Traditionally, developing small molecules for research or drug treatments has been a painstaking enterprise. Drugs work largely by binding to a target protein and modifying or inhibiting its activity, but discovering the rare compound that hits a particular protein is like, well, finding a needle in a haystack. With a specific protein target identified, scientists typically either gather compounds from nature or synthesize artificial compounds, then test them to see whether they act on the target.The birth of combinatorial chemistry in the early nineties promised to revolutionize this laborious process by offering a way to synthesize trillions of compounds at a time. Though molecules still had to be evaluated one by one, high-throughput screening technology could manage up to a million molecules a day. Despite these technological advances, few drugs have emerged from combinatorial chemistry approaches, leaving the promise largely unfulfilled.Another strategy looks to nature as a model. The immune system fights disease and infection by generating billions of antibodies, each primed to recognize a specific pathogen. Antibodies recognize antigens (protein fragments of pathogens) with an exacting specificity that develops through an iterative process. The body first produces a diverse, random collection of antibodies. Every antibody is encoded with a unique DNA blueprint in a B-cell. Antibodies that happen to bind to a pathogen are “selected” to pass on their blueprints: successful binding stimulates cell division, during which blueprints are copied, varied by mutation, and used to create a new generation of antibodies. Specificity is refined over multiple generations.Over the past fifteen years, biologists have developed techniques to recreate this process in a test tube. Today, it's common practice to “evolve” collections of as many as a quadrillion different proteins or nucleic acids to bind a molecular target. These techniques are called molecular breeding, because like traditional livestock and crop breeding techniques, they combine sets of genotypes over generations to produce a desired phenotype. Molecular breeding has so far been restricted only to applications that involve materials encoded by DNA. Drugs produced by conventional synthetic organic chemistry, for example, cannot be bred.In a series of three articles in this issue of  PLoS Biology , David Halpin et al. describe a strategy that addresses this limitation. By inventing a genetic code that acts as a blueprint for synthetic molecules, the authors show how chemical collections of nonbiological origin can be evolved. In the first article, Halpin et al. present a method for overcoming the technical challenge of using DNA to direct the chemical assembly of molecules. In the second, they demonstrate how the method works and test its efficacy by creating a synthetic library of peptides (protein fragments) and then showing that they can find the “peptide in a haystack” by identifying a molecule known to bind a particular antibody. The third paper shows how the method can support a variety of chemistry applications that could potentially synthesize all sorts of nonbiological “species.” Such compounds, the authors point out, can be used for drug discovery or as molecular tools that offer researchers novel ways to disrupt cellular processes and open new windows into cell biology. While medicine has long had to cope with the evolution of drug-resistant pathogens, it may now be possible to fight fire with fire. To learn more about the DNA display method described here, see the primer “Translating DNA into Synthetic Molecules,” also in this issue.PLoS Biology


Background The Arg-Gly-Asp (RGD) cell adhesion sequence occurs in several extracellular matrix molecules known to interact with integrin cell-surface receptors. Recently published crystal structures of the extracellular regions of two integrins in complex with peptides containing or mimicking the RGD sequence have identified the Arg and Asp residues as key specificity determinants for integrin recognition, through hydrogen bonding and metal coordination interactions. The central Gly residue also appears to be in close contact with the integrin surface in these structures.
Background
The Arg-Gly-Asp (RGD) cell adhesion sequence occurs in several extracellular matrix molecules known to interact with integrin cell-surface receptors. Recently published crystal structures of the extracellular regions of two integrins in complex with peptides containing or mimicking the RGD sequence have identified the Arg and Asp residues as key specificity determinants for integrin recognition, through hydrogen bonding and metal coordination interactions. The central Gly residue also appears to be in close contact with the integrin surface in these structures.Results When hydrogen atoms are modelled on the central Gly residue with standard stereochemistry, the interaction between this residue and a carbonyl group in the integrin surface shows all the hallmarks of Cα-H···O=C hydrogen bonding, as seen in the collagen triple helix and in many crystal structures of small organic molecules. Moreover, molecular dynamic simulations of the docking of RGD-containing fragments on integrin surfaces support the occurrence of these interactions. There appears to be an array of four weak and conventional hydrogen bonds lining up the RGD residues with main chain carbonyl groups in the integrin surface.
Results
When hydrogen atoms are modelled on the central Gly residue with standard stereochemistry, the interaction between this residue and a carbonyl group in the integrin surface shows all the hallmarks of Cα-H···O=C hydrogen bonding, as seen in the collagen triple helix and in many crystal structures of small organic molecules. Moreover, molecular dynamic simulations of the docking of RGD-containing fragments on integrin surfaces support the occurrence of these interactions. There appears to be an array of four weak and conventional hydrogen bonds lining up the RGD residues with main chain carbonyl groups in the integrin surface.Conclusions The occurrence of weak Cα-H···O=C hydrogen bonds in the RGD-integrin interaction highlights the importance of the conserved Gly residue in the RGD motif and its contribution to integrin-ligand binding specificity. Our analysis shows how weak hydrogen bonds may also play important biological roles by contributing to the specificity of macromolecular recognition.
Conclusions
The occurrence of weak Cα-H···O=C hydrogen bonds in the RGD-integrin interaction highlights the importance of the conserved Gly residue in the RGD motif and its contribution to integrin-ligand binding specificity. Our analysis shows how weak hydrogen bonds may also play important biological roles by contributing to the specificity of macromolecular recognition.

