X-ray crystallographic structural analyses of the bacterial transmembrane receptor LapD identify conserved molecular mechanisms that control biofilm formation in response to changes in the intracellular levels of the second messenger c-di-GMP. Pseudomonas fluorescens requires the transmembrane receptor LapD. LapD exhibits a conserved and widely used modular architecture containing a HAMP domain and degenerate diguanylate cyclase and phosphodiesterase domains. c-di-GMP binding to the LapD degenerate phosphodiesterase domain is communicated via the HAMP relay to the periplasmic domain, triggering sequestration of the protease LapG, thus preventing cleavage of the surface adhesin LapA. Here, we elucidate the molecular mechanism of autoinhibition and activation of LapD based on structure–function analyses and crystal structures of the entire periplasmic domain and the intracellular signaling unit in two different states. In the absence of c-di-GMP, the intracellular module assumes an inactive conformation. Binding of c-di-GMP to the phosphodiesterase domain disrupts the inactive state, permitting the formation of a trans-subunit dimer interface between adjacent phosphodiesterase domains via interactions conserved in c-di-GMP-degrading enzymes. Efficient mechanical coupling of the conformational changes across the membrane is realized through an extensively domain-swapped, unique periplasmic fold. Our structural and functional analyses identified a conserved system for the regulation of periplasmic proteases in a wide variety of bacteria, including many free-living and pathogenic species.The bacterial second messenger bis-(3′–5′) cyclic dimeric guanosine monophosphate (c-di-GMP) has emerged as a central regulator for biofilm formation. Increased cellular c-di-GMP levels lead to stable cell attachment, which in  Pseudomonas fluorescens, there are high levels of c-di-GMP within the bacterial cell when there is plentiful nutrient availability inside the cell, and the c-di-GMP levels determine stable biofilm formation outside the cell. LapD, a transmembrane receptor for intracellular c-di-GMP, communicates changing c-di-GMP levels to the outside of the cell by controlling the stability of the large adhesin protein LapA, which keeps bacteria attached to a surface or to other cells. We conducted X-ray crystallographic analyses of the structure of the intracellular and periplasmic modules of LapD that, in combination with functional studies, including those shown in an accompanying study by Newell et al., reveal the molecular mechanisms regulating receptor function. When phosphate availability is severely restricted, intracellular c-di-GMP levels are low and LapD is in held in an “off” state by an autoinhibitory interaction, which permits the proteolytic processing of LapA, its release from the cell surface, and consequently biofilm dispersal. Conversely, when there are higher phosphate levels in the growth medium, c-di-GMP increases and binds to a cytoplasmic domain of LapD, disrupting the autoinhibitory state and triggering a conformational change that sequesters the periplasmic protease responsible for cleavage of LapA, ultimately yielding stable cell attachment. By revealing key motifs for the regulation of LapD, we have identified similar systems in many other bacterial strains that may control periplasmic protein processing events in a similar fashion.Bacteria have the ability to form surface-attached communities, so-called biofilms, in both free-living environmental habitats and during pathogenic colonization in infectious diseases. Many of the cellular processes contributing to biofilm formation, for example, changes in motility, cell adhesion, and secretion, are regulated by the nucleotide-based second messenger c-di-GMP, which is unique to bacteria. In  Bacterial biofilms arise from planktonic microbial cells that attach to surfaces and form sessile multicellular communities, a process relevant to their survival in hostile habitats and for bacterial pathogenesis Vibrio choleraeP. aeruginosaXanthomonas campestrisCaulobacter crescentus and WspR of P. aeruginosa) P. aeruginosa and CdgG of V. cholerae) c-di-GMP is a monocyclic RNA dinucleotide that functions as an intracellular second messenger exerting control at the transcriptional, translational, and posttranslational levels Bacterial proteins that mediate c-di-GMP turnover and signal transduction are often composed of multiple domains, allowing for a variety of regulatory inputs, signaling events, and/or physiological responses P. fluorescens and P. putidaThe transmembrane protein LapD belongs to the last group. It contains degenerate GGDEF and EAL domains that lack catalytic activity, but it is capable of c-di-GMP binding via its divergent phosphodiesterase domain Accompanying work by Newell et al. P. fluorescens that provide models for the c-di-GMP-unbound cytoplasmic domain lacking only the HAMP domain, a c-di-GMP-bound EAL domain dimer, and the periplasmic domain. Together these structures span almost the entire receptor and elucidate molecular mechanisms that regulate LapD function. The crystal structure of the cytoplasmic module containing the GGDEF–EAL tandem domains reveals the presence of an autoinhibitory motif formed by a helical extension of the HAMP domain. In this inactive state, the GGDEF domain restricts dinucleotide access to the EAL domain module. The crystal structure of dimeric, c-di-GMP-bound EAL domains provides insight into the conformational changes resulting from dinucleotide binding. Based on the crystal structure of the periplasmic output domain of LapD, we identify functionally important residues and propose a model for the regulation of LapD activity in inside-out signal transduction. Finally, our structural studies highlight many conserved features that allow us to identify similar signaling systems in a variety of bacterial strains including common pathogens such as V. cholerae and Legionella pneumophila.Here, we present three crystal structures of LapD from P. fluorescens LapD, comprising a HAMP–GGDEF domain linker segment and the degenerate GGDEF–EAL domain module   . Based o220–648) . We willdual  was solved by single-wavelength anomalous dispersion phasing using selenomethione-substituted protein crystals , yet the overall structure of LapDdual in the two crystals is identical , which is an arginine residue in LapD (R273). Other significant changes that affect activity concern positive residues in PleD that interact with the phosphate moiety of GTP (K442 and K327). In LapD, these residues are glutamates (E388 and E392). In general, changes rendering LapD inactive for cyclase activity are comparable to those observed in FimX The overall fold of the GGDEF and EAL domains in LapD is very similar to those of other active or inactive diguanylate cyclases and phosphodiesterases, respectively  and a glutamate residue (E262), forms a particularly close interaction (450 is located just downstream of the signature EAL motif (KVL in LapD) at the center of the c-di-GMP-binding site. E262 is presented by a loop of the GGDEF domain. While E262 directly occupies the dinucleotide-binding site, the loop itself is located at its periphery, partially blocking access of c-di-GMP to the EAL domain , respectively: D239 in the loop and R316 in the GGDEF domain of LapD, D174 and R249 in WspR, and D292 and R366 in PleD The loop that connects the S helix to the GGDEF domain adopts a conformation that is identical to the linkage between active diguanylate cyclase domains and their regulatory domains . The conIn summary, the structural analysis of the cytoplasmic domain of LapD reveals that in the absence of c-di-GMP, the protein resides in a conformation incompatible with dinucleotide binding, with the GGDEF domain restricting access of c-di-GMP to the EAL domain. Dinucleotide binding would be accompanied by a major conformational change disrupting the conformation observed in the crystal structure.EAL bound to c-di-GMP   , consistl atoms) .EAL upon c-di-GMP binding occurs in the switch loop  represents a conformational change within the receptor, rather than a change in its oligomeric state. The comparative analyses described below reveal the basic properties of the cytoplasmic module of LapD, especially the correlation between c-di-GMP binding and dimerization (Mutations were introduced into LapDrization –7. Howrization  and 9.Kd) dual for c-di-GMP by almost 2-fold  has a similar effect. In contrast, replacing A602 with a glutamate residue reduced c-di-GMP binding to LapDdual both in the gel-filtration-based binding experiment and in a filter binding assay, suggesting an interdependence of dinucleotide binding and EAL domain dimerization.We employed two methods to assess c-di-GMP binding to LapD. A gel-filtration-based assay essentially measures the off rate of nucleotide from a preformed complex. The filter binding assay is a semi-quantitative assay that allows for higher throughput and the generation of titration curves yielding an apparent dissociation constant   . This medual protein elutes in a single peak from the size exclusion column with a molecular weight of 43.5 kDa, indicating a monomeric state in solution  showed more distinct shifts in molecular weight towards dimeric species upon c-di-GMP binding  are monomeric in solution, independent of the presence of dinucleotide and unaffected by the additional mutation S229D.The wild-type LapDdual and the mutants S229D and E262A, predicted to be less inhibited, incubated with c-di-GMP prior to gel filtration, may indicate a fast exchange between monomeric and dimeric species relative to the data acquisition time and/or instability of the complex. To further investigate this phenomenon, we conducted concentration-dependent experiments by subjecting LapDdual to light scattering measurements at concentrations between 20 and 320 µM with or without incubation in c-di-GMP. All samples eluted as single peaks from the gel filtration column and showed no signs of unspecific protein aggregation. Protein concentration determination across the peak volume indicated that samples were diluted consistently ∼15-fold during the chromatography. All LapDdual variants were monomeric in the absence of c-di-GMP across the entire concentration range  were insensitive to c-di-GMP addition and remained monomeric. Wild-type LapDdual showed signs of oligomerization only at the highest concentrations tested. In contrast, the molecular weight of LapDdual variants with single-point mutations S229D or E262A, predicted to disrupt autoinhibitory features, increased in a concentration-dependent manner in the presence of c-di-GMP, indicative of dimerization of the isolated cytoplasmic domain in solution.In general, the intermediate molecular weights and non-Gaussian peak shapes observed for wild-type LapDon range . LapDdua602E mutation (A602E or S229D/A602E) remained monomeric, wild-type LapDdual and the mutants S229D and E262A exhibited more pronounced dimerization in the same assay, with molecular weights close to the theoretical values for dimers calculated based their sequence. The observation that wild-type LapDdual displayed only a moderate, c-di-GMP-induced dimer formation when c-di-GMP was omitted from the mobile phase, but robust dimerization when the dinucleotide was present throughout the experiment, distinct from the behavior of the mutants S229D or E262A, indicates that the c-di-GMP-induced conformational changes and dimerization are reversible and underscores the interdependence of dinucleotide binding and EAL domain dimerization  refined to values of 400 µM  and 420 µM , respectively. Thus, each construct exhibits only a minimal propensity for dimerization, and apo-LapDdual is statistically indistinguishable from a pure monomer population. However, and again consistent with the light scattering data, in the presence of c-di-GMP, the concentration profiles of both proteins were poorly modeled unless the bound state was allowed to form dimers  and 180 nM , respectively. It is thus clear that in the presence of c-di-GMP, the propensity of the intracellular domain to form a dimer interface is several orders of magnitude stronger than that of the apo states of both proteins. Based on the nonoverlapping confidence intervals, it also appears that there may be a slight, but statistically significant, enhancement in the dimerization propensity of the S229D mutant, paralleling its increased affinity for c-di-GMP and the results from the light scattering experiments.In order to investigate the propensity for dimer interface formation under equilibrium conditions, we performed analytical ultracentrifugation experiments on wild-type LapDed value . When alm dimers . In thisKd of 13.1 ± 0.9 µM, whereas the A602E mutant showed a decreased