Sequence and conformational preferences at termini of α‐helices in membrane proteins: Role of the helix environment

α‐helices are amongst the most common secondary structural elements seen in membrane proteins and are packed in the form of helix bundles. These α‐helices encounter varying external environments (hydrophobic, hydrophilic) that may influence the sequence preferences at their N and C‐termini. The role of the external environment in stabilization of the helix termini in membrane proteins is still unknown. Here we analyze α‐helices in a high‐resolution dataset of integral α‐helical membrane proteins and establish that their sequence and conformational preferences differ from those in globular proteins. We specifically examine these preferences at the N and C‐termini in helices initiating/terminating inside the membrane core as well as in linkers connecting these transmembrane helices. We find that the sequence preferences and structural motifs at capping (Ncap and Ccap) and near‐helical (N' and C') positions are influenced by a combination of features including the membrane environment and the innate helix initiation and termination property of residues forming structural motifs. We also find that a large number of helix termini which do not form any particular capping motif are stabilized by formation of hydrogen bonds and hydrophobic interactions contributed from the neighboring helices in the membrane protein. We further validate the sequence preferences obtained from our analysis with data from an ultradeep sequencing study that identifies evolutionarily conserved amino acids in the rat neurotensin receptor. The results from our analysis provide insights for the secondary structure prediction, modeling and design of membrane proteins. Proteins 2014; 82:3420–3436. © 2014 Wiley Periodicals, Inc.     

Structure and oligomerization of the PilC type IV pilus biogenesis protein from Thermus thermophilus

Type IV pili are expressed from a wide variety of Gram‐negative bacteria and play a major role in host cell adhesion and bacterial motility. PilC is one of at least a dozen different proteins that are implicated in Type IV pilus assembly in Thermus thermophilus and a member of a conserved family of integral inner membrane proteins which are components of the Type II secretion system (GspF) and the archeal flagellum. PilC/GspF family members contain repeats of a conserved helix‐rich domain of around 100 residues in length. Here, we describe the crystal structure of one of these domains, derived from the N‐terminal domain of Thermus thermophilus PilC. The N‐domain forms a dimer, adopting a six helix bundle structure with an up‐down‐up‐down‐up‐down topology. The monomers are related by a rotation of 170°, followed by a translation along the axis of the final α‐helix of approximately one helical turn. This means that the regions of contact on helices 5 and 6 in each monomer are overlapping, but different. Contact between the two monomers is mediated by a network of hydrophobic residues which are highly conserved in PilC homologs from other Gram‐negative bacteria. Site‐directed mutagenesis of residues at the dimer interface resulted in a change in oligomeric state of PilC from tetramers to dimers, providing evidence that this interface is also found in the intact membrane protein and suggesting that it is important to its function. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

pH-induced structural change in a sodium/proton antiporter from Methanococcus jannaschii

Na+/H+ antiporters are pH-dependent membrane transport proteins that maintain the homeostasis of H+ and Na+ in living cells. MjNhaP1 from Methanococcus jannaschii, a hyperthermophilic archaeon that grows optimally at 85 degrees C, was cloned and expressed in Escherichia coli. Two-dimensional crystals were obtained from purified protein at pH 4. Electron cryomicroscopy yielded an 8 A projection map. Like the related E. coli antiporter NhaA, MjNhaP1 is a dimer, but otherwise the structures of the two antiporters differ significantly. The map of MjNhaP1 shows elongated densities in the centre of the dimer and a cluster of density peaks on either side of the dimer core, indicative of a bundle of 4-6 membrane-spanning helices. The effect of pH on the structure of MjNhaP1 was studied in situ. A major change in density distribution within the helix bundle, and an approximately 2 A shift in the position of the helix bundle relative to the dimer core occurred at pH 6 and above. The two conformations at low and high pH most likely represent the closed and open states of the antiporter.     

Structure of the hypothetical protein Ton 1535 from Thermococcus onnurineus NA1 reveals unique structural properties by a left‐handed helical turn in normal α‐solenoid protein

The crystal structure of Ton1535, a hypothetical protein from Thermococcus onnurineus NA1, was determined at 2.3 Å resolution. With two antiparallel α‐helices in a helix‐turn‐helix motif as a repeating unit, Ton1535 consists of right‐handed coiled N‐ and C‐terminal regions that are stacked together using helix bundles containing a left‐handed helical turn. One left‐handed helical turn in the right‐handed coiled structure produces two unique structural properties. One is the presence of separated concave grooves rather than one continuous concave groove, and the other is the contribution of α‐helices on the convex surfaces of the N‐terminal region to the extended surface of the concave groove of the C‐terminal region and vice versa. Proteins 2014; 82:1072–1078. © 2013 Wiley Periodicals, Inc.     