Background The Arg-Gly-Asp (RGD) sequence is one of the most easily recognised motifs in molecular biology [ 1 ]. Discovered in fibronectin in 1984 [ 2 ], this tripeptide appears to be conserved in the cell attachment sites of many proteins from the extracellular matrix (ECM). The later discovery that RGD is recognised by members of the integrin family of cell surface receptors [ 3 ], confirmed the central role of RGD and suggested that its presence in a protein sequence might be indicative of cell-adhesion functionality [ 4 ]. Integrins are ubiquitously expressed heterodimer cell surface molecules that act as receptors for ECM molecules and other cell-surface adhesins. Through these cell-matrix and cell-cell interactions integrins control diverse cell functions such as adhesion, shape, growth, differentiation and mobility, and therefore contribute to important physiological processes such as development, immune responses and cancer [ 5 ]. Integrins are complex signalling engines: their extracellular domains interact with the ECM while their cytoplasmic tails interact with the cytoskeleton and other intracellular signalling molecules. Current hypotheses suggest that conformational changes resulting from these interactions enable integrins to transmit signals across the membrane in both directions. Recent advances in the structural biology of several integrin domains and their interactions with ligands have begun to define possible working scenarios for the signalling mechanisms [ 6 - 13 ]. As a consequence of their role in so many fundamental processes, integrin defects have been implicated in many common diseases, from cancer to pathogen invasion. An ability to block a particular integrin-ligand interaction may be a possible route to the control of certain pathological states, hence it is not surprising that some integrins have become attractive targets for drug design. Understanding the molecular bases of the interaction of integrins with their ligands is therefore essential for effective protein-based design of inhibitors or activators of their function. A milestone was reached in 2002 with the determination of the crystal structure of the extracellular segment of αVβ3 integrin in complex with a cyclic peptide containing the prototypical RGD sequence [ 8 ]. In that structure, the amino acids defining the RGD sequence are seen to establish specific interactions with corresponding residues in the integrin heterodimer surface, spanning the interface between the αV and β3 subunits (Figure  1a ). Very recently, another landmark paper has reported several crystal structures of the extracellular region of the fibrinogen-binding integrin αIIbβ3 [ 12 ]. In addition to providing an improved picture of the allosteric basis of integrin signal transmission, this new set of structures shows the molecular details of the interaction between the αIIbβ3 RGD-binding site and various ligand mimetics (Figure  1b ). These interactions are remarkably consistent with those previously observed in the complex between the αVβ3 integrin fragment and the cyclic RGD peptide ( c RGD) [ 8 ]. At first glance, two interactions consistently seen in these crystal structures appear to be key in defining the specific molecular recognition between the RGD sequence in an integrin ligand and the surface of its integrin receptor: the Asp residue of the RGD triad completes the coordination of a divalent metal ion bound to the β subunit, while the Arg side chain extends in the opposite direction to form salt-bridge hydrogen bonds with one or two Asp residues in the α subunit. These two specific interactions or their equivalent are seen both in the  c RGD-αVβ3 structure and in the structures of αIIbβ3 in complex with ligand-mimetics (Figure  1 ). There are no significant hydrophobic "pockets" or exosites contributing to the binding specificity. For example, a large fraction of the  c RGD peptide does not make any contact with the αVβ3 integrin surface (Figure  1a ). In the broader context of RGD-containing ligands and their integrin receptors, it would seem that these interactions are mainly electrostatic and that the two charged residues in the RGD sequence are necessary and sufficient for attachment [ 14 ]. What about the central Gly residue? In their analysis of the  c RGD-αVβ3 crystal structure, Xiong  et al.  report that the Gly central residue makes several hydrophobic interactions with the integrin surface, including a contact with the carbonyl oxygen of residue Arg216 in the β3 integrin subunit [ 8 ]. Such contact between the Gly methylene group and a main-chain carbonyl oxygen is also observed in the crystal structure of αIIbβ3 in complex with the peptidomimetic eptifibatide (EFB) [ 12 ] (Figure  1b ), which is a cyclic heptapeptide containing a  homo Arg-Gly-Asp sequence. The particular geometry of these contacts is strikingly reminiscent of a motif previously described in the collagen triple helix: a hydrogen bonding arrangement where the α-carbon of the Gly residue acts as hydrogen bonding donor in Cα-H···O=C interactions (Figure  2 ) [ 15 ]. So-called "weak" hydrogen bonds, such as those between carbon and oxygen atoms, have been traditionally neglected in descriptions of three-dimensional structures of macromolecules. Yet, C-H···O hydrogen bonds are ubiquitous in protein structures: virtually every conventional N-H···O=C hydrogen bond in every β-sheet in every determined protein structure carries a companion Cα-H···O=C interaction [ 16 , 17 ]. This applies to both parallel and antiparallel β-sheets, and exactly the same topology is also observed in the collagen triple helix [ 15 ]. For collagen and the β-sheet structures, the occurrence of Cα-H···O=C interactions is indicative of a very tight fit between the molecules involved, a close-packed structure in which all groups participate in some form of hydrogen bonding interaction. How important are Cα-H···O=C and other weak hydrogen bonds in shaping the three-dimensional structure of proteins and macromolecular complexes? The subject has stimulated considerable debate (see [ 18 ] and [ 19 ] for reviews), although theoretical studies leave no doubt about the cohesive nature of these interactions [ 20 - 23 ]. With a strength approximately one-half of that from conventional hydrogen bonds, it seems reasonable to assume that the large numbers of weak hydrogen bonds detected in proteins may contribute to their stability. Furthermore, several biochemical functions have been linked to specific C-H···O hydrogen bonds, where position is more important that numbers. One example is the Gly-X-X-X-Gly motif, known to favour helix-helix interactions in membrane [ 24 ] and soluble proteins [ 25 ] via  position-specific Cα-H···O=C hydrogen bonds. Another is the proposed role of C-H···O hydrogen bonds from cytosine and thymine bases to amino acid side chains during DNA-protein recognition [ 26 ]. Weak C-H···O hydrogen bonds have also been surveyed at protein-protein interfaces [ 27 ], and have been reported to play specific roles in catalysis [ 28 ], and in substrate and inhibitor recognition [ 29 - 32 ]. Recently, a server to identify weak hydrogen bonding interactions in protein structures has been made publicly available [ 33 ]. The functional occurrence of weak C-H···O hydrogen bonds in protein-ligand, protein-protein, and protein-DNA recognition suggests that their presence should be examined in detail in the structures of macromolecules with biomedical or biotechnological interest. Their potential should not be neglected in rational drug design approaches [ 31 ]. With this in mind, we present here an analysis of possible Gly-Cα-H···O=C interactions between RGD motifs and the RGD-binding sites from the αVβ3 and αIIbβ3 crystal structures. We conclude that the mutual geometry of the interaction is consistent with Cα-H···O=C hydrogen bonding. We discuss the implications of these hydrogen bonds for the cell adhesion interactions between integrins and their RGD-containing ligands.
