevervia studies of the entire dimer structure, may not have been captured. Furthermore it has highlighted that the 2A subdomain of vimentin may contain parallel strands rather than the left handed coiled-coil seen elsewhere [Keratins form heterodimers; type I and type II keratin proteins intertwine in-parallel and in-register to give a central rod domain comprising, from the N- to the C-term, the 1A-L1-1B-L12-2A-L2-2B subdomains. 1A, 1B, 2A and 2B are coiled-coil subdomains, and L1, L12, L2 are the intervening linkers . The seqlsewhere ,20. The lsewhere . In the e.g. the head domains of K1 and K18 comprise 180 and 80 residues, respectively) and identity between different keratins. These domains are generally longer for the type II keratins  than the type I keratins . Epithelial keratin sequences typically possess high numbers of glycine residues and few cysteine residues, while trichocyte keratins contain relatively greater numbers of cysteine residues. Current understanding on the head and tail domains indicates that these correspond with intrinsically-disordered regions of the keratin protein [via experiment or simulation. This analysis suggested that the head domain could be divided into three subdomains, denoted as the end subdomain, E1, a variable glycine-rich subdomain, V1, and, a region of sequence homology close to the rod domain, H1 , as well as truncated dimer models (fifteen simulations). The truncated system comprised only the first half of the dimer, spanning the head-1A-L1-1B domains. Atomistic-level experimental data, obtained for closely-related systems such as vimentin, provided a crucial basis for the construction our structural models (see below). While such data for the rod domain are relatively more mature than for the head and tail domains, very recent electron paramagnetic resonance (EPR) measurements for vimentin have provided vital clues regarding the arrangement of the head domain,23. ThesTo model the K1/K10 dimer we constructed several initial starting structures and used these as a basis for further refinement during equilibration of the MD simulation. Each dimer was built by first constructing the head, rod and tail domains and then linking these three domains together to form the dimer .Since detailed structures of key regions of the rod domain (i.e. the coiled-coil domains) exist for a related IF, vimentin, we used these as a starting point for our model of the K1/K10 dimer. Comparison between vimentin and K1/K10 rod domain sequence shows that there is a 30% and 90% sequence similarity in amino acid identity and residue type, respectively, and is suggestive of similar structure. The crystal structures of the vimentin 1A (3G1E.pdb ), 1B  onto thThe head and tail domains of the K1/K10 dimer comprise 365 and 275 residues, respectively, making these the second-longest head and tail domains of any keratin. There are little direct data available on the structure of the head and tail domains of keratin or any other closely related IFs. However, recent site-directed spin labeling and EPR measurements for vimentin ,23 suggey-axis of the simulation cell). Each chain of the head domain was looped back onto the rod domain using a hairpin turn of nine residues . A force constant of 1000 kcal (Å mol)−1 and an equilibrium distance of 14.37 Å , were used for the restraint.The most challenging parts of the dimer to model, due to the unavailability of detailed experimental data, are the intrinsically disordered head and tail domains. To better sample the range of folded conformations of these domains we considered 25 different models, varying the initial configuration of these domains. In ten of our models we simulated the full dimer; for these we built the tail domain in the same configuration as the head domain. In our remaining fifteen simulations, we considered a truncated model, where we cleaved the dimer at the C-terminal end of the 1B coiled-coil subdomain and terminated each chain with a COOH group. To ensure that this artificial termination of the dimer did not cause the 1B coiled-coil subdomain to unravel during the simulation, a harmonic restraint was applied between the between the Ce.g. K1 onto K10 and vice versa); (D) each chain onto its own chain face of the rod; and, (E) fully extended away from the rod domain (i.e. not folded). A schematic of these different initial configurations is presented in There are a multitude of possible ways that each head/tail chain can be folded back on to the rod domain. The construction of our initial models of each head and tail model was based on two key degrees of freedom: (1) the direction of the turn relative to the rod domain of the dimer, and (2) the position of the turn along the sequence of the head/tail domain of each chain. Five plausible folding directions were chosen for folding the head and tail chains back on to the rod domain which covered the entire range of possible orientations for the two chains. These were to fold: (A) both chains towards the K1 face of the rod domain; (B) both chains towards the K10 face of the rod domain; (C) each chain onto the opposite chain face of the rod (n residues (denoted as n-shift) towards the rod domain . The combinations of different orientation and n-shift values produced the 25 different models. An overview of the 25 combinations used for the dimer models is given in Fig Ae in Another possible variable in our set-up of the head and tail domains was the initial position of the hairpin turn as a function of location along the chain length. We explored up to six different positions of the hairpin turn for each of the orientations (A-E). Hypothesizing that the head domain of keratin may be folded as far back as vimentin, we started by placing the hairpin turn across the residues K1:103–111 and K10:86–94  for We performed MD simulations using the NAMD simulation package . The proα atoms restrained. Next, with these restraints still present, MD was performed for 30000 steps from initially at-rest molecules, using the same Langevin thermostat. Finally, from this point, full unconstrained MD was carried out.We took two approaches to initiating the implicit solvent simulations. In the first approach, the entire structure was energy minimized using the steepest descent algorithm. The velocities were then initialized to the thermostat temperature of 305 K before proceeding to the MD. In the second approach, the backbone atoms were fixed during the energy minimization. A second minimization was then performed with only the backbone CEach dimer model was simulated for 100 ns and was determined as equilibrated, with a stable structure, when the root mean square displacement (RMSD) in residue positions had plateaued  were solvated with 250,000–300,000 TIP3P  water moα atoms were restrained and MD performed for 3000 steps, from initially at-rest molecules, in the NVT ensemble using the same Langevin thermostat as for the implicit simulations. Next, a NPT simulation was performed for 5000 steps with restraints still placed on the Cα atoms. Finally, all restraints were removed for the remainder of the simulation. Here a Langevin piston was used, with a target pressure of 1 bar, a time period of 100 fs and decay of 50 fs. Group pressure was used. The inter-particle forces were calculated using a switching distance of 10 Å, a cut-off distance of 12 Å and pair list distance of 14 Å. Particle mesh Ewald with a grid spacing of 1 Å was used to treat the long-ranged electrostatic interactions calculation. Simulations were run to obtain 25 ns of equilibrated trajectory data.The explicit solvent simulations were initiated by energy minimizing the system with the protein backbone atoms held fixed in space. Next the backbone CIn the study we use statistical analysis over all of our 25 structural models to gain insight into the most likely structural features for the keratin dimer. In this sense, we have classified the frames taken from our trajectories based on a range of geometric criteria as detailed below, and we then used these classifications to essentially coarse-grain the underlying potential energy landscape of these keratin dimer systems.pN(i) of the central axis of the α-helix of each peptide chain was calculated first , with each pN(i) calculated from the Cα positions of four conservative residues with positions i − 1, i, i + 1 and i + 2 in the α-helical chain. The path of the central axis of the coiled-coil P(i) is then the mean of the paths of the two α-helices, i.e. P(i) = (pK1(i) + pK10(i))/2.The structural properties of the coiled-coil subdomains were calculated using the same procedure as outlined in previous studies on the experimental crystal structure data of vimentin ,35,36. TR(i), of the coiled-coil can then be calculated as half the separation distance between the centers of the two peptide chains at residue i, i.e. R(i) = (|pK1(i) − P(i)| + |pK10(i) − P(i)|)/2. Next, a dihedral angle, Ψ, made between the two vectors that connect the center of the α-helix of a given chain (e.g K1 or K10) to the central path of the coiled-coil, at i − 1 and i, around the path vector of the central axis between these (as given in Strelkov and Burkhard [i and i + 1 to produce four estimates (two each from K1 and K10). An average of these estimates then defines the local change in radial angle of each peptide chain at residue i. The local pitch, λ(i), was estimated from the change in distance along the central path, from i − 1 to i + 1, and the change in dihedral angle Ψ, using The local radius, Burkhard ). CalculThe vector of the major axis of each coiled-coil was defined as the normalized displacement vector between the positions of the central axis at the 2nd residue and 3rd to last residue of the subdomain. The hinge angle, defined as that between coiled-coil subdomains, can then be calculated from d, and the radial orientation, θr, of the V1 globule subdomain of a given chain (K1 or K10), we first defined its center-of-mass using the Cα atom positions of the chain found within the V1 globule. We then defined a vector, d, as the projection of i.e. To calculate the axial displacement, e. V→⋅M→ .