Structural comparison and classification of alpha‐helical transmembrane domains based on helix interaction patterns

Structural classification of membrane proteins is still in its infancy due to the relative paucity of available three‐dimensional structures compared with soluble proteins. However, recent technological advances in protein structure determination have led to a significant increase in experimentally known membrane protein folds, warranting exploration of the structural universe of membrane proteins. Here, a new and completely membrane protein specific structural classification system is introduced that classifies α‐helical membrane proteins according to common helix architectures. Each membrane protein is represented by a helix interaction graph depicting transmembrane helices with their pairwise interactions resulting from individual residue contacts. Subsequently, proteins are clustered according to similarities among these helix interaction graphs using a newly developed structural similarity score called HISS. As HISS scores explicitly disregard structural properties of loop regions, they are more suitable to capture conserved transmembrane helix bundle architectures than other structural similarity scores. Importantly, we are able to show that a classification approach based on helix interaction similarity closely resembles conventional structural classification databases such as SCOP and CATH implying that helix interactions are one of the major determinants of α‐helical membrane protein folds. Furthermore, the classification of all currently available membrane protein structures into 20 recurrent helix architectures and 15 singleton proteins demonstrates not only an impressive variability of membrane helix bundles but also the conservation of common helix interaction patterns among proteins with distinctly different sequences. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Comparison of the transmembrane helices of bovine rhodopsin in the crystal structure and the C α template based on cryo-electron microscopy maps and sequence analysis of the G protein-coupled receptors

G protein-coupled receptors (GPCR) are activated by a diverse array of extracellular signals, ranging from light to polypeptide molecules. The receptors propagate these signals intracellularly using G protein secondary messenger pathways. A common feature in the architecture of these receptors is their seven transmembrane domains. The first crystal structure of a GPCR, bovine rhodopsin, has recently been solved at 2.8 Å. We compared the seven membrane-spanning helices (TMH) from the crystal structure of bovine rhodopsin with those from the low-resolution model of bovine rhodopsin based on the cryo-electron microscopy structure of frog rhodopsin developed by Dr Joyce Baldwin. The model developed by Baldwin used a consensus sequence approach to predict the rotational position of each helix with respect to the other six helices. Superposition of the entire helix bundle of the Baldwin model with the crystal structure gave a RMS difference (RMSD) of 3.2 Å for the 198 C f atoms which suggests a high level of similarity in the arrangement of the helices. Except for TMH IV (RMSD of 4.0 Å), the position of corresponding helices within the helix bundle overlapped well. The superposition of individual helices showed that the RMSD values over 3 Å in the global superposition were largely due to one or more of the following: (i) differences in the unraveling and kinks for these helices, (ii) translation of TMH perpendicular to the membrane and (iii) rotation of helices up to 31°, except for TMH IV in which an additional contribution to the RMSD came from the aforementioned observation. As other crystal structures of GPCRs become available, a comparison with the Baldwin consensus model may reveal larger differences than those observed here.     

Crystal structure of the secreted protein HP1454 from the human pathogen Helicobacter pylori

HP1454 is a protein of 303 amino acids found in the extracellular milieu of Helicobacter pylori. The protein structure, crystallized in the orthorhombic C222₁ space group with one molecule per asymmetric unit, has been determined using the single‐wavelength anomalous dispersion method. HP1454 exhibits an elongated bent shape, composed of three distinct domains. Each domain possesses a fold already present in other structures: Domain I contains a three‐strand antiparallel β‐barrel flanked by a long α‐helix, Domain II is an anti‐parallel three‐helix bundle, and Domain III a β‐sheet flanked by two α‐helices. The overall assembly of the protein does not bear any similarity with known structures. Proteins 2014; 82:2868–2873. © 2014 Wiley Periodicals, Inc.     