Background
The Arg-Gly-Asp (RGD) sequence is one of the most easily recognised motifs in molecular biology [ 1 ]. Discovered in fibronectin in 1984 [ 2 ], this tripeptide appears to be conserved in the cell attachment sites of many proteins from the extracellular matrix (ECM). The later discovery that RGD is recognised by members of the integrin family of cell surface receptors [ 3 ], confirmed the central role of RGD and suggested that its presence in a protein sequence might be indicative of cell-adhesion functionality [ 4 ]. Integrins are ubiquitously expressed heterodimer cell surface molecules that act as receptors for ECM molecules and other cell-surface adhesins. Through these cell-matrix and cell-cell interactions integrins control diverse cell functions such as adhesion, shape, growth, differentiation and mobility, and therefore contribute to important physiological processes such as development, immune responses and cancer [ 5 ]. Integrins are complex signalling engines: their extracellular domains interact with the ECM while their cytoplasmic tails interact with the cytoskeleton and other intracellular signalling molecules. Current hypotheses suggest that conformational changes resulting from these interactions enable integrins to transmit signals across the membrane in both directions. Recent advances in the structural biology of several integrin domains and their interactions with ligands have begun to define possible working scenarios for the signalling mechanisms [ 6 - 13 ].As a consequence of their role in so many fundamental processes, integrin defects have been implicated in many common diseases, from cancer to pathogen invasion. An ability to block a particular integrin-ligand interaction may be a possible route to the control of certain pathological states, hence it is not surprising that some integrins have become attractive targets for drug design. Understanding the molecular bases of the interaction of integrins with their ligands is therefore essential for effective protein-based design of inhibitors or activators of their function. A milestone was reached in 2002 with the determination of the crystal structure of the extracellular segment of αVβ3 integrin in complex with a cyclic peptide containing the prototypical RGD sequence [ 8 ]. In that structure, the amino acids defining the RGD sequence are seen to establish specific interactions with corresponding residues in the integrin heterodimer surface, spanning the interface between the αV and β3 subunits (Figure  1a ). Very recently, another landmark paper has reported several crystal structures of the extracellular region of the fibrinogen-binding integrin αIIbβ3 [ 12 ]. In addition to providing an improved picture of the allosteric basis of integrin signal transmission, this new set of structures shows the molecular details of the interaction between the αIIbβ3 RGD-binding site and various ligand mimetics (Figure  1b ). These interactions are remarkably consistent with those previously observed in the complex between the αVβ3 integrin fragment and the cyclic RGD peptide ( c RGD) [ 8 ].cAt first glance, two interactions consistently seen in these crystal structures appear to be key in defining the specific molecular recognition between the RGD sequence in an integrin ligand and the surface of its integrin receptor: the Asp residue of the RGD triad completes the coordination of a divalent metal ion bound to the β subunit, while the Arg side chain extends in the opposite direction to form salt-bridge hydrogen bonds with one or two Asp residues in the α subunit. These two specific interactions or their equivalent are seen both in the  c RGD-αVβ3 structure and in the structures of αIIbβ3 in complex with ligand-mimetics (Figure  1 ). There are no significant hydrophobic "pockets" or exosites contributing to the binding specificity. For example, a large fraction of the  c RGD peptide does not make any contact with the αVβ3 integrin surface (Figure  1a ). In the broader context of RGD-containing ligands and their integrin receptors, it would seem that these interactions are mainly electrostatic and that the two charged residues in the RGD sequence are necessary and sufficient for attachment [ 14 ].ccWhat about the central Gly residue? In their analysis of the  c RGD-αVβ3 crystal structure, Xiong  et al.  report that the Gly central residue makes several hydrophobic interactions with the integrin surface, including a contact with the carbonyl oxygen of residue Arg216 in the β3 integrin subunit [ 8 ]. Such contact between the Gly methylene group and a main-chain carbonyl oxygen is also observed in the crystal structure of αIIbβ3 in complex with the peptidomimetic eptifibatide (EFB) [ 12 ] (Figure  1b ), which is a cyclic heptapeptide containing a  homo Arg-Gly-Asp sequence. The particular geometry of these contacts is strikingly reminiscent of a motif previously described in the collagen triple helix: a hydrogen bonding arrangement where the α-carbon of the Gly residue acts as hydrogen bonding donor in Cα-H···O=C interactions (Figure  2 ) [ 15 ].cet al.homoSo-called "weak" hydrogen bonds, such as those between carbon and oxygen atoms, have been traditionally neglected in descriptions of three-dimensional structures of macromolecules. Yet, C-H···O hydrogen bonds are ubiquitous in protein structures: virtually every conventional N-H···O=C hydrogen bond in every β-sheet in every determined protein structure carries a companion Cα-H···O=C interaction [ 16 , 17 ]. This applies to both parallel and antiparallel β-sheets, and exactly the same topology is also observed in the collagen triple helix [ 15 ]. For collagen and the β-sheet structures, the occurrence of Cα-H···O=C interactions is indicative of a very tight fit between the molecules involved, a close-packed structure in which all groups participate in some form of hydrogen bonding interaction.How important are Cα-H···O=C and other weak hydrogen bonds in shaping the three-dimensional structure of proteins and macromolecular complexes? The subject has stimulated considerable debate (see [ 18 ] and [ 19 ] for reviews), although theoretical studies leave no doubt about the cohesive nature of these interactions [ 20 - 23 ]. With a strength approximately one-half of that from conventional hydrogen bonds, it seems reasonable to assume that the large numbers of weak hydrogen bonds detected in proteins may contribute to their stability. Furthermore, several biochemical functions have been linked to specific C-H···O hydrogen bonds, where position is more important that numbers. One example is the Gly-X-X-X-Gly motif, known to favour helix-helix interactions in membrane [ 24 ] and soluble proteins [ 25 ] via  position-specific Cα-H···O=C hydrogen bonds. Another is the proposed role of C-H···O hydrogen bonds from cytosine and thymine bases to amino acid side chains during DNA-protein recognition [ 26 ]. Weak C-H···O hydrogen bonds have also been surveyed at protein-protein interfaces [ 27 ], and have been reported to play specific roles in catalysis [ 28 ], and in substrate and inhibitor recognition [ 29 - 32 ]. Recently, a server to identify weak hydrogen bonding interactions in protein structures has been made publicly available [ 33 ].viaThe functional occurrence of weak C-H···O hydrogen bonds in protein-ligand, protein-protein, and protein-DNA recognition suggests that their presence should be examined in detail in the structures of macromolecules with biomedical or biotechnological interest. Their potential should not be neglected in rational drug design approaches [ 31 ]. With this in mind, we present here an analysis of possible Gly-Cα-H···O=C interactions between RGD motifs and the RGD-binding sites from the αVβ3 and αIIbβ3 crystal structures. We conclude that the mutual geometry of the interaction is consistent with Cα-H···O=C hydrogen bonding. We discuss the implications of these hydrogen bonds for