affinity, with an apparent Kd of 36.3 ± 5.4 µM  or no significant changes  in phenotype, roughly corresponding to their surface exposure in the autoinhibited state structure  failed to respond to phosphate starvation efficiently, showing little to no reduction in biofilm formation (262A) showed an intermediate response to phosphate starvation, suggesting that mutant receptor function is still controlled by c-di-GMP, albeit not as effectively as in wild-type LapD    and S7B.surface) .Z-score  =  5.4, rmsd of 2.5 Å) A DALI  search comparing LapD's output domain to proteins in the RSCB Protein Data Bank (PDB) revealed structural similarity of its domain-swapped arms to the periplasmic domain of the sensor histidine kinase CitA –tagged LapG, a periplasmic cysteine protease that binds to LapD's output domain in a c-di-GMP-dependent manner  125, we hypothesize that the output domain may act as a molecular ruler, with the tryptophan residues forming the tips of a caliper. Varying the angle between the arms of the V-shaped fold upon c-di-GMP-triggered HAMP domain rotation could form the basis for modulating binding of LapG in the periplasm, assuming that both tryptophan residues of the dimeric, periplasmic fold interact with LapG (monomers or dimers).What is the relevance of the unusual fold of LapD's output domain? Unlike CitA and related sensor proteins, which bind small molecules in the periplasm and relay this information to the inside of the cell, LapD sequesters a periplasmic protein upon receiving a cytosolic signal. We speculate that a domain-swapped fold would respond more efficiently and precisely in coupling conformational changes in the cytosolic domains across the membrane than canonical dimeric periplasmic domains. One may consider the periplasmic domain of LapD as a single domain given the extensive sharing of structural elements and a negligible monomer–dimer transition. Given the functional importance and the particular position of WAlthough competent for specific LapG binding, the isolated LapD output domain failed to compete for LapG sequestration with the full-length c-di-GMP-bound receptor . It is likely that the intracellular and transmembrane domains facilitate the formation of a stable, high-affinity state. In addition, removal of the domain from its native context may alter its conformation. The observation that the isolated output domain can bind LapG is consistent with a model in which the dinucleotide-free, intracellular domains hold the receptor in an autoinhibited conformation that relaxes into a LapG-binding state upon activation. Consequently, deletion of the regulatory domains would allow for the output domain to adopt the active, LapG-binding conformation. In addition, potential higher-order oligomerization of LapD into lattices may contribute to sequestering LapG over larger membrane surfaces and to the fine-tuning of the signaling system. Two crystal structures described here, of the output domain and the c-di-GMP-bound EAL domain, show some potentially relevant higher-order interactions . FurtherPseudomonas strains, including P. putida and P. aeruginosa, are likely to function in a similar fashion  show a high degree of sequence conservation and functionally rescue deletions in these genes in P. fluorescens, no biofilm phenotype has been associated with this signaling system in their native strain Legionella and various Vibrio strains. In all these bacteria, lapD and lapG homologs with conserved, functionally important residues exist within the same operon . The mechanism described for LapD may also be applicable to these systems, in which the HAMP domain and S helix could be regulatory features to control the phosphodiesterase and/or diguanylate cyclase activity in the outside-in signaling mechanism, thus leading to changes in cellular c-di-GMP levels.The GGDEF–EAL domain–containing proteins described here are degenerate with respect to their active sites, lack catalytic activity, and function as c-di-GMP receptors. A similar system has been previously described in P. fluorescens LapD, a transmembrane receptor essential for biofilm formation in this strain. Similar receptors are conserved in many bacteria where they control a LapG-type, periplasmic protease. LapD is autoinhibited with regard to c-di-GMP binding by interactions of the EAL domain with the S helix and the GGDEF domain. Receptor activation requires the concurrent release of the EAL domain from these interactions and the binding of c-di-GMP, which triggers a conformational change in the output domain from an incompetent to a competent state with regard to LapG binding Here, we elucidated the molecular mechanism underlying the function and regulation of Given the occurrence of the HAMP–GGDEF–EAL domain module in many other proteins from different free-living and pathogenic bacterial species, the results discussed here will have broad implications for receptors predicted to mediate either inside-out or outside-in signaling involving the bacterial second messenger c-di-GMP.dual; residues 220–648), the EAL domain , and the periplasmic output domain  of P. fluorescens Pf0-1 LapD were produced following standard molecular biology and liquid chromatography techniques. Crystals were obtained by hanging drop vapor diffusion, and datasets were collected using synchrotron radiation at the Cornell High Energy Synchrotron Source . Detailed protocols are provided in The dual GGDEF–EAL domain module  or WTC-015S5 (for LapDEAL) column (Wyatt Technology) equilibrated in gel filtration buffer (25 mM Tris-HCl [pH 8.4] and 250 mM NaCl). Where specified, wild-type or mutant LapD protein variants were incubated with c-di-GMP (500 µM), produced enzymatically  were subjected to size exclusion chromatography (SEC) using a WTC-030S5 . Six-sector cells were loaded with 1×, 2×, and 4× dilutions of ∼12 µM stock solutions of either wild-type or S229D LapDdual in 25 mM Tris (pH 7.5) and 150 mM NaCl, either neat or supplemented to a final concentration of 20 µM c-di-GMP. Curves collected at all three speeds for all three channels were globally fit. Protein partial specific volume  were preincubated with excess c-di-GMP (500 µM) at 4°C and separated from unbound dinucleotide via SEC. SEC-eluted protein peaks were collected, concentrated to a final concentration of 200 µM to normalize for protein content, heat-denatured, and filtered through Microcon Centrifugal Filter Units . Dinucleotide content in the resulting samples was analyzed on a C18 reverse-phase HPLC column by using a methanol-phosphate gradient  32P]-c-di-GMP to purified LapDdual or LapDEAL (1 µM) was assessed by filter binding assays as described before Y = Bmax·X/(Kd + X) in GraphPad Prism .Binding of  and 10 mM MgCl2), and pelleting debris at 15,000g for 12 min. Samples were normalized to protein concentration using the BCA kit (Pierce).LapD proteins expressed in http://www.pdb.org) under the ID codes 3pjt, 3pju, 3pjv, 3pjw, and 3pjx.Atomic coordinates and structure factors have been deposited in the RCSB Protein Data Bank , P. putida KT2440 (NP_742334), P. aeruginosa PA01 (NP_250124), Pectobacterium carotovorum subsp. brasiliensis PBR1692 (ZP_03826388), Citrobacter sp. ATCC 29220 (ZP_06355256), Polaromonas sp. JS666 (YP_547171), Rhodoferax ferrireducens T118 (YP_524995), Dechloromonas aromatica RCB (YP_286553), Cellvibrio japonicus Ueda107 (YP_001981887), L. pneumophila str. Lens (YP_126219), Geobacter sp. M18 (ZP_05313414), V. alginolyticus 12G01 (ZP_01258281), V. parahaemolyticus AQ3810 (ZP_01990882), V. harveyi HY01 (ZP_01986262), V. shilonii AK1 (ZP_01866121), V. cholerae 1587 (ZP_01950486), V. fischeri ES114 (YP_207124), and V. angustum S14 (ZP_01233947).(4.74 MB PDF)Click here for additional data file.Figure S2dual and LapDEAL•c-di-GMP.Crystal forms of LapD (A) LapDdual. Two independent crystal forms were obtained for LapDdual. The resulting structures were superimposed on the EAL domain and shown as protein backbone traces. (B) c-di-GMP-bound LapDEAL. Two independent crystal forms were obtained for LapDEAL. Both crystal lattices show the same dimeric assembly of EAL domains. Dimers were superimposed on one EAL domain and shown as protein backbone traces. (C) Stereo views. Stereo views of the structural comparisons shown in (A) and (B) are shown. In this view, the EAL domains of LapDdual and LapDEAL•c-di-GMP are shown in a similar orientation.(7.01 MB TIF)Click here for additional data file.Figure S3Comparison of the GGDEF domains from LapD and PleD. (A) Sequence alignment. Sequences of GGDEF domains with known structure were used to generate the alignment (1.79 MB TIF)Click here for additional data file.Figure S4EAL and YkuI dimers.Comparison of c-di-GMP-bound LapD (A) Sequence alignment. Sequences of EAL domains with known structure were used to generate the alignment (7.34 MB TIF)Click here for additional data file.Figure S5dual.GGDEF–EAL domain interactions and S helix–GGDEF domain linker conformation observed in apo-LapD (A) GGDEF–EAL domain interaction. Close-up views are shown for regions of direct contact between the GGDEF and EAL domains in the autoinhibited structure of LapDdual. The GGDEF and EAL domains are colored in green and orange, respectively. The S helix is colored in blue. (B) Nucleotide-binding pocket in apo-LapDdual. A close-up view of the c-di-GMP-binding pocket of LapD is shown (right panel). c-di-GMP is shown as a stick presentation after superimposing the crystal structure of LapDEAL•c-di-GMP onto the EAL domain of apo-LapDdual. The interacting residue pair R450/E262 in LapD is incompatible with c-di-GMP binding. The left panels show surface presentations of apo-LapDdual. The middle panel shows accessibility of the c-di-GMP-binding site, with c-di-GMP taken from LapDEAL•c-di-GMP after superimposition. (C) S helix–GGDEF connector. The S helix and the GGDEF domain are connected via a short loop that forms a tight turn. The loop conformation is conserved in other GGDEF domain–containing proteins, and is stabilized by the interaction between two residues D239 and R316, which are strictly conserved in many GGDEF domain–containing proteins (9.22 MB TIF)Click here for additional data file.Figure S6Structural comparion of LapD with other c-di-GMP receptors. (A) LapD. The monomoric apo-LapDdual structure is shown as a surface presentation (top). The middle panel shows the c-di-GMP-bound EAL domain of LapD in two orthogonal views. The dinucleotide-binding site is colored in red. The conformation of c-di-GMP is similar to that observed in other EAL domains such as FimX, YkuI, and BlrP1 (bottom panel) . The dimeric species is stabilized by c-di-GMP binding to the base of the regulatory receiver domain (middle panel) see also  [17],[18(7.57 MB TIF)Click here for additional data file.Figure S7output and potential mechanisms for higher-order oligomerization of LapD.Structural analysis of LapD (A) Comparison between LapDoutput protomers. The periplasmic output domain of LapD crystallized with two molecules in the asymmetric unit. The protomers were superimposed on the first two helices of the fold, revealing a minor, rigid-body rotation of one half of the molecule relative to the other half between the two protomers. The rotation occurs at the connecting loop between β2 and α3 that forms the crossing-over point in the domain-swapped dimer. (B) LapDoutput crystal packing. Domain-swapped dimers of the output domain interact predominantly via two interfaces in the crystal lattices. One involves bottom-to-bottom interaction between LapDoutput dimers via a conserved, hydrophobic patch coinciding with the putative membrane-interaction surface. The other interface involves hydrophobic interactions between the arms of the V-shaped output domain dimers. (C) Potential higher-order oligomerization based on the structure of LapDoutput. Crystal lattice contacts reveal a potential mode for higher-order assemblies of LapD. The close-up view (right panel) shows the hydrophobic contacts between output domain dimers. (D) Potential higher-order oligomerization based on the structure of LapDEAL•c-di-GMP. In the C2221 crystal lattice, EAL domains form higher-order lattices that may highlight a mode for receptor oligomerization in the membrane.(7.15 MB TIF)Click here for additional data file.Figure S8output.Surface conservation and hydrophobicity of LapD (A) Surface conservation. Based on an alignment of 18 sequences of LapD homologs Click here for additional data file.Table S1X-ray data collection and refinement statistics.(0.08 MB DOC)Click here for additional data file.Table S2Strains and plasmids.(0.05 MB DOC)Click here for additional data file.Text S1Supporting information. The file includes supplemental Materials and Methods and associated references.(0.07 MB DOC)Click here for additional data file.