θr, we constructed a plane C, defined as perpendicular to the 1A major-axis θr as the angle between the vectors i.e. To calculate the radial orientation of the chain’s V1 subdomain, A domain . From th1(P→.O→) .This procedure was repeated for the tail globule (substituting the residues of the V1 subdomain for those of V2 subdomain residues), and using the positions of the 3rd to last residue of the 2B subdomain as reference, see Finally, the position and orientation of the end termini of the head and tail domains were calculated using the above procedure and substituting the residues of the V1 subdomain for either the N-term or C-term residue of the head or tail domain, respectively  chain terminal residue  and the center of the N-term or C-term of the 1A and 2B subdomains, respectively.The radius of gyration was defined as the root mean square of the distances between individual backbone atoms of the head, tail domain or V1 subdomain and their center of mass. The end-to-end distance of the head and tail domains was defined as the distance between the Cα atoms of a pair of residues was sampled uniformly in time and considered for analysis only if this distance was less than 12 Å. Residue pairs were classed into three types of interaction based on the chains involved: K1-K1; K10-K10; K1-K10. These were then used to calculate the frequency that a particular pair of residues are found to be close in these models. The results were normalized to account for the differences in residue numbers between truncated and full dimers.From each model the distance between the CWe also measured the time average number and types of residue sidechain contacts present in each dimer model. Each specific side chain contact was classified depending on the pair of residue types present ; the α-helical propensities for these motifs are unfavorable [d-heptad position . These a- and d-heptad helical positions of the K1 and K10 chains. In practice, our simulations revealed on average 214 such contacts; of these, 80%  were hydrophobic, with 12 of these due to aromatic ring contacts. Nine further contact pairs were found between acid and basic sidechains. This contrasts with the intra-chain sidechain interactions of the α-helical chains, where each have twice the number of electrostatic attractions (compared with the inter-chain case), given there are 19 out of the 197 observed K1-K1 interactions and 19 out of the 192 observed K10-K10 interactions  interdigitation of hydrophobic residues. As a theoretical maximum, an ideal coiled-coil rod domain for keratin K1/K10 would involve 228 inter-chain side-chain interactions linking between the A key aim of our work was to obtain a comprehensive picture of the spatial extent and subdomain structure of the head and tail domains, generated via statistical analysis of their structure obtained from our 25 simulations. To indicate the detailed structure of the head domain, from each model we identified contacting pairs of residues , as marked by the black dashed lines and the black domain labels in Our contact plots showed that residues K1:61–153 and K10:31–137 were typically in close contact and indicated these residues were part of a globular subdomain V1. This sub-domain encapsulates both the glycine loop motif  in the sequence [neighbor-only’ (in sequence) rings forming separate π-stacks, the regions highlighted by rectangles in The dominant feature of the contact plots is the V1 globule, featuring a 36% (107 residues) glycine content, which has been hypothesized to confer a ‘glycine loop’ structure . Glycinesequence . Glycinesequence , and couWe identified the types of sidechain interactions in the head domain ; similarly, a negative value of d indicates the globule is located at a point below the N-term of the 1A domain  or extended away from the rod domain (and hence would be expected to be more mobile). We investigated the positional and orientational preferences of the head domain relative to the rod, and identified from the models three categories of head domain spatial position, depending on the statistical location of the head domain relative to the 1A subdomain of the rod .The most likely values of  and θr  show tha and θr . This and and θr, for the N-term residue of the E1 subdomains in K1 and K10 (To identify whether the termini of the head domain (e.g residues 1 of K1 and K10) were anchored onto the rod or were free to move, we calculated preferences for  and K10 . Unlike  and K10  showed aThe tail domain presented the same types of questions on its structure as the head domain and thus we conducted similar statistical analyses as was done (see above) for the head domain, which is shown in The central V2 globule subdomain of the tail (K1:497–607 and K10:461–558), consists chiefly of glycine loop motifs, with 6–12 mainly glycines between pairs of aromatic residues. Analysis of the sidechain interactions within the tail showed that each aromatic residue is on average paired with one other Fig F in , supportθr, and the axial displacement, d, of the V2 center-of-mass relative to the second to last residue of the 2B coiled-coil (see ) centered about the end of the rod domain. Similar analysis for the C-termini of the E2 subdomain (θr = 120°), i.e. at the tip of the rod rather than folded back. The greater variability in this distribution, relative to E1, further supports the notion that the E2-termini are held more loosely to the rod compared with E1.In Fig coil see . The poscoil see , suggestcoil see  centereubdomain  showed tOur results suggest that the K1/K10 dimer features a kinked central rod domain with a complex helical twist and is capped by a head domain that is folded back down the rod and a tail domain folded more loosely to the side. Based on our findings, we propose model structures for the head and tail domains of K1/K10. The head domain, Aggregation of aromatic residues as seen in our models, via π-stacking, may be important for maintaining and restoring the globular nature of these head and tail domains, when not under mechanical strain. Such behavior is also seen in available crystal structures of the tail domain fragments of the IF proteins lamin (1IFR.pdb) and in blood fibrin (3H32.pdb). Unique to the K1/K10 dimer is the large number of glycine residues present in the head and tail domains. These may be required to give keratin sufficient elastic properties that allow these domains to expand when under tension. Such sequences are also present to a lesser extent in the K5/K14 and K8/K18 keratins, which are also found in the skin, but not in trichocyte keratins (as found in hair) or other IF proteins which may not have such such high elasticity requirements. Another feature that is not conserved amongst the IF proteins, but is present in K1/K10, is the overall basic character of the head and tail domains. In other IF proteins the head and tail domains are highly charged, but their characters range between basic (including many of the keratin head domain), acidic and mixed charge, suggesting that these are specific for the IF.Our models of the rod domain indicate several kinks and stutters in the coiled-coil structure. These occur when the internal structure is disrupted from the interdigitation of an ideal coiled-coil. For example, in the 1B domain there are several places where a pair of opposing IF conserved, charged sidechains occupy the a- or d-heptad position of each chain . These features may be necessary for providing areas of interfacial registry between two dimers, through interlocking of the dimer rod profiles, which then induces the correct staggering of dimers within the tetramer structure.11, A22, A12 and ACN. Filaments are thought to exhibit a right-handed supertwist in the protofilaments, which are formed from tetramers [et al. [CN tetramer and hexamer  structure would also seem unsuitable candidates for such twisting. Thus, for such a model to be plausible, we suggest that this region must feature a break from the twisting between dimers, allowing the head and tail to interface . A similar kink is not required between either the 1B and 1A, or 1B and 2A subdomains  would be extremely valuable for comparison with our predicted structures for the K1/K10 rod domain, while EPR measurements, alongside our predictions, would be enormously helpful in resolving likely structures of the head and tail domains. The good level of overall residue conservation found within all IFs means that our approach and findings will translate to give detailed insights into the dimer structures of IFs in general, particularly of the rod domain. Additionally for keratins, as several sections of the sequence of the head domain are conserved within keratins, our models suggest that these regions are key to defining the emergent sub-domain structure.S1 FileSection A, Models Used. Section B, Modeling of the rod domain. Section C, Equilibration of the dimers. Section D, Ensemble properties of the dimer models. Section E, Dividing the head and tail domains into a tri-domain structure using keratin informatics data. Section F, Sidechain Contacts. Section G, Hydrogen and Hydrogen-π Interactions. Section H, Sulfide Residues.(DOCX)Click here for additional data file.S2 FileFig A, Keratin model building. Fig B, Evolution in structure of the truncated dimer models. Fig C, Evolution in structure of the full dimer models. Fig D, Statistical geometry of the dimer. Fig E, Definition of the subdomains of the head and tail. Fig F, Contacts between amino acid side chains. Fig G, Details on Hydrogen-bond interactions.(DOCX)Click here for additional data file.S3 FileAtomic coordinates of the final structures of our truncated dimer models.(TXT)Click here for additional data file.S4 FileAtomic coordinates of the final structures of our full dimer models.(TXT)Click here for additional data file.