the cell adhesion interactions between integrins and their RGD-containing ligands.Results and discussion Building standard-geometry Hα atoms on the Gly central residues of the  c RGD and EFB peptides produces the geometric arrangements shown in Figure  2 , clearly reminiscent of the hydrogen bonding pattern previously described in the collagen triple helix (Figure  2c ). The metrics of these Gly-carbonyl contacts are shown in Table  1 . The Cα···O distances in the  c RGD-αVβ3 and EFB-αIIbβ3 structures appear to be longer than the mean Cα···O distance in collagen, but are well within the observed range in crystal structures of small organic molecules (see below). Both Hα atoms from the collagen Gly residues are in hydrogen bonding position (Cα–Hα···O > 90°), and their Cα-Hα···O=C hydrogen bonds adopt a three-centred and bifurcated configuration (Figure  2c  and [ 15 ]), that is not seen in the integrin structures. Nevertheless, the central Gly residues in the  c RGD and EFB peptides appear to have one and two Hα atoms respectively in hydrogen bonding position to the carbonyl group of Arg216, a residue on the surface of the β3 subunit and directly at the interface with the αV and αIIb subunits. An obvious  caveat  to this analysis comes from the moderate resolution of the  c RGD-αVβ3 and EFB-αIIbβ3 crystal structures (3.2 Å and 2.9 Å respectively). Positional errors inevitable at that resolution may affect the precision of the fitting of the  c RGD and EFB peptides and the accuracy of the hydrogen bonding geometries for both weak and strong hydrogen bonds. For example, a close look at the salt-bridge interactions between the Arg guanidinium group from the  c RGD peptide and two Asp side chains on the αV integrin surface (Asp150 and Asp218), shows less than "ideal" hydrogen bonding orientation, especially for Asp150 (not shown). Yet, the accumulated knowledge of hydrogen bonding geometries in high-resolution crystal structures and their significant variability leaves no doubt about the existence of these strong hydrogen bonds and their contribution to the specificity of binding. A similar level of confidence can be achieved for the Gly-Cα-Hα···O=C hydrogen bond by analysing the metrics of equivalent interactions in high-resolution crystal structures of small organic and organometallic molecules. Figure  3  shows the two fragment probes used in a statistical search for Gly-Cα-Hα···O=C nonbonded interactions in the Cambridge Structural Database (see Methods). Figure  4  shows that single hydrogen bonding (only one angle Cα-Hα···O ≥ 90°) predominates over the bifurcated case, and that a broad maximum in the Cα···O distribution occurs at about 3.4 Å, which can be taken as the "hydrogen bonding distance" for this type of interaction. This value is consistent with the theoretical value of 3.34 Å for the Gly-Cα-Hα···OH 2  hydrogen bond from  ab initio  quantum calculations [ 22 ]. Figure  5  shows the distributions of Hα···O distances and Cα-Hα···O angles for the single hydrogen bond. The Hα···O distribution shows a broad maximum around 2.7 Å, whereas the angular distribution is very broad with maxima around 110° and 140°. Average parameters for single and double Gly-Cα-Hα···O=C hydrogen bonding (Table  1 ) are perfectly compatible with those calculated for the  c RGD-αVβ3 and EFB-αIIbβ3 structures respectively, even though the accuracy of the values shown in Table  1  is clearly overestimated with respect to the resolution of these crystal structures. Thus, strictly from a geometrical point of view, the contacts between the Gly residues in the  c RGD and EFB peptides and the main chain carbonyl group from Arg216 in the integrin surface bear all the characteristics of Cα-Hα···O=C hydrogen bonding. This observation is consistent with the exceptionally high frequency of intermolecular Gly-Cα-H···O=C hydrogen bonds recently reported in high resolution crystal structures of protein-ligand complexes [ 32 ]. A simple molecular docking analysis further supports the occurrence of Gly-Cα-H···O=C hydrogen bonds between RGD-containing ligands and integrin binding sites. In a first set of calculations, an RGD tripeptide was docked into the binding sites of both αVβ3 and αIIbβ3 integrins using constrained molecular dynamics (MD). Two constraints were imposed in the docking calculations: the carboxyl group from the Asp residue had to complete the metal coordination on the β3 subunit, and the Arg side chain had to form a salt bridge with appropriate Asp residues in the αV and αIIb subunits (see Methods), as observed in the crystal structures of αVβ3 and αIIbβ3 with different ligand-mimetics. Ten slightly different RGD models were obtained from the NMR structures of the adhesion domain of fibronectin [ 34 ], and were placed about 10 Å away from the integrin surface. Then these RGD models were subject to MD simulations until they docked into the integrin binding sites. In a second set of calculations, a longer peptide fragment with sequence VTGRGDSPAS from the adhesion domain of fibronectin was also docked into the binding sites of the two integrins (Figure  6 ). Again, ten different models for this peptide were obtained from fibronectin NMR structures [ 34 ]. Most of the simulations converged to models with Cα···O contact distances between the central Gly residue and the carbonyl of Arg216 in the 2.7–3.7 Å range (Figure  6b ), with either one or two Gly-Hα atoms in hydrogen bonding orientation. These models were also the most favourable energetically (Table  2 ). From these calculations it seems to emerge that the RGD binding sites of the αVβ3 and αIIbβ3 integrins are primed to place the central Gly residue in the RGD triad directly above the carbonyl group of Arg216 of the β3 subunit (as observed in the  c RGD-αVβ3 and EFB-αIIbβ3 crystal structures), forming one or two Cα-H···O hydrogen bonds that complement the main metal-coordination and salt-bridge interactions from the Asp and Arg side chains. How important are these weak C-H···O hydrogen bonds in stabilising the  c RGD-αVβ3 and EFB-αIIbβ3 complexes? A quantitative analysis of C-H···O hydrogen bonding at protein-protein interfaces has shown that they have an important contribution to the association and stability of protein complexes, accounting for about one third of the total hydrogen bonding interaction energy [ 27 ]. In fact, some of the hydrophobic or van der Waals interactions usually invoked to explain stabilising close contacts between molecules can be described better as weak C-H···O hydrogen bonds. These occupy a middle ground between the highly directional, conventional hydrogen bonds, and the directionless van der Waals interactions [ 32 ]. The recurrent appearance of some weak hydrogen bonding topologies in many structures of proteins and at protein-protein interfaces also reinforces the notion that they have a significant contribution to macromolecular stability. The most common occurrence of C-H···O hydrogen bonds in protein structures is a widespread Cα-H···O=C hydrogen bond  N -terminal to the conventional N-H···O=C hydrogen bond in β-sheets [ 16 , 17 ] and in the collagen triple helix [ 15 ]. In this structural motif (Figure  7a ), the Cα-H donor group is in the residue immediately  N -terminal to the one carrying the N-H donor group, and both share the same C = O group as acceptor, an arrangement sometimes referred as "bifurcated" hydrogen bond [ 17 , 18 , 27 ]. This bifurcated hydrogen bonding motif is also the most common occurrence of C-H···O hydrogen bond at protein-protein interfaces [ 27 ]. The situation in Figure  7b  occurs when the residue  N -terminal to the one carrying the N-H donor group is Gly, with one or two Hα from Gly being in hydrogen bonding position. The bifurcated hydrogen bond scenario also occurs in the  c RGD-αVβ3 and EFB-αIIbβ3 structures, where the N-H group from the Asp residue in the RGD peptide donates a hydrogen bond to the main chain