EMR2 is an EGF-like module containing mucin-like hormone receptor-2 precursor, a G-protein coupled receptor (G-PCR). Mutation in EMR2 causes complicated disorders like polycystic kidney disease (PKD). The structure of EMR2 shows that the fifth domain is comprised of EGF-TM7 helices. Functional assignment of EMR2 by support vector machine (SVM) revealed that along with transporter activity, several novel functions are predicted. A twenty amino acid sequence “MGGRVFLVFLAFCVWLTLPG” acts as the signal peptide responsible for posttranslational transport. Eight amino acids are involved in N-glycosylation sites and two cleavage sites are Leu517 and Ser518 in EMR2. The residue Arg241 is responsible for interaction with glycosaminoglycan and chondroitin sulfate. On the basis of structure, function and ligand binding sites, competitive EMR2 inhibitors designed may decrease the rate of human diseases like Usher's syndrome, bilateral frontoparietal polymicrogyria and PKD. G protein-coupled receptors (GPCRs) belong to the family of integral-membrane proteins (IMPs) that transduce external signals through the cell membraneEMR2 genes are predominantly expressed on dendritic cells, monocytes and macrophages. The EGF-TM7 molecules belong to a large protein family known as the long N-terminal of group B transmemberane 7 (LNB-TM7) or B2 subgroup of class B GPCRs with large extracellular N-terminal domainsEMR2 is an epidermal growth factor (EGF)-like module containing mucin-like hormone receptor-2 precursor and a cell surface protein receptor (GPCR) restricted to leukocytes and/or smooth muscle cells in humansThese EGF domains are coupled to TM7 via an extended spacer region. As a result of alternate RNA splicing, receptor isoforms possessing variable numbers of EGF domains are expressed. The EGF domains of EGF-TM7 receptors have been shown to mediate binding to cellular ligands. The domain 4 of EMR2 interacts with glycosaminoglycan (GAG) and chondroitin sulfateThe human-restricted adhesion-GPCR, EMR2, regulates neutrophil responses by potentiating the effects of a number of proinflammatory mediators and it has been shown that the transmembrane region is critical for adhesion-GPCR function. On neutrophil activation, EMR2 is rapidly translocated to membrane ruffles and the leading edge of the cellEMR2 is a myeloid cell-restricted member of the EGF-TM7 family that is closely related to CD97Mutations within this region of adhesion-GPCRs occur in a number of human diseases, including Usher's syndrome, bilateral frontoparietal polymicrogyria and PKDhttp://www.ebi.ac.uk/Tools/clustalw2/index.html)The sequence of human EMR2 protein (823 amino acids) was retrieved from NCBI. Multiple alignments of the related sequences were performed using the Clustal W program accessible through the European Bioinformatics Institute  protein of different strains of Leishmania have already been reported using the techniqueWe employed SVMProt server with support vector machine (SVM) learning techniques, which classifies a protein into functional families from its primary sequencesPocket-Finder or Q-site finder is a molecule-binding site prediction server based on Ligsite algorithmFunctional sites in eukaryotic proteins which fit the description “linear motif” are specified as patterns using regular expression rules. ELM server provides core functionality including filtering by cell compartment, phylogeny, globular domain class (using the SMART/Pfam databases) and structureLeishmania of six different strains has been reportedProtein-ligand interaction, i.e. docking, was studied by LigandFit/LigandScorehttp://igs-server.cnrs-mrs.fr/elnemo/index.html)J is the Jacobian matrix, which is a vector (the gradient) for scalar-valued functions. The full Hessian matrix can be difficult to compute in practice; in such situations, quasi-Newton algorithms have been developed that use approximations to the Hessian. The most well-known quasi-Newton algorithm is the BFGS algorithmNormal mode analysis was conducted to analyze the intrinsic motions of the EMR2 modeled structure. The elNémo online server ; similarly, the second domain consists of 69-120 residues, the third domain lies in between amino acids 121-238 and the fourth and fifth domains stretch out in amino acids 239-318 and 319-823, respectively. The GPCR proteolytic sites (GPS) occur in amino acid residues 479-529. The folding pattern of this protein consists of double-stranded β-sheet followed by a loop to a C-terminal short double-stranded sheet.st domain structure of EMR2 is 15.97 and the 2nd domain is 15.99. No invalid region was found in the model. Similarly, the three dimensional model of the 3rd domain was modeled with templates named human notch-1 ligand binding protein (pdb id: 1TOZ_A) and EGF domain 1, 2, and 5 of human EMR2, a 7-TM immune system molecule (pdb id: 2BOU) and showed high DOPE score. The three dimensional structure of the 4th domain of EGF-like module EMR2 showed good DOPE score i.e. 35.88. Dali serverth domain of EMR2 was modeled by using structural coordinates of six PDB structures i.e. hemochromatosis protein HFE complexed with transferin (pdb id: 1DE4), bovine rhodopsin (pdb id: 1U19), prostate-specific membrane antigen A (pdb id: 1Z8L), bovine rhodopsin (pdb id: 1L9H), peroxisomal acyl-coA oxidase-II (pdb id: 1IS2), and adaptor protein 2 clathrin adaptor core (pdb id: 1GW5) with a DOPE score of 124.83. The modeled structure of five different domains of EMR2 is shown in Modeler program constructed the structural models of full length EMR2 considering different templates i.e. EGF domain 1, 2, and 5 of human EMR2, a 7-TM immune system molecule in complex with barium (pdb id: 2BOU) and EGF domain 1, 2, and 5 of human EMR2, a 7-TM immune system molecule in complex with calcium (pdb id: 2BO2). The first domain 3-D structure was constructed by using three different templates i.e. EGF domain 1, 2, and 5 of human EMR2 with barium (pdb id: 2BO2), EGF-like module of human C1R (pdb id: 1APQ) and fibrillin-1CB EGF protein (pdb id: 1LMJ_A). The verify protein  score of the 1To validate the models, we further evaluated three dimensional structures of EGF-like module EMR2 with Ramachandran plot. Stereo-chemical evaluation of backbone Psi(Ψ) and Phi (Φ) dihedral angles of five modeled EMR2 domains was revealed in different percentages, i.e. 72%-90%, 3%-31% and 2%-14% residues fell within the most favored regions, additionally allowed regions and generously allowed regions and few residues are in disallowed region of Ramachandran plot, respectively (2 to COOH terminus) are found to be present in EMR2. The first domain is composed of 68 amino acids and is hydrophilic in nature. Two anti-parallel β-sheet structures have been found in the first domain of EMR2. The second domain is comprised of 51 amino acid residues, and contains one anti-parallel β- sheet and two small helical regions. The third domain of EMR2 is comprised of 117 amino acids and consists of two anti-parallel β-sheets and three small helical regions are shown in Five structural domains  are identical to six other EMR2 sequences . Non-synonymous mutations are deleterious in natureAmino acid sequence of EMR2  was downloaded and aligned with six different genes , which showed close identity. From the 1th-561st amino acids (538th-560th in few cases) are responsible for the formation of the first transmembrane region. Similarly, the 571st-591st and 600th-626th amino acids of the fifth domain participate in formation of the second and third transmembrane regions, respectively. Other amino acids 646-649, 688-710, 730-756 and 758-782 are involved in constructing the 4th, 5th, 6th, and 7th transmembrane helices of EMR2, respectively.Different transmembrane prediction programs i.e. DAS, HMMTOP, TMHMM, TMpred, TopPred and SOSUI  is shown in nd and 3rd domains, EGF has posttranslational ASX hydroxylation at the conserved aspartate or asparagine residues, forming erythro-β-hydroxyaspartic acid or erythro-β-hydroxyasparagine. In the fifth domain, APCC-binding destruction motifs are present. The BRCT domain of the fourth domain is associated with DNA damage response and recognizes and binds to specific phosphorylated serine sequences. PCSK cleavage site motif is present in the fifth domain of EMR2. A cyclin recognition motif that interacts with cyclin has also been detected in domain 5. FHA phosphopeptide ligand motif 1 is present in domain 5, and motif 2 is present in domain 1, 4 and 5. The FHA domain is a signal transduction module, which recognizes phosphothreonine-containing peptides on the ligand proteins. In domain 1, 4 and 5, ww ligand motifs are predicted that are implicated in protein-protein interactions mediated by WW domains. Casein kinase (CK1) phosphorylation motifs (recognized by CK1 and CK2 for Ser/Thr phosphorylation) are present in domain 1, 4 and 5. In domain 1, 4 and 5, GSK3 phosphorylation site is present and in domain 5, and 16 different patterns of this motif are present and shown in Posttranslational modifications are important for future localization or function of the protein. Various posttranslational modification sites have been identified in the EGF-like module protein EMR2  function.Different unknown and hidden functions of a protein (EMR2) were predicted using machine learning technique like a statistical SVM based classifier, the SVMProt  for complement, unlike the related CD97 antigen, and indicates that these very closely related proteins likely have non-redundant functions. The It is known from the literature that the fourth domain of EMR2 has three active sites. Chondroitin 4-sulphate molecules were docked in a groove of EMR2 that is on the active site. Higher ligand protein interaction has been detected up to 81.147 dock score. Ten different binding conformations have been observed during docking study in DS (Accelrys). The groove contains several positively charged side chains that interact directly with the negatively charged groups on chondroitin 4-sulphate  program was run to know the native motions  of the EMR2 modeled structure. Essential features of the top ten low-frequency normal modes, including its frequency, amplitude, collectivity of atom movements, and the overlap with observed conformational changes are summarized in in vitro and in vivo.On the basis of modeled structure, predicted binding sites and functions of EMR2, high throughput screening of various compounds may be carried out to find appropriate leads against different diseases, which can further be screened