In this work, the influence of micro- and macro-deformation profiles in GaN nanowires (NWs) on the angular intensity distribution of X-ray diffraction are studied theoretically. The calculations are performed by using kinematical theory of X-ray diffraction and assuming the deformation decays exponentially from the NW/substrate interface. Theoretical modeling of X-ray scattering from NWs with different deformation profiles are carried out. We show that the shape of the (002) 2θ/ω X-ray diffraction profile (XDP) is defined by initial deformation at the NW's bottom and its relaxation depth given by the decay depth of the exponential deformation profile. Also, we demonstrate that macro-deformation leads to XDP shift, whereas micro-deformations are the cause of XDP's asymmetry and its symmetrical broadening. A good correlation between calculated and experimental XDP from self-assembled GaN NWs on Si(111) substrate was achieved by taking into account all parameters of micro- and macro-deformation profiles. The wide range of unique properties of semiconductor GaN nanowires (NWs), along with various techniques of NW's growth, makes them promising candidates for nano-sized optoelectronics . In receOver recent years, there have been only a few significant reports devoted to this subject. In , the assIn this letter, we study the influence of macro- and micro-deformation along the GaN NW's growth axis on the X-ray diffraction peak profile. We show that macro-deformations lead to X-ray diffraction profile (XDP) shift, whereas micro-deformations are the cause of XDP's asymmetry and its symmetrical broadening. This allows distinguishing the influence of macro- and micro-deformation components on XDP and thereby to extract them separately.Kα1 radiation (λ = 1.540598 Å), a symmetric 4 × Ge(220) monochromator and a channel-cut Ge(220) analyzer.Self-induced GaN NWs were grown by plasma-assisted molecular beam epitaxy (MBE) on a Si111) substrate at approximately 760°C under highly nitrogen-rich conditions. Before the growth started, the substrate was exposed to a nitrogen flux for 30 min at the nitridation temperature of approximately 150°C. The procedure of plasma-assisted MBE growth of GaN NWs on Si(111) is described in ,11. The 1 substraA large jump of deformation occurs at the GaN/substrate interface, due to the large mismatch of the lattice parameters and coefficients of thermal expansion between GaN and conventional substrates. As GaN NWs are bounded by free surfaces, the misfit deformation that is mainly concentrated at the NW/substrate heterointerface elastically relaxes to the NW's free sides. Therefore, the deformation of a whole NW and at the heterointerface will considerably differ, i.e., deformation decreases from the bottom to the top of NW, as schematically shown in Figure ε|) and micro-deformations (ε|| and ε|||). The macro-deformation ε| is an integral deformation of a whole sample. On the other hand, deformations ε|| and ε||| occur in a limited part of a crystal and lattice unit volume, respectively. We consider that NWs are mostly deformed at their bottoms, thus they can be assumed to be free of ε|. As it is shown in Figure ε||(z) as the average lattice cells deformation along the NW diameter (see Equation ε|||(z) is the deformation fluctuation around the ε||(z)  and cmax(z) are the minimal and maximal deviation from co of the out-of-plane lattice parameter along the GaN nanowire's diameter at NW's height z.Depending on the crystal's considering volume, deformations are divided into two groups: macro-deformation (ε||(z) and ε|||(z) along the NW's growth axis are defined assuming exponential deformation decay [LR is the deformation relaxation depth, at which the deformation relaxes to 1/e of the initial values  is related to the incidence angle θ by:A simple simulation of XDP was performed to get information about the deformation distribution in GaN NWs. Here, we simulated a symmetric (002) 2θ/ω diffraction profile using the kinematical theory of X-ray diffraction. The deviations of the out-of-plane lattice parameter from the bulk value along the NW's height appear as variations in the length of the scattering vector lections , the lendl)(00 sin(θ) = nλ, we can present the length of the scattering vector proportional to the lattice parameter c(z):hkl) are Miller indices and n is the diffraction order.Taking into account the Bragg's law 2c(z) along the NW height is the origin of intensity distribution in Equation Fi is the structure factor of a plane at depth zi; N is the number of crystal planes and u(z) = 〈u(z)〉 ± δu(z). Here, 〈u(z)〉 = ε||(z) ⋅ co and δu(z) = ε|||(z) ⋅ co are the deviations of NW's lattice parameter c(z) from the bulk value co, caused by deformations ε||(z) and ε|||(z), respectively.Thus, from Equations ε||(z). We show how the magnitude of initial deformation LR (dependent on the aspect ratio of NW) contribute to the XDP. It is shown  is another source of asymmetrical XDP broadening. Moreover, when LR exceed some critical value (for a specific aspect ratio of NW), which leads to the XDP shift, we can conclude on the presence of macro-deformation ε|. Thus, the value of this deformation can be estimated directly from the magnitude of the XDP shift. It should be noted that the asymmetric broadening and shift of XDP for GaN NWs was observed experimentally in [Different types of deformation lead to specific changes in the XDP. In Figure entclass1pt{minimaentclass1pt{minimatally in ,4.Figureε|||(z) (caused by the elastic strain relaxation at the free NW's surface) on the XDP's shape. According to the definition, this unsigned deformation ε|||(z) is the deformation fluctuation around the ε||(z), and it is described by the same law (see Equation LR = const and ε|||(z). It is shown  magnitude leads only to symmetrical XDP broadening. It should be noted that coalescence of NWs and lattice distortion have a strong influence on deformation profile of ε|||(z) [ε|||(z) deformation leads to an appearance of ε||(z) deformation. Moreover, the increase of micro-deformation (ε||(z)) relaxation depth LR leads to the appearance of a macro-deformation (ε|). Thus, to fit the theoretical XDP from GaN NWs to the experimental one, the distribution of micro- and macro-deformation profiles should be taken into account.Next, we analyze the influence of only micro-deformation n Figure  that the ε|||(z) ,13. Actu00 l) 2θ/ω reflections . From the angular position of GaN(105) and Si(224) peaks on RSM  ⋅ 100 % ≈ 99 %). Here, Δθexp and Δθbulk are the differences between angular positions of GaN(105) and Si(224) peaks measured experimentally and their bulk values, respectively. On the other hand, the heterogeneous micro-deformation along the NWs c-axis and a small vertical correlation length (VCL) contribute to the broadening of the symmetrical 2θ/ω reflections in radial scan direction. These two effects have different dependencies on the diffraction vector and can be separated using Williamson-Hall (WH) plots [−4.Then, we consider the simulation of XDP from self-induced GaN NWs grown on Si(111) substrate. In order to evaluate the macro- and micro-deformation state of GaN NWs, we first used a standard approach based on measurements of reciprocal space map (RSM) and a series of symmetrical  plots ,15. The ns l = 2,,6. From D2 (where D is the NW's diameter) and the frequency of the corresponding nanowire diameter. Experimental and calculated (002) 2θ/ω XDPs of GaN NWs are shown in Figure ε|||(z) or ε||(z) is taken into account individually . It is demonstrated that in order to achieve a good fitting of experimental 2θ/ω XDPs, both components of micro-deformation should be considered. Moreover, since no peak shift is observed, we conclude that the NWs are free of macro-deformation ε|, which well correlates with results obtained from RSM. The large asymmetry of the red XDP indicates significant ε||(z) tensile micro-deformation at the bottom of the GaN NWs. The calculation provides the value of deformation relaxation depth LR to be 1.4 times larger than the NW's diameter and the magnitude of ε||(z) of the order of 10−3, which are close to values obtained in [z equal to 400 nm, which correlates well with values obtained from cross-sectional SEM image (not shown here). The average value of micro-deformation < ε|||(z) > ~ 1.7 × 10− 4 is in good agreement with the values presented in [Further, we calculated the XDP for a whole GaN nanowire ensemble taking into account the NW diameter distribution. We used the approach presented in , where tained in . Also, tented in  for GaN ε| of a whole NW leads to the angular position shift of XDP. Inhomogeneous micro-deformation ε||(z) along the NW height leads to asymmetry of the XDP. The micro-deformation ε|||(z) fluctuation around the ε||(z) causes only broadening of XDP. Thus, our theoretical approach of 2θ/ω XDP calculation can be used for quantitative analysis of GaN NWs with different shapes and deformation states.In this work, the theoretical analysis of micro- and macro-deformation parameters that influence symmetrical (002) 2θ/ω XDP from GaN NWs is presented. It is demonstrated that the macro-deformation