carbonyl group from Arg216 of β3. This hydrogen bond has very bad geometry in the  c RGD-αVβ3 structure (distance H···O 2.69 Å, angle N-H···O 133°), but looks better in the EFB-αIIbβ3 structure (distance H···O 2.39 Å, angle N-H···O 144°). These deviations from ideal hydrogen bonding geometry might be consequence of the resolution of the crystal structures, but all the MD docking simulations described above result in N-H···O=C hydrogen bonds that are slightly longer (typical H···O distances 2.4–2.5 Å) and slightly less linear (typical N-H···O angles 140°-150°) than the average hydrogen bonds between peptide groups in protein secondary structures. Automatic computational docking calculations of known integrin ligands on structural models of αVβ3 and αVβ5 consistently predict this N-H···O=C hydrogen bond to occur whenever an N-H group is present in the proximity of the carboxylate moiety [ 35 , 36 ]. Had these simulations included all nonpolar hydrogens, the companion Cα-H···O=C hydrogen bonds from the Gly residues would also have been observed. A quick inventory of hydrophobic interactions between the  c RGD and EFB peptides and the αVβ3 and αIIbβ3 surfaces suggests an additional candidate for classification as C-H···O=C hydrogen bond, between the Cβ-Hβ group from the Asp residue of the  c RGD and EFB peptides and the main chain C = O group from Asn215 in the β3 subunit. This weak hydrogen bond is adjacent to the stronger, conventional hydrogen bond between the main chain N-H group from Asn215 and Oδ2 from the Asp residue in the RGD motif. Thus, a total of four hydrogen bonds, weak and conventional, aligns the bottom of the  c RGD and EFB peptides against the integrin surfaces (Figure  8 ), and complements the main interactions from the Asp carboxyl and Arg guanidinium groups to provide a higher binding specificity. It is clear from the  c RGD-αVβ3 and EFB-αIIbβ3 structures that any side chain other than Gly in the RGD triad would not allow it to fit snugly within the integrin binding site, with the resulting weakening of hydrogen bonding and van der Waals interactions. Furthermore the main chain conformation for the central Gly residue in the  c RGD-αVβ3 structure falls in a region of the Ramachandran map that is not allowed to any L-amino acid residue. Thus, Gly residues at the centre of the RGD motif are essential for being small, for being able to adopt specific main chain conformations, and for being able to interact closely with the integrin surface via Cα-H···O=C hydrogen bonds. All three characteristics contribute to the integrin-binding specificity of Gly residues at the centre of RGD motifs. Inasmuch as the  c RGD-αVβ3 and EFB-αIIbβ3 structures remain valid models for the structural basis of integrin-RGD ligand-binding specificity, it is reasonable to assume that the weak Cα/β-H···O=C hydrogen bonds depicted in Figure  8  will also occur in RGD-based cell-adhesion interactions. A special feature of the integrin surface at the RGD-binding site is the presence of two main chain carbonyl groups exposed to the solvent in the β3 subunit: Asn215 and Arg216. In absence of ligands these groups will probably interact with water molecules through conventional hydrogen bonding interactions (as seen for example in the crystal structure of the cacodylate-bound form of αIIbβ3, PDB accession code 1TXV [ 12 ]). Upon ligand binding, the RGD residues will displace these waters and place one amide and two methylene groups in hydrogen bonding position to carbonyl groups, increasing the specificity of the RGD-integrin interaction through multipoint recognition (Figure  8 ). This strategy will obviously be exploited by many competitive inhibitors for the integrin RGD-binding site. For example it is possible to substitute the weaker Cα-H donors from the Gly residue by a conventional N-H group (Figure  7c ). This strategy has been exploited already in the design of aza-peptide and azacarba-peptide RGD mimetics [ 37 - 39 ], several of them with nanomolar activity. Molecular modelling of the interaction of these peptides with αVβ3 and αVβ5 RGD binding sites predicts the hydrogen bonding topology shown in Figure  7c [ 36 ]. It is interesting to notice that even in the absence of a conventional hydrogen bonding donor, the carbonyl group Arg216 in the β3 subunit still may be acceptor for weak hydrogen bonds. In the crystal structure of αIIbβ3 in complex with tirofiban [ 12 ], a non-peptidomimetic inhibitor derived from L-tyrosine, the Cδ1 atom from the substituted Tyr ring is some 3.01 Å away from the carbonyl oxygen of the very same Arg216. If a hydrogen atom is built with standard geometry on Cδ1, the calculated Hδ1···O distance is 2.01 Å and the Cδ1-Hδ1···O angle is 172°, again hydrogen bonding-like metrics. How should this Cδ1···O=C contact be called? We think that a description in terms of weak C-H···O hydrogen bonding is in this case more accurate than referring to this interaction as simply hydrophobic.
Results and discussion
Building standard-geometry Hα atoms on the Gly central residues of the  c RGD and EFB peptides produces the geometric arrangements shown in Figure  2 , clearly reminiscent of the hydrogen bonding pattern previously described in the collagen triple helix (Figure  2c ). The metrics of these Gly-carbonyl contacts are shown in Table  1 . The Cα···O distances in the  c RGD-αVβ3 and EFB-αIIbβ3 structures appear to be longer than the mean Cα···O distance in collagen, but are well within the observed range in crystal structures of small organic molecules (see below). Both Hα atoms from the collagen Gly residues are in hydrogen bonding position (Cα–Hα···O > 90°), and their Cα-Hα···O=C hydrogen bonds adopt a three-centred and bifurcated configuration (Figure  2c  and [ 15 ]), that is not seen in the integrin structures. Nevertheless, the central Gly residues in the  c RGD and EFB peptides appear to have one and two Hα atoms respectively in hydrogen bonding position to the carbonyl group of Arg216, a residue on the surface of the β3 subunit and directly at the interface with the αV and αIIb subunits.cccAn obvious  caveat  to this analysis comes from the moderate resolution of the  c RGD-αVβ3 and EFB-αIIbβ3 crystal structures (3.2 Å and 2.9 Å respectively). Positional errors inevitable at that resolution may affect the precision of the fitting of the  c RGD and EFB peptides and the accuracy of the hydrogen bonding geometries for both weak and strong hydrogen bonds. For example, a close look at the salt-bridge interactions between the Arg guanidinium group from the  c RGD peptide and two Asp side chains on the αV integrin surface (Asp150 and Asp218), shows less than "ideal" hydrogen bonding orientation, especially for Asp150 (not shown). Yet, the accumulated knowledge of hydrogen bonding geometries in high-resolution crystal structures and their significant variability leaves no doubt about the existence of these strong hydrogen bonds and their contribution to the specificity of binding.caveatcccA similar level of confidence can be achieved for the Gly-Cα-Hα···O=C hydrogen bond by analysing the metrics of equivalent interactions in high-resolution crystal structures of small organic and organometallic molecules. Figure  3  shows the two fragment probes used in a statistical search for Gly-Cα-Hα···O=C nonbonded interactions in the Cambridge Structural Database (see Methods). Figure  4  shows that single hydrogen bonding (only one angle Cα-Hα···O ≥ 90°) predominates over the bifurcated case, and that a broad maximum in the Cα···O distribution occurs at about 3.4 Å, which can be taken as the "hydrogen bonding distance" for this type of interaction. This value is consistent with the theoretical value of 3.34 Å for the Gly-Cα-Hα···OH 2  hydrogen bond from  ab initio  quantum calculations [ 22 ]. Figure  5  shows the distributions of Hα···O distances and Cα-Hα···O angles for the single hydrogen bond. The Hα···O distribution