Specific peptide ligand recognition by modular interaction domains is essential for the fidelity of information flow through the signal transduction networks that control cell behavior in response to extrinsic and intrinsic stimuli. Src homology 2 (SH2) domains recognize distinct phosphotyrosine peptide motifs, but the specific sites that are phosphorylated and the complement of available SH2 domains varies considerably in individual cell types. Such differences are the basis for a wide range of available protein interaction microstates from which signaling can evolve in highly divergent ways. This underlying complexity suggests the need to broadly map the signaling potential of systems as a prerequisite for understanding signaling in specific cell types as well as various pathologies that involve signal transduction such as cancer, developmental defects and metabolic disorders. This report describes interactions between SH2 domains and potential binding partners that comprise initial signaling downstream of activated fibroblast growth factor (FGF), insulin (Ins), and insulin-like growth factor-1 (IGF-1) receptors. A panel of 50 SH2 domains screened against a set of 192 phosphotyrosine peptides defines an extensive potential interactome while demonstrating the selectivity of individual SH2 domains. The interactions described confirm virtually all previously reported associations while describing a large set of potential novel interactions that imply additional complexity in the signaling networks initiated from activated receptors. This study of pTyr ligand binding by SH2 domains provides valuable insight into the selectivity that underpins complex signaling networks that are assembled using modular protein interaction domains. Every cell in our body is an immensely powerful computational device capable of integrating vast amounts of data from intrinsic and extrinsic cues and responding with remarkable fidelity. What underlines this computational power are not static wires, but dynamic interactions that leverage the finite number of genes to generate an almost infinite number of combinatorial interactions between protein components. In the post-genomics era, mapping these interactions represents a next frontier. The sum total of all permitted interactions is referred to as the potential interactome. In any given cell, only a subset of potential interactions will be enabled and this defines the selective differences in signalling between tissues. Understanding the whole provides insight into the information processing power of the system and may suggest new avenues for therapeutic intervention to treat diseases caused by faults in signal processing mechanisms. This study outlines the potential interactome for initial signalling events from the insulin receptor, insulin-like growth factor receptor and all four members of the fibroblast growth factor receptor family. These systems are essential for human development and dysfunctional signalling has been implicated in a wide range of human diseases including diabetes, many cancers, Alzheimer's disease, many developmental disorders and even aging. Binary connections are reported between 50 SH2 domain-containing proteins and 192 phosphopeptide nodes on 13 signal-initiating proteins. This verified almost every interaction described in the past 25 years and adds an extensive new data, providing a step towards fathoming the intricacies of differential cell communication between various tissues and disease states.Signaling immediately downstream of receptor tyrosine kinases (RTKs) is accomplished in large part by the recruitment of phosphotyrosine (pTyr) interacting proteins to sites of tyrosine phosphorylation on the activated receptors and their associated scaffold proteins -3. A givDespite the importance of SH2-mediated signaling in human disease, our understanding of their interactions remains far from complete. Direct experimental measurement of binding partners has typically focused on specific interactions driven by hypotheses relating to the precise signaling events under investigation. This yields a set of high quality, but inevitably sparse data. Certain pTyr proteins and SH2 domains are extensively studied while others are more arcane. Nonetheless, the SH2-mediated interactions reported over 25 years of intensive study provide a solid foundation for validating high-throughput datasets.a priori knowledge of the specific target proteins or peptides. Profiling signaling using SH2 domains has direct implications to diagnosis and guiding therapeutic decisions as the patterns obtained can be used to classify tumors [SH2 domain interactions are almost always phosphorylation dependent as roughly half of the binding energy is devoted to pTyr recognition ,18. Despy tumors . The ligy tumors ,28,29, oy tumors ,30 and py tumors . Informay tumors ,21.Recruitment of SH2 domain proteins to phosphorylated sites is a dynamic process and is by no means predetermined by the phosphorylation event alone. Each tyrosine site on a scaffold (including sites on receptors that recruit SH2 domains) can be phosphorylated or unphosphorylated. The phosphorylated site can either be free or occupied by one of its potential binding partners. Each possible assembly of interaction partners on a given scaffold represents an interaction microstate -35. The http://www.sh2domain.org.Herein, we describe a potential interactome obtained using addressable peptide arrays consisting of 192 physiological peptides from the insulin (Ins), insulin growth factor 1 (IGF-1) and fibroblast growth factor (FGF) signaling pathways to identify interactions with 50 SH2 domains. This set represents a broad sampling of the SH2 domains extant in the human genome. The results of this study map a range of potential phosphotyrosine-dependent interactions within the FGF and Ins/IGF-1 pathways. These signaling systems have relevance to understanding complex multi-tissue pathologies such as diabetes and cancer as well as in normal physiology and development. This study confirms 44 of 54 previously described interactions. In addition, we report an extensive set of novel interactions. Validation of 60 binary interaction pairs was conducted using the orthogonal method of solution binding measured by fluorescence polarization. The binding motifs obtained for each SH2 domain closely match those reported in a number of independent studies. Protein co-precipitation experiments, or endogenous phosphorylation upon receptor stimulation, were further used to validate a number of interactions. The results of this study highlight the available pool of potential SH2-mediated interactions with these 13 major signaling proteins and serve as a first step in understanding signaling microstate variations. Interactive figures and additional information may be found at D) values span a range from low nM to 50 μM were incorporated to aid in validating the results. These control peptides provide a reference and establish the empirical cut-off for designated binding interactions , while array negative interactions were demonstrably lower affinity (median KD > 30 μM)  Figure . This suProbing of arrays individually with each of 50 SH2 domains provides a snapshot of SH2 specificity Figure . As we hD values. In addition, we noted 43 interactions with the 13 signaling proteins represented on the arrays reported in UniHI [D values stronger than 4.1 μM. Thus, this control set suggests an approximate threshold of binding in the range of 10 μM ± 5 μM. Of the 43 database-reported interactions, most were array positive and of those that were not array-positive, a number were just sub-threshold and judged to be indeterminate , likely due to the synthesis efficiency and accessibility of these particular peptides and the semi-quantitative nature of the system. Indeed, many of the peptide-SH2 interactions that fall in the indeterminate set are likely to be real binders. Some surprising differences between SH2 domains can be reconciled this way. For instance, comparing between the Abl1 and Abl2 SH2 domains there is a significant difference in array positive interactions between the two. This is surprising considering the sequence similarity between the two domains. Because of the heterogeneities inherent in this study design as indicated above and the similarities between the two proteins, discrepancies of this sort likely represent false negatives. In total, the limited number of incongruities between the current data set and the literature are thus largely reconcilable.A high-throughput binding study reported interactions between a large set of SH2 domains and phosphopeptides within four receptor tyrosine kinases (including IGF-1R and FGFR1) overlaps with the present study . Our datD values reported by Kaushansky et al. appear inconsistent with direct measurements conducted using well controlled solution binding measured by fluorescence polarization [D of 175nM for the interaction between Rasa1-N-SH2 and FGFR1 pY-463 while we measured a KD of 1.54 μM by fluorescence polarization  low affinity interactions; 2) near misses that are array-indeterminate and thus just below threshold; or 3) cases where orthogonal measurement confirms no interaction at the level of the individual SH2 domain and 11-mer phosphopeptide. Comparison with an SH2 domain array study reveal limitations in that technique and suggest that SH2 domain arrays on glass substrates may suffer from a high rate of false positive and false negative interactions. This is consistent with results from the same group investigating PDZ domain binding using a similar protein microarray method which concluded that the technique resulted in a false positive rate of approximately 50%, and poor correspondence between array-estimated and solution-binding measured equilibrium-dissociation values -62.Probing arrays with 50 SH2 domains identifies a total of 529 array-positive interactions, together with 5949 array-negative and 1122 indeterminate SH2-ligand pairs. Array-positive interactions between SH2 domains and pTyr sites map the potential SH2 interactome. The connections between SH2 domains and InsR, IGF-1R, IRS-1, IRS-2, FGFR1, FGFR2, FGFR3, FGFR4, FRS2 and FRS3 together with p130Cas, PLCγ1 and p62DOK1 highlight a wide range of putative SH2 interactions within the immediate FGF and Ins/IGF-1 signaling networks  based on the array-positive peptides. PWMs such as the position-specific scoring matrix (PSSM)  are a weBy expressing differences between peptides that bind specifically and the peptide set as a whole, the EDSM attempts to compensate for any inherent bias arising in the relatively small set of non-random peptides drawn from physiological proteins. The EDSM for each SH2 in this study is visualized using sequence logos  the available set of SH2 proteins expressed in specific cells; and 2) the capacity of phosphorylated receptor and scaffold sites to recruit those SH2 proteins. The 111 SH2 domain proteins extant in the human genome vary extensively in their tissue and cell specific expression ,36. In sEven in normal physiological circumstances of healthy tissues, the potential interactome may inform our understanding of tissue-specific signaling events. A variety of tissues can respond to insulin stimulation, including adipose, muscle, pancreas, liver, brain etc. ,76. SH2 in vivo, our interaction maps are best used in conjunction with the expanding mass spectrometry literature and their associated databases.The interaction data also reveals the specificity of 50 SH2 domains for a set of physiological peptides. Typical binding motifs for SH2 domains describe the residues at positions +1 to +4 C-terminal of the essential phosphotyrosine -79. SH2 Broadly speaking, the SH2 consensus binding motifs identified from interactions observed using addressable arrays of physiological peptides are remarkably similar to the motifs described using peptide library approaches Table . Yet binIn our analysis we find that peptides reported to be phosphorylated in PhosphoSite are significantly more likely to have one or more SH2 domain-binding partners than peptide nodes that are not currently known to be phosphorylated. This is not surprising given that evolutionary pressure may be exerted to conserve critical binding sites. Conversely, given the specificity of SH2 domains, the chances of an SH2-interacting peptide occurring by chance within a