In this study, we use a chimaeric approach to model how an intra-domain interaction between domain 1 and domain 2 of the norovirus NS6 protease influences cleavage at specific non-structural substrate boundaries. in vitro assays using Escherichia coli-expressed, purified enzyme. The NS6 protease is formed of two distinct domains joined by a linking loop. Structural data suggest that domain 2 of the protease possesses substantial substrate binding pockets which form the bulk of the interactions with the NS boundaries and largely dictate boundary specificity and cleavage. We have constructed chimaeric murine norovirus (MNV) genomes carrying individual domains from the HNV protease and demonstrated by cell transfection that chimaeric HNV proteases have functional activity in the context of the full-length ORF1 polyprotein. Although domain 2 primarily confers boundary specificity, our data suggest that an inter-domain interaction exists within HNV NS6 protease which influences cleavage of specific substrates. The present study also shows that chimaeric MNVs provide improved models for studying HNV protein function in the context of a full ORF1 polyprotein.The norovirus NS6 protease is a key target for anti-viral drug development. Noroviruses encode a 2200 amino acid polyprotein which is cleaved by this critical protease at five defined boundary substrates into six mature non-structural (NS) proteins. Studies of the human norovirus (HNV) NS6 protease, in the context of a full ORF1 polyprotein, have been severely hampered because HNVs are not culturable. Thus, investigations into the HNV NS6 protease have been largely restricted to  Noroviruses are members of the calicivirus family of positive-sense, single-stranded RNA viruses. Human noroviruses (HNVs) are the leading cause of acute non-bacterial gastroenteritis, with epidemics common in semi-enclosed communities such as hospitals, schools and cruise ships. Currently, there is no vaccine or anti-viral therapy for treating HNV infection.A major factor limiting the progress of vaccine and anti-viral development is the lack of a robust cell culture system for studying the molecular mechanisms of HNV infection and pathogenesis, with most recent studies using murine norovirus (MNV) which can be cultured, as a surrogate for studying HNV biology.in cellulo have been severely hampered due to the limited systems available for studying HNVs. NS6-mediated scissile bond cleavage of the ORF1 polyprotein occurs at five NS boundaries, defined by Q-G, E-G or E-A at the cleavage junctions, with the addition of Q-N in the case of the MNV [The norovirus genome is translated into three open reading frames (ORFs), ORF1, ORF2 and ORF3 ,5. ORF1  the MNV –21. Clea the MNV ,16,22–25139), histidine (His30) and either a glutamic acid (Glu54) or aspartic acid (Asp54) residue in HNV and MNV respectively [Previous studies have reported the crystal structures of HNV and MNV proteases, either alone or in complex with natural substrate peptides and peptide-like inhibitors ,26–29. Tectively ,28,30–32Specific subsites or ‘pockets’ within the protease, which interact with the substrate boundary upstream of the cleavage junction, are well defined and named S5–S1 according to the standard nomenclature for proteases . Crystalin cellulo, within the context of a full ORF1 polyprotein.Since domain 2 of the protease contains the majority of the residues which form the S5–S1 pockets and interact with the cleavage boundary, we hypothesized that domain 2 dominates boundary specificity to dictate cleavage of the ORF1 polyprotein. To investigate this hypothesis, we made chimaeric MNV constructs by exchanging individual domains from the MNV NS6 protease, or the protease in its entirety, with the equivalent portion from the HNV protease. In doing so, we demonstrate that chimaeric MNV/HNV proteases show functional activity and that the HNV protease is able to process the MNV ORF1 polyprotein. In addition, results presented in the present study suggest that although domain 2 may confer the majority of the boundary specificity, an inter-domain interaction within HNV NS6 influences cleavage at specific NS boundaries. Furthermore, we propose that these ‘humanized’ MNV constructs, which carry a HNV NS6 protease in place of the MNV protease, provide an additional model for further understanding of HNV protease function The polII based MNV plasmid expression construct, pMNV*, from which all pMNV* mutants were derived, has been described previously . The seqTo introduce the entire Southampton virus (SV) NS6 sequence mutagenic PCR was used, with first round amplification using primers SV3C_Fwd + SVD2_MNV3CDP6-P2′_Rvs with template pT7-SV3BCD  , to amplGeneration of the domain swap mutants was performed by standard overlapping 2-step PCR mutagenesis . PrimersThe AK29TT and D54E coding changes were introduced using standard two-step PCR mutagenesis, with first round reactions using primers MNV3CD1_AK29TT_Fwd or MNV3CD1_D54E_Fwd with MNV_6R and MNV3CD1_AK29TT_Rvs or MNV3CD1_D54E_Rvs with MNV_4F, with template pMNV*-SV3CD1. The second round PCR products, generated using the products from the first round and primers MNV_4F + MNV_6R, were digested with AflII and cloned into AflII-digested pMNV*. The same strategy was used to introduce the TT29AK and E54D mutations into pMNV*-SV3CD2 with mutagenic PCR primers MNVSV3CD1_TT29AK_Fwd + MNVSV3CD1_TT29AK_Rvs and MNVSV3CD1_E54D_Fwd + MNVSV3CD1_E54D_Rvs.Escherichia coli expression construct, pSV3C (pT7-7/SV9), referred to in the present study as pT7-SV3BCD, has been previously described [The T7-based escribed . Brieflyescribed  to ampliThe similar MNV construct, pRSETA-MNV3BCD, was generated by PCR, using primers NS6_Fwd + NS6_Rvs with template pMNV*, to amplify the entire NS6 sequence including the flanking 123 bp of MNV* sequence at both ends. The amplified PCR product was digested with BamHI and EcoRI and introduced into the expression vector pRSETA (Invitrogen) at the BamHI and EcoRI sites. For generating the pRSETA-derived constructs expressing the domain swap chimaeric proteases, the unique SalI–Bsu36I fragment from pRSETA-MNV3BCD, which covers the entire protease gene and upstream/downstream flanking regions, was exchanged with the equivalent SalI–Bsu36I fragment from the pMNV* domain swap vectors.E. coli overexpression of pRSETA based constructs and subsequent protease purification was carried out as previously described [escribed . FollowipNA (Peptide Protein Research) which contains an acetylated (Ac) N-terminus and a C-terminal p-nitroaniline (pNA) group to provide a spectrophotometric output. The rate of cleavage of this substrate  by NS6 protease at a final concentration of 0.1 mg/ml was monitored at 415 nm using a iMark plate reader (Bio-Rad) over a 10 min time period. Results show mean kinetic values of three independent experiments.Kinetic studies on purified NS6 proteases were performed as previously described , using tSDS/PAGE and Western blot were carried out as previously described . PrimaryHEK-293T cells  were grown in Dulbecco's modified Eagle's medium supplemented with 10% FBS and GlutaMAX-1 (Invitrogen). Transfection of HEK-293T cells was performed as previously described  using 1 Models of the three-dimensional structures of the two domain swapped chimaeric proteases were produced by least-squares superposition of the SV and MNV protease structures ,28. FollThe structure of MNV and HNV NS6 protease in complex with boundary substrates and substrate-like inhibitors strongly suggests that domain 2 is primarily responsible for recognizing all five NS protein boundaries and dictating cleavage specificity ,28,29. T71 and Cys77. It was therefore decided to splice the domains within the lpeI loop between residues 72 and 73  and are predicted to form a total of seven hydrogen bonds and three salt bridges. Likewise, the two domains of the chimaeric protease carrying SV domain 2, are estimated to interact with a similar ΔG  and form 11 hydrogen bonds and five salt bridges. Both of these values compare very favourably with the equivalent values of −19.5 kcal/mol  for the two domains of the wild-type SV protease and −21.1 kcal/mol  for the wild-type MNV protease, strongly suggesting the chimaeric enzymes are likely to be as stable as the wild-type proteases.Alignment of the MNV and SV NS6 20-residue lpeI loop, which connects the two protease domains, shows that the greatest sequence identity exists between Met2 and 73 . Compute2 and 73 . Indeed,E. coli expression system. This system, developed in our laboratory using wild-type SV protease, uses a T7 vector to express MNV NS6 flanked by 41 residues of upstream NS5 sequence and 41 residues of downstream NS7 sequence to produce a ‘mini’ NS5–NS7 precursor. Fused upstream of the NS5 sequence are 36 amino acids of vector sequence containing a His tag and Xpress epitope  [Having established that all the chimaeric NS6 proteases were able to self-excise from the ‘mini’ NS5–NS7 precursor to yield mature NS6, we were able to produce highly purified protease from all five constructs and conduct a comparative kinetic analysis. The five NS6 proteases were purified from whole gure S3) . A majorgure S3) ,28,43,44pNA peptide Ac-DEFQLQ-pNA, a synthetic substrate representing the SV NS3NS4 cleavage boundary . Compared with wild-type protease, the double domain swap chimaera (MNV/SVD1+D2 NS6) demonstrated a less than 2-fold reduction in activity in terms of the kcat and a minor reduction in Km, resulting in overall a less than 2-fold reduction in activity in terms of specificity constant (kcat/Km). The chimaeric protease carrying SV domain 1 (MNV/SVD1 NS6) also showed a reduction in kcat of approximately 3-fold compared with the wild-type proteases. However, a less than 2-fold reduction in Km resulted in only a 2-fold overall reduction in activity in terms of the specificity constant. In contrast, the chimaeric protease carrying SV domain 2 (MNV/SVD2 NS6) demonstrated no proteolytic activity on this substrate during the assay period. Furthermore, increasing the assay period for up to 4 h resulted in no increase in