Current status of membrane protein structure classification

For over 2 decades, continuous efforts to organize the jungle of available protein structures have been underway. Although a number of discrepancies between different classification approaches for soluble proteins have been reported, the classification of membrane proteins has so far not been comparatively studied because of the limited amount of available structural data. Here, we present an analysis of α‐helical membrane protein classification in the SCOP and CATH databases. In the current set of 63 α‐helical membrane protein chains having between 1 and 13 transmembrane helices, we observed a number of differently classified proteins both regarding their domain and fold assignment. The majority of all discrepancies affect single transmembrane helix, two helix hairpin, and four helix bundle domains, while domains with more than five helices are mostly classified consistently between SCOP and CATH. It thus appears that the structural constraints imposed by the lipid bilayer complicate the classification of membrane proteins with only few membrane‐spanning regions. This problem seems to be specific for membrane proteins as soluble four helix bundles, not restrained by the membrane, are more consistently classified by SCOP and CATH. Our findings indicate that the structural space of small membrane helix bundles is highly continuous such that even minor differences in individual classification procedures may lead to a significantly different classification. Membrane proteins with few helices and limited structural diversity only seem to be reasonably classifiable if the definition of a fold is adapted to include more fine‐grained structural features such as helix–helix interactions and reentrant regions. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Crystal structure of vinculin in complex with vinculin binding site 50 (VBS50), the integrin binding site 2 (IBS2) of talin

The cytoskeletal protein talin activates integrin receptors by binding of its FERM domain to the cytoplasmic tail of β‐integrin. Talin also couples integrins to the actin cytoskeleton, largely by binding to and activating the cytoskeletal protein vinculin, which binds to F‐actin through the agency of its five‐helix bundle tail (Vt) domain. Talin activates vinculin by means of buried amphipathic α‐helices coined vinculin binding sites (VBSs) that reside within numerous four‐ and five‐helix bundle domains that comprise the central talin rod, which are released from their buried locales by means of mechanical tension on the integrin:talin complex. In turn, these VBSs bind to the N‐terminal seven‐helix bundle (Vh1) domain of vinculin, creating an entirely new helix bundle that severs its head‐tail interactions. Interestingly, talin harbors a second integrin binding site coined IBS2 that consists of two five‐helix bundle domains that also contain a VBS (VBS50). Here we report the crystal structure of VBS50 in complex with vinculin at 2.3 Å resolution and show that intramolecular interactions of VBS50 within IBS2 are much more extensive versus its interactions with vinculin. Indeed, the IBS2‐vinculin interaction only occurs at physiological temperature and the affinity of VBS50 for vinculin is about 30 times less than other VBSs. The data support a model where integrin binding destabilizes IBS2 to allow it to bind to vinculin.     

A method for α-helical integral membrane protein fold prediction

Integral membrane proteins (of the α-helical class) are of central importance in a wide variety of vital cellular functions. Despite considerable effort on methods to predict the location of the helices, little attention has been directed toward developing an automatic method to pack the helices together. In principle, the prediction of membrane proteins should be easier than the prediction of globular proteins: there is only one type of secondary structure and all helices pack with a common alignment across the membrane. This allows all possible structures to be represented on a simple lattice and exhaustively enumerated. Prediction success lies not in generating many possible folds but in recognizing which corresponds to the native. Our evaluation of each fold is based on how well the exposed surface predicted from a multiple sequence alignment fits its allocated position. Just as exposure to solvent in globular proteins can be predicted from sequence variation, so exposure to lipid can be recognized by variable-hydrophobic (variphobic) positions. Application to both bacteriorhodopsin and the eukaryotic rhodopsin/opsin families revealed that the angular size of the lipid-exposed faces must be predicted accurately to allow selection of the correct fold. With the inherent uncertainties in helix prediction and parameter choice, this accuracy could not be guaranteed but the correct fold was typically found in the top six candidates. Our method provides the first completely automatic method that can proceed from a scan of the protein sequence databanks to a predicted three-dimensional structure with no intervention required from the investigator. Within the limited domain of the seven helix bundle proteins, a good chance can be given of selecting the correct structure. However, the limited number of sequences available with a corresponding known structure makes further characterization of the method difficult. © 1994 John Wiley & Sons, Inc.     