non-phosphorylated peptide may be assumed to be relatively low. The more residues that must be specified to stipulate binding, the lower the probability is that this will occur spontaneously within a non-phosphorylated sequence. If only one key residue supported by one of two secondary residues was capable of allowing an SH2 domain to bind, then the chances of randomly generating an SH2 binding site centered around a given tyrosine residue are less than one in a hundred. Given the specificity observed for SH2 domains in this study, the likelihood of a random sequence encoding an SH2 domain ligand appears rather limited. The appearance of a small number of highly connected peptide nodes on sites not currently known to be phosphorylated raises the question of whether SH2 domain-binding might serve as means of predicting phosphorylation. Perhaps highly connected peptide hubs such as IRS1 Y-151, IRS2 Y-184, FRS3 Y-287 and FRS3 Y-322 predict phosphorylation. ScanSite predicts the first three of these sites as kinase substrates, while the sequence surrounding FRS3 Y-322 is identical to a known phosphorylation site on FRS2, suggesting that these may indeed turn out to be phosphorylated under appropriate conditions.A high degree of selectivity for physiological ligands may itself be an outcome of evolutionary pressures, as has been noted for yeast SH3 domains. The Sho1 SH3 domain recognizes a binding peptide in Pbs1, and no other SH3 domain in the yeast genome cross-reacts with the Pbs1 peptide. SH3 domains from other species that have not been under evolutionary pressure to ignore this site exhibit less selectivity for the Pbs1 peptide . A high D values are likely inaccurate. This is consistent with other studies suggesting that protein microarray data is semi-quantitative and subject to false-positive results [In the quarter century since the SH2 domain was first described ,84, hund results , particuin vitro binding studies may yield erroneous results. The present study used only 50 SH2 domains that have previously been shown to express well and exhibit good solubility and reproducible binding. A second issue relates to validation by orthologous method, to which the current study examines 60 binary pairs by the orthogonal method of solution phase fluorescence polarization binding, as well as a smaller set by GST-pulldown. A third consideration is agreement between HTP datasets and existing literature. Well-controlled studies reporting peptide-binding motifs for SH2 domains provide a wealth of data. SH2 domains bind to relatively specific motifs [Several lessons may be taken from such results and suggest a set of standards that could be universally applied in future high throughput studies of protein-peptide interactions and these are explored in detail elsewhere . First, c motifs ,29, and In examining SH2 domain interactions, we followed a systematic approach for systems-level interactome studies using orthologous validation and literature curation as a means of enhancing confidence in the experimental dataset. This results in a large set of high-confidence interactions that outline the potential interactome between 50 SH2 domains and 192 phosphopeptide sequences covering 13 proteins involved in FGF, Insulin, and IGF-1 signaling. The development of a detailed potential interactome for this set of signaling components represents an early step towards a more detailed understanding of cell-specific signaling networks. This stands to deepen our understanding of tissue-specific and disease-specific signaling networks that are predicated upon the varying and inevitably complex interpretation of the potential interactome by the available expressed interaction partners.E. coli strain BL21 (Stratagene) at 37°C overnight and induced with 1 mM IPTG for 3 hours. Cells were centrifuged, resuspended in PBS and lysed by sonication. The cellular fractions were incubated with glutathione sepharose (Thermo Scientific) and washed with PLC lysis buffer . SH2 proteins were eluted using 10 mM glutathione, 50 mM Tris HCl pH 8.0 and purified using the NAP-10 (Amersham Pharmacia) column system.A comprehensive list of 121 SH2 domains contained in 111 human proteins  served a2O cocktail for three hours. Phosphotyrosine incorporation was assessed by incubation with anti-phosphotyrosine antisera 4 G10 (Upstate) and pY20 (Santa Cruz). Additional file The peptide libraries were synthesized onto an acid hardened amino-PEG500 cellulose membrane #UC540  using an Intavis Multipep as described . The estAll steps were carried out at room temperature unless otherwise specified. The SPOTs membrane was first blocked with 5% nonfat milk in TBS-T (0.1 M TrisHCl (pH 7.4), 150 mM NaCl, and 0.1% Tween 20) overnight at 4°C. GST alone or GST fusion proteins (0.25 μM) were incubated with the SPOTs membrane in the same buffer containing 1 mM DTT for 1Â½ hours at room temperature and then washed with TBS-T. Anti-GST (Amersham) antibodies were used to detect GST fusion proteins and then incubated with anti-Goat Alexa-Fluor-680 (Molecular Probes). The array membrane was subsequently washed four times with TBS-T for 10 min. Peptides that bound the domain of interest were visualized by Li-Cor Odyssey using the 700 nm channel. Intensities were calculated using a grid with 192 circular features of 2 mm diameter, each centered around a peptide spot to avoid scoring SPOTs with halo or rings. For each feature, the average (integrated) intensity was used for downstream analysis.Peptides were synthesized using FMOC-chemistry onto pre-loaded tenta-gel resins. Peptides were then labeled with Rhodamine B (Abbey Color) and then cleaved using trifluoroacetic acid. Peptides were lyophilized and then purified using a LC/MS (Agilent 2100). Dissociation constants were measured using the Beacon 2000 (Invitrogen) as previously described .All analysis steps were performed as previously described . Peptidehttp://www.rcsb.org). Surface accessible tyrosines were solved using the Gerstein algorithm (http://helixweb.nih.gov/structbio/). The phosphorylation status of the 192 sites was identified using the protein modification resource, Phosphosite (http://www.phosphosite.org).The structures files of InsR , IGF-1R (1P4O), IRS-1 , FGFR1 (1FGK), FGFR2 (2PVF), FRS2 (1XR0), p62DOK1 (2 V76), PLCG1  collected from Protein Data Bank (PBD) (For each SH2 domain a position specific scoring matrix (PSSM) was calculated for the array-positive peptides (posPSSM). A second PSSM was calculated for all peptides, excluding those judged to be non-specific, as the expected distribution of amino acids represented on the array (exPSSM). Subtracting exPSSM from posPSSM yields the expectation deviation scoring matrix or EDSM. The EDSM for each SH2 domain was visualized as a logo of positive and negative factors using Weblogo .The unbiased position specific expectation deviation scoring matrix was expanded into a hyper-dimensional vector representation, and the Euclidean distances between vectors was computed. The resulting N-by-N distance matrix was then clustered using the Fitch-Margoliash method in the Phylip package . The unrReported peptide interactions were collected by searching HPRD and literature. Reported protein interactions were collected from the major protein-protein interaction databases of MINT , BIND 5, HPRD 5, and DIP4) with protease inhibitors . Pre-cleared lysates were incubated with GST-SH2 domains immobilized on glutathione beads and rocked for 3 hours at 4°C. Activated InsR and IRS-1 were detected using anti-phosphotyrosine 4 G10 (Upstate).Chinese Hamster Ovary (CHO) cells stably overexpressing insulin receptor (InsR) and IRS-1 were graciously provided by Xiao Jian Sun (UChicago). CHO cells were grown in DMEM/F12 supplemented with 10% fetal bovine serum, penicillin and streptomycin. CHO cells were serum starved for 24 hours and treated with and without insulin (100 nM) for 5 mins. Cells were lysed in HNTG . Information regarding whether the peptide spot is considered “non-specific” is indicated. SH2 domains that bound >3× and between 2× and 3× the mean are also listed.Click here for fileSupplementary materials includes a detailed description of GST background removal, supplemental tables and supplemental figures.Click here for fileComplete list of SH2 domains tested onto the SPOT arrays.Click here for file


Deinococcus geothermalis amylosucrase (DgAS) is more stable than Neisseria polysaccharea amylosucrase (NpAS). Based on our results, there are two main factors that contribute to the superior thermostability of DgAS. On the one hand, DgAS holds some good structural features that may make positive contributions to the thermostability. On the other hand, the contacts among residues of DgAS are thought to be topologically more compact than those of NpAS. Furthermore, the dynamics and unfolding properties of the two AS were also explored by the gauss network model (GNM) and the anisotropic network model (ANM). According to the results of GNM and ANM, we have found that the two AS could exhibit a shear-like motion, which is probably associated with their functions. What is more, with the discovery of the unfolding pathway of the two AS, we can focus on the weak regions, and hence designing more appropriate mutations for the sake of thermostability engineering. Taking the results on structure, dynamics and unfolding properties of the two AS into consideration, we have predicted some novel mutants whose thermostability is possibly elevated, and hopefully these discoveries can be used as guides for our future work on rational design.Amylosucrase (AS) is a kind of glucosyltransferases (E.C. 2.4.1.4) belonging to the Glycoside Hydrolase (GH) Family 13. In the presence of an activator polymer, in vitro, AS is able to catalyze the synthesis of an amylose-like polysaccharide composed of only α-1,4-linkages using sucrose as the only energy source. Unlike AS, other enzymes responsible for the synthesis of such amylose-like polymers require the addition of expensive nucleotide-activated sugars. These properties make AS an interesting enzyme for industrial applications. In this work, the structures and topology of the two AS were thoroughly investigated for the sake of explaining the reason why  Neisseria perflava as early as 1946 et al. Neisseriae species, and later an extracellular Neisseria polysaccharea AS was discovered of NeisseriaDeinococcusAlteromonasNeisseria polysaccharea amylosucrase (NpAS) is the only AS for which several structures, alone or in complex with sucrose substrate or products are available to date 8-barrel catalytic A-domain, a B-domain between β3 and α3, and a C-terminal domain consisting of a sandwich of two Greek key motifs. In addition to these common structural features of GH13, AS also possesses two unique domains: an α-helical N-terminal domain and a B′-domain between β7 and α7 in the catalytic core, which has been suggested to be involved in the polymerase activity of this enzyme. The B and B′-domains contribute largely to the formation of the active site pocket Amylosucrase (AS) is a kind of glucosyltransferase (E.C. 2.4.1.4) belonging to the Glycoside Hydrolase (GH) Family 13 according to the Carbohydrate-Active EnZymes classification in vivo is undoubtedly the extension of glycogen-like oligosaccharides, which is clearly demonstrated by the formidable increase in kcat observed when glycogen is present Unlike many other enzymes of the GH13, which mainly degrade polyglucan, AS