OD above background levels for this enzyme .Measurements of the initial rate of cleavage were performed for each chimaeric protease (and wild-type controls) over a 10 min time period using the chromogenic E. coli on the NS5NS6 and NS6NS7 MNV boundaries, no functional activity was detected on the synthetic substrate representing the SV NS3NS4 cleavage boundary, suggesting this enzyme has restricted boundary recognition and therefore limited proteolysis. However, the in vitro kinetic analysis was limited to a single synthetic boundary outside the context of a full ORF1 polyprotein and not in the context of a eukaryotic background. Therefore, to investigate any potential variations in boundary proteolysis in a full-length ORF1 polyprotein, where all five MNV cleavage boundaries are present, all three chimaeric proteases were introduced into MNV*, a construct expressing the entire MNV cDNA under control of a tTA responsive polII promoter. Transfection of MNV* constructs into permissive cell lines allows for export of capped viral transcripts to the cell cytoplasm and expression of the full ORF1 polyprotein where cleavage of the NS boundaries can be easily accessed by Western blot. The chimaeric MNV* constructs and controls were co-transfected into HEK-293T cells with ptTA, a plasmid expressing the tetracycline trans-activator allowing for maximal promoter activity, and NS protein cleavage was assayed by Western blot  showed complete processing at the NS1/2NS3 boundary, yielding mature NS1/2 and NS3. Both chimaeric constructs carrying either SV domain 1 or SV domain 2  demonstrated little or no processing at the NS4NS5 and NS5NS6 boundaries, yielding mainly uncleaved NS4–NS6 and NS5NS6 precursors and a moderate amount of fully cleaved NS4. Interestingly, the double domain swap chimaera (MNV/SVD1 + D2) showed partial processing at both the NS4NS5 and NS5NS6 boundaries, resulting in a moderate amount of mature NS4 and NS6. Due to the unavailability of an anti-NS5 antibody capable of detecting the relatively low abundance of protein in this system, assignment of NS5 containing precursors was done by elimination.Our original hypothesis was that domain 2 alone interacts with the boundary sequence to define cleavage specificity. Thus, the cleavage profile observed by exchanging solely domain 2 should be the same as exchanging both domains of the protease. Contrary to this expectation, although the double domain swap chimaera demonstrated partial cleavage at the NS5NS6 boundary, the chimaeric protease carrying SV domain 2 showed a complete block in NS5NS6 processing. This suggests that boundary specificity is not conferred by domain 2 alone, and domain 1 must contribute some key residues which influence cleavage at certain NS boundaries.30 of the catalytic triad), which are changed from two threonines in SV protease to an alanine and a lysine in the MNV protease  had no effect on NS boundary cleavage. However, introduction of the TT29AK mutations (MNV/SVD1/TT29AK) moderately increased cleavage at the NS5NS6 boundary yielding a limited amount of mature NS6 protease. The chimaeric construct carrying the SV domain 2 showed a limitation in cleavage at the NS4NS5 and NS5NS6 boundaries (yielding approximately 37±3% NS4-6 and 62±3% NS5NS6 by densitometry), in addition to limited processing of the NS1/2NS3 boundary. Introduction of the AK29TT mutations within MNV domain 1 of this construct (MNV/SVD2/AK29TT) resulted in no change in NS boundary cleavage (approximately 38±0.5% NS4–NS6 and 62±1% NS5NS6 by densitometry). However, mutation of the aspartic acid residue at position 54 to a glutamic acid (MNV/SVD2/D54E) resulted in increased cleavage at the NS5NS6 boundary, yielding mature NS6 to levels similar to that observed in the double domain swap chimaeric construct . Although it is worth noting that this D54E mutant protease (MNV/SVD2/D54E) still demonstrated a greater proportion of uncleaved NS4-6 and less NS5NS6  when compared with the double domain swap chimaeric construct .E. coli lysates (results not shown), and measurements of the initial rate of cleavage performed, as before, with wild-type MNV and SV proteases included as internal controls . Purified protease was produced from whole controls .kcat/Km values not significantly different to those observed in kcat when compared with either wild-type protease, and as a result showed a 10-fold reduction in the specificity constant (kcat/Km). Conversely, introduction of the D54E mutation within the chimaeric NS6 carrying SV domain 2 (MNV/SVD2/D54E NS6), restored a minimal level of proteolytic activity on this substrate, albeit with a 2.5-fold greater Km and a 30-fold reduction in kcat, resulting in approximately a 70-fold reduced specificity constant (kcat/Km) when compared with wild-type protease.Little difference was observed in the kinetic activities of the wild-type MNV or SV NS6 proteases on this substrate which showed 139 has been identified as the active site nucleophile and His30 as the general base catalyst which is protonated on the imidazole ring. The acidic third residue of the triad, Glu54 in HNV and Asp54 in MNV is suggested to hydrogen bond with His30 to orientate and stabilize the imidazole ring. The S1, S2 and S4 binding pockets appear to be the most critical for substrate binding, the S2 and S4 subsites both forming particularly deep hydrophobic pockets which overlap and share residues [Release of the NS proteins from the ORF1 polyprotein in noroviruses is facilitated by the NS6 protease, which cleaves at five defined scissile bond dipeptides to yield six mature NS proteins ,16,17. Tresidues –28,31,32in vitro assays and in the context of a full-length ORF1 polyprotein. These observations led to our second hypothesis, that domain 1 contributes critical residues which facilitate cleavage of specific NS protein boundaries.We sought to investigate whether individual protease domains conferred specificity to cleavage sites within the MNV ORF1 polyprotein. Our initial hypothesis was that domain 2 of the protease was the main contributor to substrate specificity, dictating boundary cleavage. Accordingly, we constructed simple domain swap chimaeric proteases and re-examined polyprotein processing. First, it was surprising that proteolytic activity is retained with all the chimaeric domain swaps, considering the evolutionary distance between MNV and HNV  and the 30, in HNV is changed to TAK in MNV. Furthermore, both Glu54 and the triple neutral-polar stretch are conserved across all HNV sequences (the latter being either TTT or TST depending on isolate). We therefore hypothesized that some of these differences, within domain 1 of the protease, could influence cleavage at some NS protein boundaries. In agreement with these predictions, we found that introduction of a TT29AK mutation within SV domain 1 of the chimaeric protease carrying MNV domain 2, increased cleavage at the NS5NS6 boundary, despite showing a 10-fold reduction in kinetic activity in vitro. Moreover, the D54E mutation within MNV domain 1 of the chimaeric protease carrying SV domain 2, restored cleavage at the NS5NS6 boundary to levels equivalent to the double domain swap chimaera, and restored minimal activity in vitro on the NS3NS4 substrate.Comparison of the domain 1 residues in MNV and HNV that are predicted to lie close to the boundary substrate and active site highlighted two strong potential candidate residues that could influence boundary cleavage. First, the acidic third residue of the catalytic triad is different between MNV and HNV. Secondly, a triple neutral-polar stretch of TTT at positions 27–29, just prior to His54 within domain 1 can salt-bridge with Arg112 within the flexible lpbII loop of domain 2. Furthermore, computer modelling suggests that this interaction would be weaker in the chimaeric protease carrying SV domain 2 (which contains MNV domain 1 and thus Asp54) due to a slightly shorter side chain. Although it should be noted that the interaction between Glu54 and Arg112 is somewhat variable in the available crystal structures, and indeed the structure in which these residues come the closest together has a partially disrupted catalytic triad, it is conceivable that the Glu54–Arg112 interaction is of some physiological importance, not least because this particular glutamate is the closest negatively charged group to the guanidinium of Arg112. It would also be anticipated that re-introduction of Glu54 into MNV domain 1 of the chimaeric protease carrying SV domain 2, might strengthen this salt bridge interaction. It is possible that this salt bridge interaction is important for proteolysis of particular substrates, such as the NS5NS6 boundary (KINFE-AP). Recently, Muhaxhiri et al. [54–Arg112 salt bridge interaction is required for HNV NS6-mediated cleavage of KINFE-AP substrates, it is conceivable that the Glu54–Arg112 salt bridge interaction is key in stabilizing the more ‘open’ protease structure to allow cleavage of boundaries with larger P2 residues when glutamic acid is found in the P1 position. Previous reports have shown that mutation of the acidic Glu54 to a basic residue (lysine or arginine) is greatly detrimental to protease stability, and introduction of many hydrophobic residues at this position restricts cleavage at certain substrate boundaries [54 residue to several alternatives, including aspartic acid, had no effect on processing of the NS5NS6 boundary, despite showing a 4–5-fold reduction in kinetic activity on a NS3NS4 boundary substrate [in vitro studies were conducted outside the context of the full-length polyprotein, where secondary factors such as protein