Idiosyncrasy and identity in the prokaryotic Phe-system: crystal structure of E. coli phenylalanyl-tRNA synthetase complexed with phenylalanine and AMP

The crystal structure of Phenylalanyl‐tRNA synthetase from E. coli (EcPheRS), a class II aminoacyl‐tRNA synthetase, complexed with phenylalanine and AMP was determined at 3.05 Å resolution. EcPheRS is a (αβ)₂ heterotetramer: the αβ heterodimer of EcPheRS consists of 11 structural domains. Three of them: the N‐terminus, A1 and A2 belong to the α‐subunit and B1‐B8 domains to the β subunit. The structure of EcPheRS revealed that architecture of four helix‐bundle interface, characteristic of class IIc heterotetrameric aaRSs, is changed: each of the two long helices belonging to CLM transformed into the coil‐short helix structural fragments. The N‐terminal domain of the α‐subunit in EcPheRS forms compact triple helix domain. This observation is contradictory to the structure of the apo form of TtPheRS, where N‐terminal domain was not detected in the electron density map. Comparison of EcPheRS structure with TtPheRS has uncovered significant rearrangements of the structural domains involved in tRNA^Phe binding/translocation. As it follows from modeling experiments, to achieve a tighter fit with anticodon loop of tRNA, a shift of ～5 Å is required for C‐terminal domain B8, and of ～6 to 7 Å for the whole N terminus. EcPheRSs have emerged as an important target for the incorporation of novel amino acids into genetic code. Further progress in design of novel compounds is anticipated based on the structural data of EcPheRS.     

Conformation of membrane‐bound proteins revealed by vacuum‐ultraviolet circular‐dichroism and linear‐dichroism spectroscopy

Knowledge of the conformations of a water‐soluble protein bound to a membrane is important for understanding the membrane‐interaction mechanisms and the membrane‐mediated functions of the protein. In this study we applied vacuum‐ultraviolet circular‐dichroism (VUVCD) and linear‐dichroism (LD) spectroscopy to analyze the conformations of α‐lactalbumin (LA), thioredoxin (Trx), and β‐lactoglobulin (LG) bound to phosphatidylglycerol liposomes. The VUVCD analysis coupled with a neural‐network analysis showed that these three proteins have characteristic helix‐rich conformations involving several helical segments, of which two amphiphilic or hydrophobic segments take part in interactions with the liposome. The LD analysis predicted the average orientations of these helix segments on the liposome: two amphiphilic helices parallel to the liposome surface for LA, two hydrophobic helices perpendicular to the liposome surface for Trx, and a hydrophobic helix perpendicular to and an amphiphilic helix parallel to the liposome surface for LG. This sequence‐level information about the secondary structures and orientations was used to formulate interaction models of the three proteins at the membrane surface. This study demonstrates the validity of a combination of VUVCD and LD spectroscopy in conformational analyses of membrane‐binding proteins, which are difficult targets for X‐ray crystallography and nuclear magnetic resonance spectroscopy. Proteins 2016; 84:349–359. © 2016 Wiley Periodicals, Inc.     

FoldGPCR: Structure prediction protocol for the transmembrane domain of G protein‐coupled receptors from class A

Building reliable structural models of G protein‐coupled receptors (GPCRs) is a difficult task because of the paucity of suitable templates, low sequence identity, and the wide variety of ligand specificities within the superfamily. Template‐based modeling is known to be the most successful method for protein structure prediction. However, refinement of homology models within 1–3 Å Cα RMSD of the native structure remains a major challenge. Here, we address this problem by developing a novel protocol (foldGPCR) for modeling the transmembrane (TM) region of GPCRs in complex with a ligand, aimed to accurately model the structural divergence between the template and target in the TM helices. The protocol is based on predicted conserved inter‐residue contacts between the template and target, and exploits an all‐atom implicit membrane force field. The placement of the ligand in the binding pocket is guided by biochemical data. The foldGPCR protocol is implemented by a stepwise hierarchical approach, in which the TM helical bundle and the ligand are assembled by simulated annealing trials in the first step, and the receptor‐ligand complex is refined with replica exchange sampling in the second step. The protocol is applied to model the human β₂‐adrenergic receptor (β₂AR) bound to carazolol, using contacts derived from the template structure of bovine rhodopsin. Comparison with the X‐ray crystal structure of the β₂AR shows that our protocol is particularly successful in accurately capturing helix backbone irregularities and helix‐helix packing interactions that distinguish rhodopsin from β₂AR. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Modeling of halorhodopsin and rhodopsin based on bacteriorhodopsin