is a very exceptional one since its main role is to catalyze the produce of the insoluble polysaccharides. In the presence of an activator polymer i.e. glycogen, in vitro, AS is able to catalyze the synthesis of an amylose-like polysaccharide composed of only α-1,4-linkages using sucrose as the only energy source kcat = 1·s−1) and a weak thermostability (t1/2[50°C] = 3 min), limiting its industrial development Deinococcus geothermalis (DgAS) Deinococcus radiodurans (DrAS) −1 at the optimal temperature of 50°C, the recombinant DgAS is the most thermostable AS characterized to date Among the AS discovered so far, NpAS is the one which is studied most. Although of great potential for industrial applications, NpAS suffers from a low catalytic efficiency on sucrose alone  step and reaching biologically meaningful time scales, i.e. >106 seconds, can be challenging for real proteins. In comparison with MD simulation, elastic network models (ENM) that use coarse-graining at the amino acid-level with normal mode analysis (NMA) proved powerful in deciphering biologically meaningful motions For thermostability elevation, understanding of the structural and dynamics differences between the mesophilic and the thermophilic proteins are of great importance. MD simulation is a powerful method and especially useful when some specific residues play a role in protein function and activity Two main problems we want to solve in the work are what constitute the superior thermostability of DgAS and how we can utilize the features of one AS to engineer another? To figure out the two problems, the GNM and ANM methods are applied to gain insight into the dynamics and unfolding properties of NpAS and DgAS. Furthermore, the structures of these two proteins are carefully and thoroughly investigated, for the purpose of finding out the structural basis lying behind the differences in thermostability of the two proteins. Hopefully the results revealed in the current work can be used as guides for practical developments on the AS in the further.http://www.rcsb.org) Searching Protein Data Bank , and the other ten's resolutions range from 1.66 to 2.2 Å. All of these PDB files, except 3UEQ contain 628 (1–628) residues. By scrutinizing the PDB file of 3UEQ, the first four residues are merely the residual expression tag. 3UEQ, therefore, is also of the same length of the other ten structures of NpAS. In this work, 1G5A 647EAPA650, at the tail of the C-domain. These missing residues were added to complete the DgAS structure with MODELER The tertiary structure of DgAS was just published on the PDB by the end of 2011. To date, only two structures are deposited in the PDB. Both 3UCQ and 3UER contain 651 residues, form −4 to 646, and the first five residues in the two structures are the residual expression tag. 3UCQ was used in this work as the input, and the residual expression tag was removed to construct a more reasonable model. Besides, there are four missing residues, i.e. α atoms connected by harmonic springs within a certain cutoff distance. The cutoff distance is usually taken as 7.0 Å, based on the radius of the first coordination shell around residues observed in PDB structures ith and jth Cα atoms and α atom and the cross-correlation fluctuations between different atoms are in proportion to the diagonal and off-diagonal elements of the pseudo inverse of the Kirchhoff matrix. The pseudo inverse of the Kirchhoff matrix can be decomposed as:ith and jth residues are given by:i = j, Eq. (4) stands for the mean-square fluctuations of the ith residue. The Debye-Waller or B-factor, which is correlated to the mean-square fluctuation, can be calculated with the following expression:ith residue associating with the kth mode is given by:x, y and z represent the coordinates of atoms.The GNM was first proposed by Bahar et al. The traditional GNM can only explore the dynamics of the target protein with a specific conformation. By breaking each “weak” contact between nodes step by step, we have shown the unfolding processes of proteins can be predicted by the iterative GNM, with a reasonable accuracy In this work, the iterative GNM is employed to predict the unfolding behavior of the two AS. When applying the iterative GNM, the interactions between residues are divided into the covalent and the non-covalent ones. In contrast to the traditional GNM, the interactions between the covalent and the non-covalent bonded ones are treated differently. The spring constants between all pairs of non bonded residues within the cutoff distance are treated equally, i.e. a single force constant i and j can be calculated by with i and j, respectively.The mean-square fluctuation in the distance vector As the temperature of the system is gradually increased, the native contacts among residues are expected to break in a fluctuation-dependent manner The fluctuations in the distance between all residues are calculated based on Eq.(13). The nonlinear elasticity during protein unfolding is considered through iterative normal mode calculations, thus we can mimic the protein unfolding process by using the following procedure:The mean-square fluctuations of the distance in all residue pairs are calculated based on the native structure topology with Eq. (13).The contact in the residue pair with the largest distance fluctuation is broken. Then, a new matrix The mean-square fluctuations of the distance in all residue pairs are recalculated based on the new matrix The above two steps are repeated until all the non-covalent contacts are broken.All the topologies of different conformation during protein unfolding are obtained and the unfolding pathway can be derived from the above obtained data.In order to evaluate the thermodynamics stability of the wild type (WT) AS and mutants, the folding free energy changes are estimated by the FoldX program The predictive power of FoldX forcefield has been tested on a very large set of point mutants (1088 mutants) spanning most of the structural environments found in proteins. In a recent comparison study on the accuracy and applicability of several energy functions, the overall performance of FoldX beat many other functions. The performance of FoldX on predictive accuracy is quite impressive, whose accuracy is as high as 70% when the crystal structure is available α atoms between the two input structures is 1.14 Å for the 563 common positions. The largest difference between our sequence alignment and the one provided by Guérin et al.16TPEQRAGI23, in the N-domain of NpAS. Besides, one thing should be mentioned here is that the alignment for the residues at the very beginning of NpAS and DgAS are not associated well with the structure information, since the local structures can not be superimposed well.As has been stated above, the PDB files of NpAS and DgAS used in this study contains 628 and 650 residues, receptively. Since there are many insertions and deletions in the two AS comparing with each other, the normal sequence alignment method available in most software can not guarantee to give a completely correct alignment. To overcome this problem, we firstly superimposed the structure of DgAS onto that of NpAS with the Iterative Magic Fit (IMF) method provided in the Swiss-PDBViewer A verity of differences in structures have been identified between thermophilic and mesophilic proteins sharing the similar functions, which are believed to be, at least partially, responsible for the differences in their thermostability. Here the thermostability is synonymous with the heat resistance of a protein, and commonly measured by the half life at a specific temperature or the half inactivation temperature of the protein. A thermostable protein is generally believed to have larger hydrophilic solvent accessible surface areas (SASA), more H-bonds and ion-pairs, higher proline and lower non-positive-ϕ glycine proportions. For the sake of gaining insight into the differences in structure between the two AS, the factors mentioned above were calculated under same conditions. The results are listed in 2. If we exclude this part from calculation, the SASA of the hydrophobic residues of DgAS is in the same level of that of the NpAS. This result, thereby, suggests that the solvation energy of DgAS is not necessarily worse than that of the NpAS. In order to figure out this problem, the unfolding free energies of the two AS were estimated with the FoldX program. The absolute values of the free energies are meaningless since the FoldX forcefield is not scaled to fit the real free energy, but the relative difference between calculations is meaningful. According to the calculation . Unsurprisingly, the B-domain of DgAS still holds the highest proline proportion comparing with the other domains. According to −1 lower than that of the WT NpAS according to calculation, thus implicating D427P may elevate the thermostability of NpAS. The V438 of DgAS is corresponding to the P435 of NpAS. According to calculation, substituting V438 with P438 lower the folding free energy of DgAS by 0.5 kcal mol−1. This suggests that the thermostability of DgAS could be elevated by using the structural features of NpAS as reference.As to the B′-domain, the proline proportions for the two AS are of little difference. From 12LDIYTPEQRAGIE24 peptide of the N-domain, is identified 16TPEQRAGI23 insertion in NpAS. The P41 of NpAS is closely corresponding to the E36 of DgAS, and this the only match we can identified in structure. Based on calculation, the E36P substitution lower the folding free energy of DgAS by 1.1 kcal·mol−1 . Since the modes with low frequency correspond to functional motions and those with high frequency correspond to localized motions, only the slow modes are used to improve the signal/noise ratio. Here the first 40 modes are used for both the NpAS and the DgAS. Altogether, they can explain over 50% of the total motions. The results for NpAS and DgAS are depicted in According to the crystal structures of the two AS, the flexible loops of the B and B′ and A-domains constitutes the geometrical door of the main active site. In order to access the binding pocket, the sucrose molecule has to break through the hindrance formed by these loops. From both the experimental and the calculated B-factors of the two AS (data not shown), we can see that these loops are more flexible than the other parts in the same domain. Based on the cross correlation map , the looThe fast modes correspond to geometric irregularity in the local structure and the fluctuations associated with fast modes are accompanied by a decrease in entropy larger than that for slow modes 2, and is regarded as a possible warm spot if its SF of the in the fastest 10 modes is larger than 0.01 but less than 0.03 Å2. According to this criterion, NpAS possesses 8 hot spots and 6 warm spots; DgAS possesses 7 hot spots and 5 warm spots. These spots are marked in In this work, a residue is regarded as a possible hot spot if its square fluctuation (SF) of the fastest 10 modes is larger than 0.03 ÅIn spite of these differences, two conserved hot regions are discovered. The first hot region (residues 431–456 in NpAS and residues 434–459 in DgAS) is located at the B′-domain and the second one is located around the long loop connecting β8 and α8 of the A-domain (A3 part). The second region is geometrically close to the first region. Since the loops of B, B′ and A-domain are considered to be related with the functions of AS, the