shows a broad maximum around 2.7 Å, whereas the angular distribution is very broad with maxima around 110° and 140°. Average parameters for single and double Gly-Cα-Hα···O=C hydrogen bonding (Table  1 ) are perfectly compatible with those calculated for the  c RGD-αVβ3 and EFB-αIIbβ3 structures respectively, even though the accuracy of the values shown in Table  1  is clearly overestimated with respect to the resolution of these crystal structures. Thus, strictly from a geometrical point of view, the contacts between the Gly residues in the  c RGD and EFB peptides and the main chain carbonyl group from Arg216 in the integrin surface bear all the characteristics of Cα-Hα···O=C hydrogen bonding. This observation is consistent with the exceptionally high frequency of intermolecular Gly-Cα-H···O=C hydrogen bonds recently reported in high resolution crystal structures of protein-ligand complexes [ 32 ].ab initioccA simple molecular docking analysis further supports the occurrence of Gly-Cα-H···O=C hydrogen bonds between RGD-containing ligands and integrin binding sites. In a first set of calculations, an RGD tripeptide was docked into the binding sites of both αVβ3 and αIIbβ3 integrins using constrained molecular dynamics (MD). Two constraints were imposed in the docking calculations: the carboxyl group from the Asp residue had to complete the metal coordination on the β3 subunit, and the Arg side chain had to form a salt bridge with appropriate Asp residues in the αV and αIIb subunits (see Methods), as observed in the crystal structures of αVβ3 and αIIbβ3 with different ligand-mimetics. Ten slightly different RGD models were obtained from the NMR structures of the adhesion domain of fibronectin [ 34 ], and were placed about 10 Å away from the integrin surface. Then these RGD models were subject to MD simulations until they docked into the integrin binding sites. In a second set of calculations, a longer peptide fragment with sequence VTGRGDSPAS from the adhesion domain of fibronectin was also docked into the binding sites of the two integrins (Figure  6 ). Again, ten different models for this peptide were obtained from fibronectin NMR structures [ 34 ]. Most of the simulations converged to models with Cα···O contact distances between the central Gly residue and the carbonyl of Arg216 in the 2.7–3.7 Å range (Figure  6b ), with either one or two Gly-Hα atoms in hydrogen bonding orientation. These models were also the most favourable energetically (Table  2 ). From these calculations it seems to emerge that the RGD binding sites of the αVβ3 and αIIbβ3 integrins are primed to place the central Gly residue in the RGD triad directly above the carbonyl group of Arg216 of the β3 subunit (as observed in the  c RGD-αVβ3 and EFB-αIIbβ3 crystal structures), forming one or two Cα-H···O hydrogen bonds that complement the main metal-coordination and salt-bridge interactions from the Asp and Arg side chains.cHow important are these weak C-H···O hydrogen bonds in stabilising the  c RGD-αVβ3 and EFB-αIIbβ3 complexes? A quantitative analysis of C-H···O hydrogen bonding at protein-protein interfaces has shown that they have an important contribution to the association and stability of protein complexes, accounting for about one third of the total hydrogen bonding interaction energy [ 27 ]. In fact, some of the hydrophobic or van der Waals interactions usually invoked to explain stabilising close contacts between molecules can be described better as weak C-H···O hydrogen bonds. These occupy a middle ground between the highly directional, conventional hydrogen bonds, and the directionless van der Waals interactions [ 32 ].cThe recurrent appearance of some weak hydrogen bonding topologies in many structures of proteins and at protein-protein interfaces also reinforces the notion that they have a significant contribution to macromolecular stability. The most common occurrence of C-H···O hydrogen bonds in protein structures is a widespread Cα-H···O=C hydrogen bond  N -terminal to the conventional N-H···O=C hydrogen bond in β-sheets [ 16 , 17 ] and in the collagen triple helix [ 15 ]. In this structural motif (Figure  7a ), the Cα-H donor group is in the residue immediately  N -terminal to the one carrying the N-H donor group, and both share the same C = O group as acceptor, an arrangement sometimes referred as "bifurcated" hydrogen bond [ 17 , 18 , 27 ]. This bifurcated hydrogen bonding motif is also the most common occurrence of C-H···O hydrogen bond at protein-protein interfaces [ 27 ]. The situation in Figure  7b  occurs when the residue  N -terminal to the one carrying the N-H donor group is Gly, with one or two Hα from Gly being in hydrogen bonding position. The bifurcated hydrogen bond scenario also occurs in the  c RGD-αVβ3 and EFB-αIIbβ3 structures, where the N-H group from the Asp residue in the RGD peptide donates a hydrogen bond to the main chain carbonyl group from Arg216 of β3. This hydrogen bond has very bad geometry in the  c RGD-αVβ3 structure (distance H···O 2.69 Å, angle N-H···O 133°), but looks better in the EFB-αIIbβ3 structure (distance H···O 2.39 Å, angle N-H···O 144°). These deviations from ideal hydrogen bonding geometry might be consequence of the resolution of the crystal structures, but all the MD docking simulations described above result in N-H···O=C hydrogen bonds that are slightly longer (typical H···O distances 2.4–2.5 Å) and slightly less linear (typical N-H···O angles 140°-150°) than the average hydrogen bonds between peptide groups in protein secondary structures. Automatic computational docking calculations of known integrin ligands on structural models of αVβ3 and αVβ5 consistently predict this N-H···O=C hydrogen bond to occur whenever an N-H group is present in the proximity of the carboxylate moiety [ 35 , 36 ]. Had these simulations included all nonpolar hydrogens, the companion Cα-H···O=C hydrogen bonds from the Gly residues would also have been observed.NNNccA quick inventory of hydrophobic interactions between the  c RGD and EFB peptides and the αVβ3 and αIIbβ3 surfaces suggests an additional candidate for classification as C-H···O=C hydrogen bond, between the Cβ-Hβ group from the Asp residue of the  c RGD and EFB peptides and the main chain C = O group from Asn215 in the β3 subunit. This weak hydrogen bond is adjacent to the stronger, conventional hydrogen bond between the main chain N-H group from Asn215 and Oδ2 from the Asp residue in the RGD motif. Thus, a total of four hydrogen bonds, weak and conventional, aligns the bottom of the  c RGD and EFB peptides against the integrin surfaces (Figure  8 ), and complements the main interactions from the Asp carboxyl and Arg guanidinium groups to provide a higher binding specificity.cccIt is clear from the  c RGD-αVβ3 and EFB-αIIbβ3 structures that any side chain other than Gly in the RGD triad would not allow it to fit snugly within the integrin binding site, with the resulting weakening of hydrogen bonding and van der Waals interactions. Furthermore the main chain conformation for the central Gly residue in the  c RGD-αVβ3 structure falls in a region of the Ramachandran map that is not allowed to any L-amino acid residue. Thus, Gly residues at the centre of the RGD motif are essential for being small, for being able to adopt specific main chain conformations, and for being able to interact closely with the integrin surface via Cα-H···O=C hydrogen bonds. All three characteristics contribute to the integrin-binding specificity of Gly residues at the centre of RGD motifs.ccInasmuch as the  c RGD-αVβ3 and EFB-αIIbβ3 structures remain valid models for the structural basis of integrin-RGD ligand-binding specificity, it is reasonable to assume that the weak Cα/β-H···O=C hydrogen bonds depicted in Figure  8  will also occur in RGD-based cell-adhesion interactions. A special feature of the integrin surface at the RGD-binding site is the presence of two main chain