Bacteriorhodopsin (BR), halorhodopsin (HR), and rhodopsin (Rh) all belong to the class of seven-helix membrane proteins. For BR, a structural model at atomic resolution is available; for HR, diffraction data are available only down to a resolution of 6 Å in the membrane plane, and for Rh, down to 9 Å. BR and HR are closely related proteins with a sequence homology of 34%, while Rh does not share any sequence homology with BR. An atomic model for HR is derived that is based on sequence alignment and the atomic model for BR and is improved by molecular dynamics simulations. The model structure obtained accounts well for the experimentally observed difference between HR and BR in the projection map, where HR exhibits a higher density in the region between helices D and E. The reason for this difference lies partially in the different side chains and partially in slightly different helix tilts. The scattering amplitudes and phases of the model structure are calculated and agree with the experimental data down to a resolution of about 8 Å. If the helix positions are adopted from the projection map for HR and used as input in the model, this number improves to 7 Å. Analogously, an atomic model for Rh is derived based on the atomic model for BR and subjected to molecular dynamics simulations. Optimal agreement with the experimental projection map for Rh is obtained when the entire model structure is rotated slightly about two axes in the membrane plane. The agreement with the experimental projection map is not as satisfactory as for HR, but the results indicate that even for a nonhomologous, but structurally related, protein such as Rh, an acceptable model structure can be derived from the structure of BR. © 1996 Wiley-Liss, Inc.     

Assembly of transmembrane helices of simple polytopic membrane proteins from sequence conservation patterns

The transmembrane (TM) domains of most membrane proteins consist of helix bundles. The seemingly simple task of TM helix bundle assembly has turned out to be extremely difficult. This is true even for simple TM helix bundle proteins, i.e., those that have the simple form of compact TM helix bundles. Herein, we present a computational method that is capable of generating native-like structural models for simple TM helix bundle proteins having modest numbers of TM helices based on sequence conservation patterns. Thus, the only requirement for our method is the presence of more than 30 homologous sequences for an accurate extraction of sequence conservation patterns. The prediction method first computes a number of representative well-packed conformations for each pair of contacting TM helices, and then a library of tertiary folds is generated by overlaying overlapping TM helices of the representative conformations. This library is scored using sequence conservation patterns, and a subsequent clustering analysis yields five final models. Assuming that neighboring TM helices in the sequence contact each other (but not that TM helices A and G contact each other), the method produced structural models of Calpha atom root-mean-square deviation (CA RMSD) of 3-5 A from corresponding crystal structures for bacteriorhodopsin, halorhodopsin, sensory rhodopsin II, and rhodopsin. In blind predictions, this type of contact knowledge is not available. Mimicking this, predictions were made for the rotor of the V-type Na(+)-adenosine triphosphatase without such knowledge. The CA RMSD between the best model and its crystal structure is only 3.4 A, and its contact accuracy reaches 55%. Furthermore, the model correctly identifies the binding pocket for sodium ion. These results demonstrate that the method can be readily applied to ab initio structure prediction of simple TM helix bundle proteins having modest numbers of TM helices.     

Conformational dynamics is more important than helical propensity for the folding of the all α‐helical protein Im7

Im7 folds via an on‐pathway intermediate that contains three of the four native α‐helices. The missing helix, helix III, is the shortest and its failure to be formed until late in the pathway is related to frustration in the structure. Im7H3M3, a 94‐residue variant of the 87‐residue Im7 in which helix III is the longest of the four native helices, also folds via an intermediate. To investigate the structural basis for this we calculated the frustration in the structure of Im7H3M3 and used NMR to investigate its dynamics. We found that the native state of Im7H3M3 is highly frustrated and in equilibrium with an intermediate state that lacks helix III, similar to Im7. Model‐free analysis identified residues with chemical exchange contributions to their relaxation that aligned with the residues predicted to have highly frustrated interactions, also like Im7. Finally, we determined properties of urea‐denatured Im7H3M3 and identified four clusters of interacting residues that corresponded to the α‐helices of the native protein. In Im7 the cluster sizes were related to the lengths of the α‐helices with cluster III being the smallest but in Im7H3M3 cluster III was also the smallest, despite this region forming the longest helix in the native state. These results suggest that the conformational properties of the urea‐denatured states promote formation of a three‐helix intermediate in which the residues that form helix III remain non‐helical. Thus it appears that features of the native structure are formed early in folding linked to collapse of the unfolded state.     