stability of these domains would be of great importance to the activity of the enzyme. Indeed, many functionally important residues of NpAS, such as R446, D394, D393, are in the close vicinity of these hot spots. Taking all of these results into consideration, it is speculate these conserved hot spot residues may act as folding nucleus, and thereby are critical for the activity of AS. This speculation is supported by results revealed by the iterative GNM (see the following section).Understanding the unfolding process of a protein is of great importance for the rational design of kinetic stability. Kinetic stability, in the context discussed here, is a measure of how rapidly a protein unfolds. It is a particularly important consideration for proteins that unfold very slowly or denature irreversibly, such as aggregation or proteolytic degradation. In cases such as these, it is not the free energy difference between the folded and unfolded state that is important. That will only influence the equilibrium between the folded and unfolded state, however, it is not an equilibrium process anymore. The important thing is the free energy difference between the folded and the transition states (activation energy), for it is the magnitude of this difference that determines the rate of unfolding and hence inactivation. Clearly, it would be very helpful for the rational design if we could gain some information of the unfolding sequence of the two AS ahead of the time consuming design and validation cycles. With the priori knowledge of the unfolding properties of the two AS, one can reinforce the weak parts specifically, thus slowing down the unfolding paces and hence the inactivation tendency. In order to figure out the unbinding processes of the two AS, the iterative GNM was performed as described in the method section. For the sake of clarity, the connections of between nodes are decomposed into two types: the intra-domain connections and the inter-domain connections. Obviously, the broken of the intra-domain connections in one domain mainly describe the unfolding of the domain itself, say, the unfolding of secondary structures. In contrast, the broken of the inter-domain connections stand for the unfolding around the interface between domains. Here, the unfolding curves for each domain of NpAS and DgAS are displayed in Clearly, the overall unfolding property of NpAS is quite different form that of DgAS based on the comparison between According to To explain why the two AS all began to unfold from the N-domain, we calculated the native contact number for each domain of the two AS, and the strength of the contact in each domain can be roughly measured by the value of NCPR  = 100, 200 and 500 states of NpAS and DgAS are given by Since the contacts within each secondary structure are relatively strong than those between two close secondary structures, most weak regions of each domain are found to be located around the interface between two independent secondary structures. Understanding the information about these weak regions ahead of time will be quite helpful for our following design and validation cycles. The kinetically weak regions of NpAS and DgAS are depicted by According to the contact map of the native state of NpAS , it is fAnother weak region of NpAS is located around the interface between the B-domain and the A-domain (A2 part). In the native state, the 221YDRTLR226 peptide of the B-domain direct interacts with the 295MGTSC299 peptide of the A-domain (region 3), however, these connects disappeared around 110th step mainly because of the deformation of the B-domain itself. The situation in DgAS is similar and the corresponding weak region is around 214FEATLP219 peptide of B-domain and the 293LETDC297 peptide of A-domain (region 3). As the core domain, the interfaces among secondary structures in the A-domain of NpAS are relatively strong, nonetheless, three exceptional regions were still found , and theIn this work, the structure, dynamics and unfolding properties of the two AS were thoroughly investigated for the sake of explaining the reason why DgAS is more stable than NpAS and revealing novel insight into rational design on thermostability. Based on the results addressed above, there are two main factors that contribute to the superior thermostability of DgAS. Firstly, DgAS holds some good features in structure that may make positive contributions to the thermostability. Secondly, the contacts among residues of DgAS are thought to be topologically more compact than those of NpAS owing to its higher NCPR value.Among these merits in structure, the number of H-bonds, salt-bridges, and the proline proportion are of great importance to the thermostability, especially the proline proportion. DgAS possesses more backbone-backbone H-bonds and salt-bridges than NpAS does, suggesting the interactions among residues of DgAS are stronger. Owing to the high proline proportion, the conformational entropy of DgAS in both of the folded and the unfolded states is highly decreased, thus stabilizing the enzyme. When investigating the distribution of proline residues over each domain of NpAS and DgAS , howeverUnlike other researches that focus on the structural differences between the thermophilic and the mesophilic proteins, in the current study, we also explored the dynamics and unfolding property of the two AS. According to the results of GNM and ANM, we have found that the two AS could exhibit a shear-like motion, which is probably associated with their functions. What is more, with the discovery of the unfolding pathway of the two AS, we can focus on the weak regions, and hence designing more appropriate mutations for the sake of thermostability engineering. Based on the comparisons between the structures of the two AS, the appropriate positions for proline substitution have been revealed and discussed above. In total, there are 13 and 8 possible positions for proline substitutions in NpAS and DgAS, respectively. Among the 13 possible substitutions in NpAS, 9 of them were predicated to be stabilized and the others are believed to be destabilized because of large steric conflicts . CheckinIn conclusion, the factors that constitute the superior thermostability of DgAS have been discovered through a combinational method of structural and topology analyses. These factors can at least partially explain the reason why DgAS is much more thermostable than NpAS, which may be helpful for the rational design of mutants with higher thermostability. Besides, the dynamics and unfolding property of the two AS have been discussed in great details, and hopefully these discoveries can be used as guides for our future work on rational design.Figure S1The detailed unfolding pathway for NpAS.(TIF)Click here for additional data file.Figure S2The detailed unfolding pathway for DgAS.(TIF)Click here for additional data file.Table S1The Ala-scan for glycine residues of NpAS and DgAS.(DOC)Click here for additional data file.Table S2The Ala-scan for proline residues of NpAS and DgAS.(DOC)Click here for additional data file.Table S3The free energy calculation for the proline residues of NpAS and DgAS.(DOC)Click here for additional data file.


Streptococcus pneumoniae, comprises four immunoglobulin (Ig)-like domains, each with a common CnaB topology. The three C-terminal domains are each stabilized by internal Lys-Asn isopeptide bonds, formed autocatalytically with the aid of an essential Glu residue. The structure and orientation of the crucial N-terminal domain, which provides the covalent linkage to the next pilin subunit in the shaft, however, remain incompletely characterised. We report the crystal structure of full length RrgB, solved by X-ray crystallography at 2.8 Å resolution. The N-terminal (D1) domain makes few contacts with the rest of the RrgB structure, and has higher B-factors. This may explain why D1 is readily lost by proteolysis, as are the N-terminal domains of many major pilins. D1 is also found to have a triad of Lys, Asn and Glu residues in the same topological positions as in the other domains, yet mass spectrometry and the crystal structure show that no internal isopeptide bond is formed. We show that this is because β-strand G of D1, which carries the Asn residue, diverges from β-strand A, carrying the Lys residue, such that these residues are too far apart for bond formation. Strand G also carries the YPKN motif that provides the essential Lys residue for the sortase-mediated intermolecular linkages along the pilus shaft. Interaction with the sortase and formation of the intermolecular linkage could result in a change in the orientation of this strand, explaining why isopeptide bond formation in the N-terminal domains of some major pilins appears to take place only upon assembly of the pili.The surface of the pneumococcal cell is adorned with virulence factors including pili. The major pilin RrgB, which forms the pilus shaft on pathogenic  Streptococcus pneumoniae (Pneumococcus) is a major human pathogen responsible for a several life-threatening conditions in both industrialized nations and developing countries Pili from Gram-negative bacteria have been well characterised and are formed from bundles of non-covalently associated proteins. They perform a variety of roles including adherence, motility and transfer of genetic information S. pneumoniaeStreptococcus pyogenesStreptococcus agalactiaeCorynebacterium diphtheriaeBacillus cereusS. pyogenes Spy0128, three for S. agalactiae GBS80 and C. diphtheriae SpaA and four for B. cereus BcpA and S. pneumoniae RrgB. Similar Ig-like domains are used to make up the basal pilins Crystal structures of the pilin subunits utilized by several Gram-positive bacteria show that this modular mechanism of Gram-positive pilus assembly is also reflected in the individual subunits B. cereus BcpA the requisite Lys, Asn and Glu residues are present but do not form an isopeptide bond in the recombinant protein A striking feature of these Gram-positive pili is the widespread use of internal covalent cross-links that provide great stability to the pilin subunits in which they are found S. pneumoniae the genes required for pilus formation are located in the 12 kb rlrA islet rlrA islet are the genes encoding three sortases SrtB, SrtC and SrtD and a positive regulator RlrA In As for most of the Gram-positive pili, except for a few that are built from only two components The 665-residue major pilin RrgB is solely responsible for the extended nature of the pilus, forming its polymeric shaft, which typically comprises 100–200 tandem RrgB molecules. A crystal structure has been determined for a large fragment of RrgB that comprises domains D2 to D4 of this four-domain protein, and can be fitted into the cryo-EM envelope of the pilus shaft Here we present the full-length crystal structure of the pneumococcal major pilin RrgB. We show that domains D2–D4 are arranged identically in the full-length structure as they are in the D2–D4 fragment. We further show that absence of an internal isopeptide bond in the D1 domain arises because the β-strand carrying the Asn residue is displaced away from the position required for bond formation. This strand also carries the YPKN pilin motif that provides the Lys residue for the linkage to the next subunit, and we trace the displacement of this strand to the proline in this motif. The structure further suggests that flexibility in the N-terminal domain may be important