carbonyl groups exposed to the solvent in the β3 subunit: Asn215 and Arg216. In absence of ligands these groups will probably interact with water molecules through conventional hydrogen bonding interactions (as seen for example in the crystal structure of the cacodylate-bound form of αIIbβ3, PDB accession code 1TXV [ 12 ]). Upon ligand binding, the RGD residues will displace these waters and place one amide and two methylene groups in hydrogen bonding position to carbonyl groups, increasing the specificity of the RGD-integrin interaction through multipoint recognition (Figure  8 ). This strategy will obviously be exploited by many competitive inhibitors for the integrin RGD-binding site. For example it is possible to substitute the weaker Cα-H donors from the Gly residue by a conventional N-H group (Figure  7c ). This strategy has been exploited already in the design of aza-peptide and azacarba-peptide RGD mimetics [ 37 - 39 ], several of them with nanomolar activity. Molecular modelling of the interaction of these peptides with αVβ3 and αVβ5 RGD binding sites predicts the hydrogen bonding topology shown in Figure  7c [ 36 ]. It is interesting to notice that even in the absence of a conventional hydrogen bonding donor, the carbonyl group Arg216 in the β3 subunit still may be acceptor for weak hydrogen bonds. In the crystal structure of αIIbβ3 in complex with tirofiban [ 12 ], a non-peptidomimetic inhibitor derived from L-tyrosine, the Cδ1 atom from the substituted Tyr ring is some 3.01 Å away from the carbonyl oxygen of the very same Arg216. If a hydrogen atom is built with standard geometry on Cδ1, the calculated Hδ1···O distance is 2.01 Å and the Cδ1-Hδ1···O angle is 172°, again hydrogen bonding-like metrics. How should this Cδ1···O=C contact be called? We think that a description in terms of weak C-H···O hydrogen bonding is in this case more accurate than referring to this interaction as simply hydrophobic.cConclusions We have analysed in detail recently published structural data on the interaction between the extracellular regions of two integrins and peptides containing or mimicking the RGD sequence [ 8 , 12 ]. From this analysis we conclude that Cα-H···O=C hydrogen bonds from the central Gly residue also contribute to the specificity of binding. Weak hydrogen bonds are traditionally overlooked when describing protein structures, although they probably contribute to their stability. We think that our analysis provides one of the most interesting examples of C-H···O hydrogen bonds playing an important biological role, and may contribute to reverse the current trend of neglect of these interactions. In a recent paper, Sarkhel and Desiraju suggest that Nature may take advantage of the weaker C-H···O hydrogen bonds to optimise the efficiency of protein-ligand interactions, with a larger number of interactions coming into play even at the expense of the strength of the individual interactions [ 32 ]. By using more interactions, they suggest, specificity of recognition is increased, and because individual interactions are weaker, reversibility is possible. Our analysis of the interaction between the  c RGD and EFB peptides and the αVβ3 and αIIbβ3 integrin surfaces would seem to corroborate this suggestion.
Conclusions
We have analysed in detail recently published structural data on the interaction between the extracellular regions of two integrins and peptides containing or mimicking the RGD sequence [ 8 , 12 ]. From this analysis we conclude that Cα-H···O=C hydrogen bonds from the central Gly residue also contribute to the specificity of binding. Weak hydrogen bonds are traditionally overlooked when describing protein structures, although they probably contribute to their stability. We think that our analysis provides one of the most interesting examples of C-H···O hydrogen bonds playing an important biological role, and may contribute to reverse the current trend of neglect of these interactions. In a recent paper, Sarkhel and Desiraju suggest that Nature may take advantage of the weaker C-H···O hydrogen bonds to optimise the efficiency of protein-ligand interactions, with a larger number of interactions coming into play even at the expense of the strength of the individual interactions [ 32 ]. By using more interactions, they suggest, specificity of recognition is increased, and because individual interactions are weaker, reversibility is possible. Our analysis of the interaction between the  c RGD and EFB peptides and the αVβ3 and αIIbβ3 integrin surfaces would seem to corroborate this suggestion.cMethods Integrin binding sites and hydrogen building The following crystal structure coordinates were downloaded from the Protein Data Bank [ 40 ]: αVβ3 integrin in complex with a cyclic RGD peptide ( c RGD), PDB accession code 1L5G [ 8 ]; αIIbβ3 integrin structure at 2.7 Å resolution, PDB accession code 1TXV [ 12 ]; αIIbβ3 in complex with eptifibatide (EFB), PDB accession code 1TY6 [ 12 ]; αIIbβ3 in complex with tirofiban, PDB accession code 1TY5. Models for integrin RGD-binding sites on αVβ3 and αIIbβ3 were obtained by selecting coordinates from integrin residues within 10 Å from the bound peptides. For the αIIbβ3 binding site, coordinates of the corresponding residues in the 1TXV structure were used, as this crystal structure has a better resolution. Coordinates for metal ions and structural waters present in the RGD-binding sites but not interfering with the binding of  c RGD or EFB were also maintained. Hydrogen atoms were built with standard stereochemistry for the  c RGD and EFB peptides and for the integrin RGD-binding sites as defined above, using the program  REDUCE  [ 41 ]. For the purpose of the analysis presented here all hydrogen atoms discussed in this paper could be positioned with satisfactory accuracy and predictable orientation. Molecular docking calculations For the molecular docking calculations, conformational models for RGD and VTGRGDSPAS peptides were obtained from the NMR structures of the adhesion domain in fibronectin [ 34 ]. Ten conformational models were used for each peptide. Each model was first manually docked approximately into the coordinates of the binding sites of αVβ3 and αIIbβ3 integrins, using the  c RGD-αVβ3 and EFB-αIIbβ3 structures for guidance. Then each docked model was pulled away to about 10 Å from the integrin surfaces, and was docked back into the integrin binding site via molecular dynamics (MD) simulations using the program  CNS  [ 42 ]. Five simulations were run for each model, to a total of 50 MD simulations for each peptide-integrin pairing. A set of distance restraints was applied to the docking MD simulations, as observed on the  c RGD-αVβ3 and EFB-αIIbβ3 structures. The side chain of the Asp residue group was restrained to coordinate the bound metal ion in the RGD-binding sites and to receive a hydrogen bond from the amide group of Asn215, in the β3 subunit. The side chain of the Arg residue was restrained to form hydrogen bonds with residues Asp150 and Asp218 on the αV subunit or residue Asp224 on the αIIb subunit. Additional restraints were imposed in the MD simulations with the VTGRGDSPAS peptide: the ring of Pro172 was restrained to hydrophobic contact with the side chain of Lys125, in the β3 subunit, and the Cα atoms of the  N - and  C -terminal residues in the peptide model were restrained not to separate more than 5 Å from each other. The coordinates of the integrin binding sites were kept fixed in all the simulations, and only the peptides were allowed to refine by restrained MD and energy minimisation. All molecular models were analysed with the program CHAIN [ 43 ] in a Silicon Graphics workstation. Analysis of hydrogen bonding geometry in crystal structures of organic molecules A survey in the Cambridge Structural Database [ 44 ] (July 2003 release), was carried out for Cα···O contacts between glycine-like fragments and carbonyl groups (Figure  3 ).