folding intermediates are likely to play a role in substrate proteolysis. This is further highlighted by the observation that although the purified NS6 carrying SV domain 2 showed no kinetic activity in vitro, on a synthetic SV NS3NS4 boundary peptide, it was able to cleave the MNV NS3NS4 boundary when in the context of a full ORF1 polyprotein. Comparison of the SV and MNV NS3NS4 boundaries show little difference in their sequences, being EFQLQ-GK and DFGLQ-NK, respectively. It is, however, possible that the difference in the residues at the P3 and P1′ position of these boundaries may account for this difference in cleavage, given the characterized interactions between domain 2 of HNV and the P3 residue [in vitro assays, such as the use of synthetic boundary peptides with purified proteases, to draw conclusions about eukaryotic virus biology.The X-ray structure of wild-type HNV protease shows that Glui et al.  have obsundaries . Howeverubstrate ,32. This residue . Taken t162 and the P3 residue is likely to be conserved in MNV [cis–trans cleavage requirements of MNV and HNV or the presence of different functional NS precursors in HNV. Both stable and/or transient NS protein precursors have been shown or suggested to have roles in the replication of several positive strand RNA viruses [in vitro studies with HNV and in cellulo studies with MNV, and the exact order of ORF1 processing for either norovirus and thus the precursors formed, has not yet been defined with a natural template in cellulo.All of the chimaeric constructs were able to efficiently process the NS3NS4 and NS6NS7 boundaries to completion. This suggests a preference of both enzymes to cleave substrates with glutamine in the P1 position, even though structural studies suggest that the protease makes the same interactions with either glutamine or glutamic acid in the P1 position . Despited in MNV ,26. Alte viruses –49. The  viruses –53. Even viruses ,55, and To date, none of the crystal structures of norovirus proteases in complex with boundary substrates or peptide mimics contained any residues on the P′ side, downstream of the cleavage junction. Alignment of the amino acid sequences on the P′ side of the boundary reveals no clear amino acid conservation beyond the preference for smaller residues  in the P1′ position and lysine, glycine, glutamine or proline in the P2′ position, suggesting the P’ side may have relatively little influence in dictating boundary cleavage. Indeed, a recent study with HNV protease showed that substitution of proline for glycine at the P2′ position resulted in a only a 3-fold decrease in cleavage efficiency . Despitein vitro based assays and the few available replicon models in that it allows for assay of protease cleavage upon multiple boundaries in the context of a full ORF1 polyprotein, which is temporally expressed in mammalian cells in a transient system which does not require the laborious process of establishing stable replicon cell lines. The technological advancements offered by this new system, in combination with the current models, offers greater insights and a deeper understanding of the multi-functional nature of this apparently simple enzyme which has many complex interactions.The lack of a robust cell culture system for HNV has severely limited the ability to dissect properties of the HNV NS6 protease in an eukaryotic background and within the context of a complete ORF1 polyprotein. In the present work, we have established a new system to study core functions of the NS6 protease. This system offers significant advantages over the current


Saccharomyces cerevisiae Fkh1 protein has roles in cell-cycle regulated transcription as well as a transcription-independent role in recombination donor preference during mating-type switching. The conserved FHA domain of Fkh1 regulates donor preference by juxtaposing two distant regions on chromosome III to promote their recombination. A model posits that this Fkh1-mediated long-range chromosomal juxtaposition requires an interaction between the FHA domain and a partner protein(s), but to date no relevant partner has been described. In this study, we used structural modeling, 2-hybrid assays, and mutational analyses to show that the predicted phosphothreonine-binding FHA domain of Fkh1 interacted with multiple partner proteins. The Fkh1 FHA domain was important for its role in cell-cycle regulation, but no single interaction partner could account for this role. In contrast, Fkh1’s interaction with the Mph1 DNA repair helicase regulated donor preference during mating-type switching. Using 2-hybrid assays, co-immunoprecipitation, and fluorescence anisotropy, we mapped a discrete peptide within the regulatory Mph1 C-terminus required for this interaction and identified two threonines that were particularly important. In vitro binding experiments indicated that at least one of these threonines had to be phosphorylated for efficient Fkh1 binding. Substitution of these two threonines with alanines (mph1-2TA) specifically abolished the Fkh1-Mph1 interaction in vivo and altered donor preference during mating-type switching to the same degree as mph1Δ. Notably, the mph1-2TA allele maintained other functions of Mph1 in genome stability. Deletion of a second Fkh1-interacting protein encoded by YMR144W also resulted in a change in Fkh1-FHA-dependent donor preference. We have named this gene FDO1 for Forkhead one interacting protein involved in donor preference. We conclude that a phosphothreonine-mediated protein-protein interface between Fkh1-FHA and Mph1 contributes to a specific long-range chromosomal interaction required for mating-type switching, but that Fkh1-FHA must also interact with several other proteins to achieve full functionality in this process.The  Specific chromosomal interactions between distal regions of the genome allow for DNA transactions necessary for normal cell function, but the protein-protein interfaces that regulate such interactions remain largely unknown. The budding yeast Fkh1 protein uses its evolutionarily conserved phosphothreonine-binding FHA domain to regulate a long-range DNA transaction called mating-type switching that allows yeast cells to switch their sexual phenotype. In this study, another conserved nuclear protein, the Mph1 DNA repair helicase, was shown to interact directly with the FHA domain of Fkh1 to regulate mating-type switching. The Fkh1-Mph1 interaction required two phosphorylated threonines on Mph1 that were dispensable for many other Mph1-protein interactions and other Mph1 chromosomal functions. Thus a discrete protein-protein interface between two multifunctional chromosomal proteins helps define a long-range chromosomal interaction important for controlling cell behavior. Saccharomyces cerevisiae Fkh1 (forkhead homolog 1) protein is a member of the FOX (forkhead box) family of proteins defined by their winged-helix DNA binding domains. The FOX family proteins are best known for their transcriptional roles in regulating the cell cycle and differentiation [CLB2-cluster genes required for the proper execution of M-phase events [FKH1 and FKH2, but not either gene alone, causes severe cell-cycle dysfunction. However, its molecular functions and the mechanisms by which Fkh1 participates in this process remain poorly understood [The e events . Fkh1 apderstood ,13. Accuderstood ,15. In aderstood ,17, but MAT locus that contains either a- or alpha- mating-type genes. This break is repaired through homologous recombination using donor template sequences located at the silent mating-type loci, HML or HMR, at the opposite ends of the same chromosome as MAT  or a genes (HMRa), respectively. Productive mating-type switching requires the proper choice between these two donor loci so that the opposite mating-type gene is inserted at MAT. Thus MATa cells favor recombination with HMLα ~90% of the time, while MATα cells choose HMRa as a donor ~90% of the time , a chromosomal region located between the MAT and HML loci [MATa cells and establishing a strong preference for HMLα for repair  caused a defect in donor preference during mating-type switching similar to that caused by mph1Δ. However, mph1-2TA cells did not share other cellular defects caused by mph1Δ, such as sensitivity to MMS or an elevated rate of mutation. Because MPH1 could only partially explain Fkh1-FHA’s role in mating-type switching, we examined the role of a second Fkh1-interacting protein identified in our screen, encoded by YMR144W. A ymr144WΔ also altered mating-type switching directionality, and ymr144WΔ mph1Δ reduced the efficiency of this process beyond that of either mutation alone. We have named this gene FDO1 for Forkhead one interacting protein involved in donor preference. Thus we have delineated a specific cellular role for Fkh1 and Mph1 mediated by an FHA-phosphothreonine interaction, and provided evidence that Fkh1-FHA bound to the RE likely must recognize several proteins at the DSB for full function in mating-type switching directionality.To address these issues, we performed a 2-hybrid interaction screen that identified five Fkh1-interacting proteins. Domain analyses revealed that Fkh1 interacted with each of these proteins via its FHA domain. Mutation of key residues within this domain revealed that it was important for Fkh1’s role in cell-cycle regulation, though no single interacting partner could account for this role. In addition, our genetic analyses indicate that functions of the FHA domain outside of its phosphopeptide binding activity contribute to Fkh1’s cell cycle role. Focusing on one Fkh1 binding partner, Mph1, we found that its loss altered