for pilus assembly, and offers an explanation for the observation that the N-terminal domains of some pilins do not form isopeptide bonds until they are incorporated into the pilus.12121 and the structure was then solved at 2.8 Å resolution by molecular replacement, using the D2–D4 fragment as a search model. The molecular replacement solution was extended to incorporate electron density corresponding to the D1 domain, with only one small loop between residues 57–61 together with the C-terminal residue 628 not modeled due to inadequate density. This model gives almost complete coverage of the mature form of the protein from signal sequence cleavage site to immediately prior to the sortase cell wall anchor motif. Two essentially identical RrgB molecules (root-mean square (rms) difference in Cα positions 0.61 Å) are present in the asymmetric unit. Full details of the data collection, refinement and structure validation are in RrgB is encoded as a 665-residue protein that includes a predicted N-terminal signal peptide of 29 residues Staphylococcus aureusC. diphtheriae SpaA 2) of B. cereus BcpA The crystal structure shows that the D1 domain of RrgB, like the other three domains D2–D4, has an Ig-like fold with the reverse IgG topology, first seen in the repetitive B domains of the collagen-binding adhesin Cna from 2) than for D2–D4 (22.8 Å2).A notable difference from the other major pilins so far characterized is that in RrgB domains D2 and D3 are packed side-by-side instead of end-on-end. Domain D1 is located above D2, with D3 packed laterally against D2, and D4 located at the base of the monomer, closest to the cell wall . The arrThe asymmetric unit contains two molecules that pack side-by-side in antiparallel fashion. In the crystal this generates antiparallel chains of RrgB molecules arranged head-to-tail. While superficially similar to the pilus-like chains seen in the crystals of SpaA and Spy0128 3 molecule, eliminated when the Lys ε-amino group bonds to the Asn carboxyamide group Mass spectrometry was utilized to measure the intact mass of recombinant RrgB and check for the presence of internal isopeptide bonds; each bond should result in a 17 Da mass difference due to the loss of an NHBoth the electron density  and massStructural comparisons with other CnaB-type pilin domains that do contain isopeptide bonds show that the key difference is that in RrgB D1, the strands that carry Lys41 and Asn184 diverge prior to the Lys residue; the last hydrogen bond is between 40 N and 180 O. In contrast, in those CnaB domains that contain an isopeptide bond, these two parallel β-strands remain fully hydrogen bonded to beyond the Asn residue. The consequence of this divergence is that Asn184 is located ∼4 Å away from residues Lys41 and Glu143, preventing bond formation with the Lys side chain . DivergeModeling studies based on fitting the D2–D4 structure, together with a modeled D1 domain, into the cryo-EM envelope for the pneumococcal pilus suggested that Lys183, part of the YPKN “pilin motif” might be too far distant from the C-terminus of the preceding monomer for this residue to form the inter-subunit isopeptide linkage The pili expressed by Gram-positive bacteria are unique examples of covalent biological polymers. They are long and thin, typically 1–5 µm in length but only ∼30–60 nm in width (no more than one molecule wide), but their strength and integrity are maintained by a remarkable sequence of covalent cross-links, both between the individual pilin subunits and within them. In both cases, these cross-links take the form of isopeptide bonds. The intermolecular linkages are catalyzed by the action of the sortase enzymes, which join the C-terminal carboxylate of one subunit to a specific lysine side chain on the next S. pyogenes. This protein comprises two Ig-like domains, both of the CnaB-type and each containing an internal Lys-Asn isopeptide bond. Spy0128 also possesses a lysine residue, Lys161, on a surface ω-loop that is extruded from the final strand (G) of its N-terminal domain, and through which it becomes linked to the next molecule in the pilus We focus here on the major pilins, or backbone pilins (BPs), which form the polymeric shaft of the pilus, comprising several hundred tandem BP subunits, arranged like beads on a string. The simplest of these BPs is that from Group A Streptococcus, exemplified by Spy0128 from the M1 SF370 strain of C. diphtheriae and GBS80 from S. agalactiae each having three domains B. cereus and RrgB from S. pneumoniae four domains Studies on the GAS major pilin Spy0128 first brought to light the paradigm that underlies the structure and stability of Gram-positive pili, although Spy0128 is in several respects atypical of most other major pilins. All the major pilins are modular proteins, made up of multiple Ig-like domains. Spy0128 is unique, however, in having only two Ig-like domains, both CnaB-type and both containing internal isopeptide bonds. In contrast, most of the other major pilins are larger, with SpaA from The larger BPs such as RrgB share several characteristic differences from Spy0128. Firstly, the critical lysine residue used to form the covalent intermolecular linkage that creates the polymeric shaft is present in a conserved YPKN motif, referred to as the pilin motif The present structure enables us to address these issues. RrgB, like BcpA, has an N-terminal domain that contains the appropriate residues for isopeptide bond formation  yet this bond is not present in the recombinant protein. In both RrgB and BcpA the Asn residue of the triad belongs to the YPKN motif, and is immediately preceded by the Lys residue used to form the sortase-mediated intermolecular linkage to the next molecule in the shaft. As we show here, this motif comprises residues 181–184 and is located on the final β-strand (G) of the N-terminal domain , just prStructural comparisons of the RrgB D1 domain with other CnaB-type pilin domains show a key difference. In all cases in which a Lys-Asn isopeptide bond is formed, the two parallel β-strands A and G, carrying the Lys and Asn residues respectively, are stitched together with C = O…HN interchain hydrogen bonds along their whole length, from before the Lys to after the Asn. In contrast, in RrgB this hydrogen bonding ceases prior to Lys41, at a position where the NH group of residue 182 should hydrogen bond to the carbonyl oxygen of residue 40. Residue 182 is the proline of the YPKN pilin motif, however, and cannot form such a hydrogen bond. Strand G then diverges away from strand A and hydrogen bonds instead to strand F, the edge strand of the other β-sheet .What are the implications for isopeptide bond formation? In domains containing a YPKN-type pilin motif the proline residue disrupts the hydrogen bonding between strands A and G. We propose that although this is not in itself sufficient to cause the strands to diverge, it does destabilize the structure, in a similar way to that shown previously for an Ig-like titin domain. In the latter, single-site proline substitutions that disrupt the hydrogen bonding between the A and G strands significantly alter mechanical stability Ig-like domains of this type are evidently sensitive to destabilization, and the interactions between the A and G strands appear to be particularly important. Loss of the isopeptide bond linking strands A and G in the N-domain of Spy0128 causes an ∼30°C reduction in melting temperature What are the implications for pilus assembly and stabilization? Assembly of the polymeric shaft requires that a pilin-specific sortase cleaves the C-terminal LPXTG motif between Thr and Gly to form a thioester intermediate, then ligates the Thr carboxyl group to a specific lysine on the next subunit. This must require some recognition of the appropriate lysine by the sortase. In all major pilins that have been characterized to date, this lysine is presented by the N-terminal domain, and is usually part of the YPKN pilin motif. Protein-protein recognition often requires some plasticity in one of the partners. As shown by Gentile et al. S. pneumoniae TIGR4 genomic DNA encoding residues 30–628 was amplified by a polymerase chain reaction (PCR) using the following primers, 5′ - GGCAGCGGCGCGGCTGGGACGACAACAACATCT - 3′ and 5′ - GAAAGCTGGCTAGATAGTGATTTTTTTGTTGACTACTT - 3′. Nested PCR (GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGAAAACCTGTATTTTCAGGGCAGCGGCGCG -3′ and 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTG -3′) was used to include a TEV-protease site and permit cloning into Gateway vector pDEST17 (Invitrogen), providing an N-terminal His6 tag. This vector was transformed by electroporation into BL21(DE3) chemically competent cells. Cells were incubated in Magnificent Broth  at 18°C until the OD600 reached 0.6, at which point selenomethionine was added to a final concentration of 1 g l−1. Protein expression was induced by addition of IPTG to 1 mM. Cells were grown for further 24 h at 18°C before harvesting by centrifugation and storage at −20°C until required. Bacterial cell pellets were resuspended in 50 ml of buffer A  and lysed with a French press, after which the soluble fraction was collected by centrifugation. This soluble fraction was loaded on to a 15 ml Ni-NTA column, washed with buffer A until A280 reached a baseline value, and then eluted with buffer B . The protein was incubated at 4°C with recombinant TEV protease at an approximate ratio of 10∶1. Following cleavage, the protein/TEV mixture was passed through a freshly stripped and recharged Ni-NTA column to remove cleaved His6 tags and uncleaved protein. The protein was then concentrated by centrifugation and purified by gel filtration on a Superdex 75 column into crystallization buffer . Purified RrgB was concentrated to 100 mg ml−1 and aliquots stored at −80°C until required.−1 RrgB solution and 100 nl of precipitant solution. Initial hits from a precipitant comprising 2.0 M ammonium sulfate, 100 mM Tris-HCl pH 8.0 were very small, poor quality crystals that proved difficult to reproduce or optimize. A crystal suitable for data collection was obtained by scaling up the initial conditions into a 24-well hanging drop plate with a reservoir volume of 500 µl and drop comprising 2 µl 75 mg ml−1 RrgB solution and 2 µl precipitant solution .Initial crystallization trials were carried out with a Cartesian nanolitre dispensing robot, using sitting drop vapor diffusion in 96-well Intelliplates  and an in-house 480-component screen A crystal of RrgB was flash-cooled to 100K using paraffin oil as a cryoprotectant 12121, with unit cell dimensions a = 84.37, b = 107.93, c = 142.45 Å, α = β = γ = 90.0° and 2 molecules in the asymmetric unit . Radiation damage to the crystals prevented the use of selenomethionine anomalous diffraction for phasing. Instead, the structure was solved by molecular replacement, using PHASER cryst = 0.217 and Rfree = 0.281 . The final model comprised two molecules of RrgB (590 residues each) with only residues 57–61 and the C-terminal residue 628 missing. Validation with Molprobity Diffraction data were processed using XDS −1 was diluted 150-fold in 50% acetonitrile, 0.1% formic acid in water. Samples were scanned from m/z 1000–2500 and the resulting multiply charged spectra were deconvoluted using the Bayesian Protein Reconstruct Tool within Analyst QS .All samples were analyzed by infusion-MS using a QSTAR XL Quadrupole Time of Flight mass spectrometer . RrgB at a concentration of 100 mg ml