Length‐dependent stability of α‐helical peptide upon adsorption to single‐walled carbon nanotube

Earlier studies have shown that the helical content of α‐helical peptide decreases upon its interaction with carbon nanotube (CNT). Further, the length of the α‐helix varies from few residues in the small globular protein to several number of residues in structural and membrane proteins. In structural and membrane proteins, helices are widely present as the supercoil i.e., helical bundles. Thus, in this study, the length‐dependent interaction pattern of α‐helical peptides with CNT and the stability of isolated α‐helical fragment versus supercoiled helical bundle upon interaction with CNT have been investigated using classical molecular dynamics (MD) simulation. Results reveal that the disruption in the helical motif on interaction with CNT is directly proportional to the length of the helix. Also it is found that the shorter helix does not undergo noticeable changes in the helicity upon adsorption with CNT. On the other hand, helicity of longer peptides is considerably affected by its interaction with CNT. In contrast to the known fact that the stability of the helix increases with its length, the disruption in the helical peptide increases with its length upon its interaction with CNT. Comparison of results shows that structural changes in the isolated helical fragment are higher than that in supercoiled helix. In fact, helical chain in supercoiled bundle does not undergo significant changes in the helicity upon interaction with CNT. Both the length of the helical peptide and the inherent stability of the helical unit in the supercoiled helix influence the interaction pattern with the CNT. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 357–369, 2013.     

The three-dimensional structure of the cytoplasmic domains of EpsF from the type 2 secretion system of Vibrio cholerae

The type 2 secretion system (T2SS), a multi-protein machinery that spans both the inner and the outer membranes of Gram-negative bacteria, is used for the secretion of several critically important proteins across the outer membrane. Here we report the crystal structure of the N-terminal cytoplasmic domain of EpsF, an inner membrane spanning T2SS protein from Vibrio cholerae. This domain consists of a bundle of six anti-parallel helices and adopts a fold that has not been described before. The long C-terminal helix α6 protrudes from the body of the domain and most likely continues as the first transmembrane helix of EpsF. Two N-terminal EpsF domains form a tight dimer with a conserved interface, suggesting that the observed dimer occurs in the T2SS of many bacteria. Two calcium binding sites are present in the dimer interface with ligands provided for each site by both subunits. Based on this new structure, sequence comparisons of EpsF homologs and localization studies of GFP fused with EpsF, we propose that the second cytoplasmic domain of EpsF adopts a similar fold as the first cytoplasmic domain and that full-length EpsF, and its T2SS homologs, have a three-transmembrane helix topology.     

Structural basis of intramitochondrial phosphatidic acid transport mediated by Ups1‐Mdm35 complex

Ups1 forms a complex with Mdm35 and is critical for the transport of phosphatidic acid (PA) from the mitochondrial outer membrane to the inner membrane. We report the crystal structure of the Ups1‐Mdm35‐PA complex and the functional characterization of Ups1‐Mdm35 in PA binding and transfer. Ups1 features a barrel‐like structure consisting of an antiparallel β‐sheet and three α‐helices. Mdm35 adopts a three‐helical clamp‐like structure to wrap around Ups1 to form a stable complex. The β‐sheet and α‐helices of Ups1 form a long tunnel‐like pocket to accommodate the substrate PA, and a short helix α2 acts as a lid to cover the pocket. The hydrophobic residues lining the pocket and helix α2 are critical for PA binding and transfer. In addition, a hydrophilic patch on the surface of Ups1 near the PA phosphate‐binding site also plays an important role in the function of Ups1‐Mdm35. Our study reveals the molecular basis of the function of Ups1‐Mdm35 and sheds new light on the mechanism of intramitochondrial phospholipid transport by the MSF1/PRELI family proteins.     

Intermolecular versus intramolecular interactions of the vinculin binding site 33 of talin

The cytoskeletal proteins talin and vinculin are localized at cell‐matrix junctions and are key regulators of cell signaling, adhesion, and migration. Talin couples integrins via its FERM domain to F‐actin and is an important regulator of integrin activation and clustering. The 220 kDa talin rod domain comprises several four‐ and five‐helix bundles that harbor amphipathic α‐helical vinculin binding sites (VBSs). In its inactive state, the hydrophobic VBS residues involved in binding to vinculin are buried within these helix bundles, and the mechanical force emanating from bound integrin receptors is thought necessary for their release and binding to vinculin. The crystal structure of a four‐helix bundle of talin that harbors one of these VBSs, coined VBS33, was recently determined. Here we report the crystal structure of VBS33 in complex with vinculin at 2 Å resolution. Notably, comparison of the apo and vinculin bound structures shows that intermolecular interactions of the VBS33 α‐helix with vinculin are more extensive than the intramolecular interactions of the VBS33 within the talin four‐helix bundle.