donor preference during mating-type switching. Using multiple approaches, we defined a peptide within Mph1 that interacted directly and efficiently with purified Fkh1 To identify proteins that interact with Fkh1, we used a 2-hybrid interaction screen in which a Fkh1-Gal4 DNA binding domain (Fkh1-GBD) fusion protein served as bait and a library of Gal4 activation domain (GAD) fusions served as prey . This FkTo define how Fkh1 interacts with the proteins identified in our screen, we tested which regions of Fkh1 interacted with Mph1, the yeast homolog of the human FANCM helicase ,37. The Next, we examined whether the predicted phosphothreonine binding ability of Fkh1-FHA was required for binding Mph1. To this end, we performed homology modeling  using puBased on this structural and alignment information we engineered several single amino acid substitutions in Fkh1-FHA and assessed their ability to interact with Mph1-Ct in 2-hybrid assays. We found that several amino acids predicted to be on the phosphopeptide binding surface, as well as a more distal residue (S155), were important for interaction with Mph1 . For exaTo test whether the FHA domain of Fkh1 is also involved in interacting with other proteins recovered from our 2-hybrid screen, we examined their binding to Fkh1-FHA and the mutant constructs described above in the 2-hybrid assay . Fkh1-FHFKH1 and FKH2, but not either gene alone, causes cell-cycle dysfunction that leads to a pseudohyphal-like growth that produces rough, chalky colonies that scar solid agar medium [FKH2 function [FKH1 function in these phenotypes has not been reported. Therefore, we determined whether mutant versions of Fkh1 examined above (referred to as fkh1-m) resulted in these defects in a fkh2Δ background , when combined with fkh2Δ, produced the same phenotype as fkh1Δ and fkh1-dbdΔ  display a preference for particular amino acids at the pT +3 residue, while other FHA domains have a preference for particular amino acids at other positions ,52. As aE. coli and its ability to bind an 18-residue peptide representing Mph1(772–789) was assessed by fluorescence anisotropy . In addition, and consistent with the effects observed in the 2-hybrid assays in Next, we tested whether Fkh1 interacted directly with Mph1 through the region containing T776 and T785 and if this interaction was controlled by phosphorylation of these threonines. To this end, recombinant Fkh1-6xHis was purified from isotropy . The pepfinities . The nonMAT and the donor locus HML. In a previous study, the N-terminal region of Fkh1 containing the FHA domain was shown to be sufficient to direct RE function [a-mating-type genes located at HMR were replaced by MATα sequences that contained a unique BamHI restriction site (HMRα-B), such that repair of a DSB generated by the HO endonuclease at MATa will always result in a MATα cell, and those using the HMRα-B donor sequence can be cut by BamHI, while those using HMLα cannot. Thus donor preference can be examined by testing the relative abundance of the two different repair products through a PCR reaction that amplifies MATα sequences followed by a BamHI restriction digest , recombination between MATa and HML was reduced to less than 20%  did not alter donor preference as drastically as deletion of MPH1 or the mph1-2TA allele, although it did have a statistically small effect. This donor preference defect caused by mph1-2TA was specific to this allele because, unlike mph1Δ cells, mph1-2TA cells did not exhibit sensitivity to MMS  and promoting recombination between an HO-induced DSB at function . This pofunction . In thisn digest . Consistthan 20% . We founof cells . While ty to MMS  or an iny to MMS . Thus thHML usage in mph1-2TA strains is less than that in cells expressing LexA-FHA-R80A, suggesting there must be other Fkh1 partners required for its role in mating-type switching. To address a role for additional Fkh1-FHA partner proteins, we examined the switching profile in cells lacking Fdo1. We found that deletion of FDO1 reduces HML usage to ~80%, a 10% reduction relative to the wild type control similar to the level of reduction caused by deletion of MPH1 , both of which abolish Fkh1-FHA-Mph1 interactions? The simplest explanation is that Mph1 is only one of several proteins bound to the DSB at MAT that the Fkh1 FHA domain uses to locate this lesion. It makes sense for Fkh1 to bind several different proteins at the DSB with relatively weak affinities—in this way the RE remains close to MAT long enough to increase the opportunity for strand invasion into HML. At the same time Fkh1 is not bound so tightly to any one partner or the DSB region itself to inhibit strand invasion and the protein/DNA remodeling necessary to drive the recombination event. Therefore, we propose that there must exist other Fkh1-FHA partner proteins at the HO-induced DSB at MAT that contribute to the RE’s ability to direct the MAT locus to HML. The multi-partner model for Fkh1 FHA function in donor preference may represent a general mechanism by which Fkh1 FHA performs its other biological functions in transcription and replication. This type of mechanism may allow for relatively high specificity but low affinity  interactions that may be important to these complex chromosomal processes.While our data were consistent with the model for Fkh1 bound to the recombination enhancer (RE) guiding the B at MAT , they alFDO1, a gene encoding another Fkh1-FHA interaction partner identified in our screen, also reduced donor preference to a degree similar to that of mph1-2TA (or mph1Δ). Moreover, a deletion of both genes to create an fdo1Δ mph1Δ cell reduced preference for HML to a degree greater than deletion of either gene alone. However, a substantial amount of Fkh1-FHA-dependent donor preference remained intact even in cells carrying null mutations in both of these genes, suggesting that another protein or proteins at the DSB must interact with Fkh1-FHA. Many proteins, in addition to Mph1, bind to DSBs and would be good candidates for additional Fkh1-FHA interaction partners that regulate donor preference [Based on this idea and data reported in a previous study, the CK2 kinase likely phosphorylates many Fkh1-interacting proteins involved in donor preference . In thiseference –57. Whileference . It willfkh1-fha single residue substitution (fkh1-m) mutants abolished interaction with all protein partners uncovered here and reduced Fkh1’s ability to function in cell-cycle regulation with Fkh2, even though deletion of no single gene encoding an interaction partner had an effect. Based on the results with donor preference, it seems likely that multiple different Fkh1-FHA interaction partners will be needed to fully explain Fkh1-FHA’s role in cell cycle regulation. A deletion of the entire FHA domain of Fkh1 (fkh1-fhaΔ) phenocopied a fkh1Δ mutation in cell cycle regulation as measured by both mitotic cell division rates and pseudohyphal-like growth and agar scarring when combined with a fkh2Δ allele. Because the established role of FHA domains is to bind phosphopeptides, it was perhaps unexpected that amino acid substitutions in the FHA domain predicted to abolish FHA-phosphopeptide interactions only slowed mitotic cell division in fkh2Δ cells without causing pseudohyphal-like growth. The Fkh1 FHA domain may play roles in Fkh1 function in addition to phosphopeptide binding by providing as yet undefined interaction surfaces for other regulators of transcription. Alternatively, the fkh1-fhaΔ allele used in this study lacked coding information for an additional ~30 amino acids outside of the alignment-defined FHA domain that may provide surfaces for additional protein-protein interactions. Regardless, these data raise new questions about whether Fkh1’s roles in regulating cell proliferation rate and suppressing pseudohyphal growth are completely separable, or whether a certain threshold of reduced transcription/altered transcriptional regulation must be met before pseudohyphal growth is also observed.Our data provided evidence that the Fkh1 FHA domain may be controlling most, if not all, Fkh1-mediated biology in yeast. Indeed, many Our data provided evidence that several Fkh1-FHA interaction partners that can direct Fkh1 cellular roles remain unidentified. As we have shown, determining the role of any particular Fkh1-protein interaction is difficult through mutation of Fkh1-FHA itself, as the same FHA residues participate in multiple Fkh1-protein interactions and Fkh1 processes. For this reason, it will be important to identify other Fkh1-FHA-partner proteins and engineer mutations that specifically abolish their ability to interact with Fkh1, as we did for Mph1 in this study, to isolate the discrete mechanisms and pathways influenced by Fkh1.Saccharomyces cerevisiae strain w303 unless otherwise noted. Standard methods were used for yeast growth, strain and plasmid construction. Strains used in this study are listed in Strains used in this study were derived from the HIS3 and ADE2. The original screen was performed using a Fkh1-GBD fusion protein in which the entire DNA binding domain was precisely replaced with the GBD. This GBD-Fkh1 fusion activated transcription of the HIS3 reporter gene. Therefore colonies harboring potential Fkh1-interacting partners were identified on minimal media lacking both histidine and adenine.2-hybrid assays were performed in the PJ69-4A strain as described in . The strA predicted structure for the Fkh1 FHA domain was generated using the N-terminal Rad53 FHA domain as a template using SWISS-MODEL –45. Aminfkh1-m/+ fkh2Δ/+ diploids expressing Fkh1 mutants were dissected and scanned