Methods
Integrin binding sites and hydrogen building The following crystal structure coordinates were downloaded from the Protein Data Bank [ 40 ]: αVβ3 integrin in complex with a cyclic RGD peptide ( c RGD), PDB accession code 1L5G [ 8 ]; αIIbβ3 integrin structure at 2.7 Å resolution, PDB accession code 1TXV [ 12 ]; αIIbβ3 in complex with eptifibatide (EFB), PDB accession code 1TY6 [ 12 ]; αIIbβ3 in complex with tirofiban, PDB accession code 1TY5. Models for integrin RGD-binding sites on αVβ3 and αIIbβ3 were obtained by selecting coordinates from integrin residues within 10 Å from the bound peptides. For the αIIbβ3 binding site, coordinates of the corresponding residues in the 1TXV structure were used, as this crystal structure has a better resolution. Coordinates for metal ions and structural waters present in the RGD-binding sites but not interfering with the binding of  c RGD or EFB were also maintained. Hydrogen atoms were built with standard stereochemistry for the  c RGD and EFB peptides and for the integrin RGD-binding sites as defined above, using the program  REDUCE  [ 41 ]. For the purpose of the analysis presented here all hydrogen atoms discussed in this paper could be positioned with satisfactory accuracy and predictable orientation.
Integrin binding sites and hydrogen building
The following crystal structure coordinates were downloaded from the Protein Data Bank [ 40 ]: αVβ3 integrin in complex with a cyclic RGD peptide ( c RGD), PDB accession code 1L5G [ 8 ]; αIIbβ3 integrin structure at 2.7 Å resolution, PDB accession code 1TXV [ 12 ]; αIIbβ3 in complex with eptifibatide (EFB), PDB accession code 1TY6 [ 12 ]; αIIbβ3 in complex with tirofiban, PDB accession code 1TY5. Models for integrin RGD-binding sites on αVβ3 and αIIbβ3 were obtained by selecting coordinates from integrin residues within 10 Å from the bound peptides. For the αIIbβ3 binding site, coordinates of the corresponding residues in the 1TXV structure were used, as this crystal structure has a better resolution. Coordinates for metal ions and structural waters present in the RGD-binding sites but not interfering with the binding of  c RGD or EFB were also maintained. Hydrogen atoms were built with standard stereochemistry for the  c RGD and EFB peptides and for the integrin RGD-binding sites as defined above, using the program  REDUCE  [ 41 ]. For the purpose of the analysis presented here all hydrogen atoms discussed in this paper could be positioned with satisfactory accuracy and predictable orientation.cccREDUCEMolecular docking calculations For the molecular docking calculations, conformational models for RGD and VTGRGDSPAS peptides were obtained from the NMR structures of the adhesion domain in fibronectin [ 34 ]. Ten conformational models were used for each peptide. Each model was first manually docked approximately into the coordinates of the binding sites of αVβ3 and αIIbβ3 integrins, using the  c RGD-αVβ3 and EFB-αIIbβ3 structures for guidance. Then each docked model was pulled away to about 10 Å from the integrin surfaces, and was docked back into the integrin binding site via molecular dynamics (MD) simulations using the program  CNS  [ 42 ]. Five simulations were run for each model, to a total of 50 MD simulations for each peptide-integrin pairing. A set of distance restraints was applied to the docking MD simulations, as observed on the  c RGD-αVβ3 and EFB-αIIbβ3 structures. The side chain of the Asp residue group was restrained to coordinate the bound metal ion in the RGD-binding sites and to receive a hydrogen bond from the amide group of Asn215, in the β3 subunit. The side chain of the Arg residue was restrained to form hydrogen bonds with residues Asp150 and Asp218 on the αV subunit or residue Asp224 on the αIIb subunit. Additional restraints were imposed in the MD simulations with the VTGRGDSPAS peptide: the ring of Pro172 was restrained to hydrophobic contact with the side chain of Lys125, in the β3 subunit, and the Cα atoms of the  N - and  C -terminal residues in the peptide model were restrained not to separate more than 5 Å from each other. The coordinates of the integrin binding sites were kept fixed in all the simulations, and only the peptides were allowed to refine by restrained MD and energy minimisation. All molecular models were analysed with the program CHAIN [ 43 ] in a Silicon Graphics workstation.
Molecular docking calculations
For the molecular docking calculations, conformational models for RGD and VTGRGDSPAS peptides were obtained from the NMR structures of the adhesion domain in fibronectin [ 34 ]. Ten conformational models were used for each peptide. Each model was first manually docked approximately into the coordinates of the binding sites of αVβ3 and αIIbβ3 integrins, using the  c RGD-αVβ3 and EFB-αIIbβ3 structures for guidance. Then each docked model was pulled away to about 10 Å from the integrin surfaces, and was docked back into the integrin binding site via molecular dynamics (MD) simulations using the program  CNS  [ 42 ]. Five simulations were run for each model, to a total of 50 MD simulations for each peptide-integrin pairing. A set of distance restraints was applied to the docking MD simulations, as observed on the  c RGD-αVβ3 and EFB-αIIbβ3 structures. The side chain of the Asp residue group was restrained to coordinate the bound metal ion in the RGD-binding sites and to receive a hydrogen bond from the amide group of Asn215, in the β3 subunit. The side chain of the Arg residue was restrained to form hydrogen bonds with residues Asp150 and Asp218 on the αV subunit or residue Asp224 on the αIIb subunit. Additional restraints were imposed in the MD simulations with the VTGRGDSPAS peptide: the ring of Pro172 was restrained to hydrophobic contact with the side chain of Lys125, in the β3 subunit, and the Cα atoms of the  N - and  C -terminal residues in the peptide model were restrained not to separate more than 5 Å from each other. The coordinates of the integrin binding sites were kept fixed in all the simulations, and only the peptides were allowed to refine by restrained MD and energy minimisation. All molecular models were analysed with the program CHAIN [ 43 ] in a Silicon Graphics workstation.cCNScNCAnalysis of hydrogen bonding geometry in crystal structures of organic molecules A survey in the Cambridge Structural Database [ 44 ] (July 2003 release), was carried out for Cα···O contacts between glycine-like fragments and carbonyl groups (Figure  3 ).
Analysis of hydrogen bonding geometry in crystal structures of organic molecules
A survey in the Cambridge Structural Database [ 44 ] (July 2003 release), was carried out for Cα···O contacts between glycine-like fragments and carbonyl groups (Figure  3 ).List of abbreviations ECM, extracellular matrix; RGD, Arg-Gly-Asp sequence;  c RGD, cyclic pentapeptide with sequence Arg-Gly-Asp-D-Phe-N(Me)-Val; EFB, eptifibatide; PDB, Protein Data Bank; CSD, Cambridge Structural Database; MD, molecular dynamics.
List of abbreviations
ECM, extracellular matrix; RGD, Arg-Gly-Asp sequence;  c RGD, cyclic pentapeptide with sequence Arg-Gly-Asp-D-Phe-N(Me)-Val; EFB, eptifibatide; PDB, Protein Data Bank; CSD, Cambridge Structural Database; MD, molecular dynamics.cAuthors' contributions J.B. conceived the study and carried out the analysis of the structural data and molecular docking calculations. Both authors participated in the design, coordination and writing of the manuscript. Both authors read and approved the final manuscript.
Authors' contributions
J.B. conceived the study and carried out the analysis of the structural data and molecular docking calculations. Both authors participated in the design, coordination and writing of the manuscript. Both authors read and approved the final manuscript.