after three days growth. Agar scarring was assessed by gently patching haploid strains onto YPD and washing with H2O after three days. Growth curves were generated by growing to saturation in YPD media, diluting to an OD600 of 0.1 in a 96-well plate, and monitoring growth by measuring the OD600 every three minutes over a 24 hour period in a Biotek Synergy 2 plate reader shaking at 30°C. Doubling times were calculated by exponential regression of data generated from growth curves during log-phase [Heterozygous og-phase .Cell extracts for western blotting were prepared as described in . Cell exCo-immunoprecipitation of Fkh1 and FLAG-tagged Mph1 (modified from ) were peE. coli. E. coli were broken with modified B-PER (Thermo Fisher) diluted 1:1 in wash buffer ) with 1 mM EDTA. His-tagged Fkh1 protein was purified using nickel chromatography (Qiagen) and eluted in buffer . Peptides (synthesized by the University of Wisconsin-Madison and the Tufts University Core Facility) were labeled on the N-terminus with 5-carboxy fluorescein and an aminohexanoic acid linker. Peptides  were mixed with titrations of purified Fkh1-6xHis protein in binding buffer ). Polarization at each concentration was measured in triplicates in 384-well polystyrene black microplates (Thermo Fisher Scientific #262260) by a Biotek Synergy H4 multimode plate reader . Fraction bound (Fb) at each concentration was calculated based on the corresponding polarization values (P): Fbc = (Pc—Pmin) / (Pmax—Pmin), where Pmin is the polarization value of the no-protein control and Pmax is the polarization value of the saturation value for that peptide. Dissociation constants (Kd) were derived by KaleidaGraph (version 4.1.3) using the following equation:C-terminally His-tagged full length Fkh1 protein was expressed from a pET28b expression vector in Rosetta 600 is 0.5 and 10-fold serial dilutions were spotted onto YPD plates containing the indicated concentration of MMS. MMS plates were poured fresh on the day of each experiment. Plates were imaged three days after plating.Mutation rates were calculated by fluctuation analysis as in . BrieflyMATα sequences using primers Yalpha105F and MAT-dist4R [BamHI and the resulting digest was run on an agarose gel. Relative densities of the different bands were determined using ImageJ [HML usage) was calculated using the formula MATα / (MATα+MATα-B).Donor preference during mating-type switching was determined by a PCR-based method as described in . BrieflyT-dist4R . 700 ng g ImageJ , and donS1 FigA) QMEAN4 scores of homology models generated of the Fkh1 FHA domain. QMEAN4 scores provided by SWISS-MODEL [B) Predicted residue error of every amino acid residue (in Ångström) in the Fkh1 homology model as assessed by the QMEAN scoring function. Provided by SWISS-MODEL [(SS-MODEL . (B) PreSS-MODEL .(TIF)Click here for additional data file.S2 FigA-C) Yeast 2-hybrid assays using different mutant forms of Fkh1 bait. The FHA domain is defined as amino acids 50–202. GAD constructs contain the segment of each protein identified in the original 2-hybrid screen (listed in ((TIF)Click here for additional data file.S3 FigA) Model of electrostatic potential. Blue indicates positively charged regions. Red indicates negatively charged regions. (B) The positively charged region on the phosphopeptide interaction surface contains residues R80, K107, R111, K112, and R132. See ((TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Leishmania major has been determined. The structure identifies a single domain dominated by a right-handed β-helix and comparisons suggest key residues involved in stimulating the GTPase activity of β-tubulin.The crystal structure of the C-terminal fragment of tubulin-binding cofactor C from LmTBCC-C.The structure of  •Leishmania major is reported.The crystal structure of the C terminal fragment of tubulin binding cofactor C from •The structure identifies a single domain dominated by a right handed β helix.•Comparisons suggest key residues involved in stimulating the GTPase activity of β tubulin. Leishmania major based on single wavelength anomalous dispersion measurements targeting a selenomethionine derivative. Although previously predicted to consist of two domains the structure is best described as a single domain dominated by a right-handed β-helix of five turns that form a triangular prism. One face of the prism is covered by the C-terminal residues leaving another face solvent exposed. Comparisons with an orthologous human GTPase activating protein match key residues involved in binding nucleotide and identify the face of the β-helix fold likely involved in interacting with the β-tubulin:GTP complex.Tubulin-binding cofactor C stimulates GTPase activity and contributes to the release of the heterodimeric α/β-tubulin from a super-complex of tubulin monomers and two ancillary cofactors. We have determined the 2.2 Å resolution crystal structure of the C-terminal domain of tubulin-binding cofactor C from  The cytoskeleton of trypanosomatids is highly enriched in microtubules. They form a network of subpellicular network beneath the cell membrane and maintain the overall parasite shape. Microtubules are abundant in the flagellum, an organelle important for pathogenicity as well as motility, for attaching the parasite to the salivary gland of the insect vector prior to the infection of a new host and also contributing to the evasion of the host immune system There are five TBCs, termed A–E. TBCB and E bind α-tubulin whilst A and D interact with β-tubulin, to deliver each tubulin subunit into a super-complex comprising the α/β-tubulin heterodimer and cofactors D and E L. major . We describe the crystallographic analysis of the C-terminal domain of LmTBCC-C. Recombinant forms of the full-length protein from Trypanosoma brucei and L. major  were prepared but proved recalcitrant to structural studies. In particular, speedy degradation of the polypeptides was noted. Limited proteolysis of the L. major protein, using trypsin, followed by mass spectrometry finger printing matched to the identification of a C-terminal fragment. A recombinant form of this domain, comprising residues 152–355 with a Leu223Met mutation was prepared (LmTBCC-C) to allow the production of selenomethionine (SeMet) derivative protein. The strategy behind the mutation was to enhance the chances of obtaining a good anomalous dispersion signal by placement of selenium into the hydrophobic core of the protein fold at a position unlikely to influence the structure. Sequence comparisons (not shown) indicated that at positions corresponding to 223 a leucine, isoleucine (as in human TBCC PDB code 2yuh) or methionine is observed. This form was crystallized and the structure determined at 2.2 Å resolution by exploiting the anomalous dispersion X-ray scattering properties of selenium. Crystallographic details are presented in Here we concentrate on TBCC from . Non-crystallographic symmetry was not restrained during the refinement and it is noteworthy that the two molecules adopt a similar structure with an r.m.s.d for all atoms of just 0.77 Å between residues 158–325, reducing to just 0.18 Å when only the main chain atoms are considered. It is therefore only necessary to detail one molecule.Two polypeptides constitute the asymmetric unit and in each there are two segments, residues 155–157 and 326–355, which could not be modeled due to disorderhttp://smart.embl-heidelberg.de/smart/do_annotation.pl?DOMAIN=CARP). The rest of the polypeptide is catalogued as the TBCC domain . The overall result is that the so-called “TBCC domain” is actually an extended structure that forms a shield covering one side of the molecule leaving another to potentially interact with binding partners. The extended conformation and overall structure of residues 243–355 suggests this is not in itself a bone fide domain but rather contributes to an overall C-terminal domain.The N-terminal segment, residues 158–242, forms a right-handed parallel β-helix barrel consisting of five coils or layers. The helical barrel is shaped as an approximate triangular prism, with faces and strands labeled A, B, C A. The siLmTBCC-C is no exception. A stack of four cysteine residues are positioned within the core of the helix in the middle of the βB strands 2 to 5, while nearby on βC strands 2 and 3 another two cysteine residues form a minimal stack, the sulfur atoms being 3.8–4.7 Å apart vertically within a stack separated by a distance of 5.2 Å horizontally between the two stacks. Aliphatic hydrophobic residues dominate this part of the structure and form the core of the β-helix.A common feature of the right-handed β-helical fold is the existence of stacks of hydrophobic residues within the barrel LmTBCC-C domain matches to the human retinitis pigmentosa 2 (RP2) protein with an r.m.s.d. of 2.3 Å and a sequence identity of about 25% over 139 residues  and Gln212. Nearby, on the other side of the arginine, a basic patch is formed on both proteins, Arg120 in RP2, Lys216 in LmTBCC. On the strand below Arg118/214 (A3), and directed onto the surface of the A-face, lie conserved serine  and phenylalanine  residues. These are positioned such that they would seem likely to be involved in protein–protein interactions.The RP2 protein is not involved in tubulin heterodimerization but like TBCC it does facilitate the GTPase activity of tubulin in the presence of TBCD LmTBCC-C in a stable recombinant form can now support and inform further studies to elucidate the precise contributions that these residues make to the final stage processing of the α/β-tubulin heterodimer.Our structural data and the availability of