Journal of Molecular Biology
Regular articleThe atomic structure of protein-protein recognition sites1
Introduction
Many biological processes are carried out, or regulated, through the interactions between preformed proteins. The importance of such interactions in biology has made the protein recognition process an area of considerable interest. Here, we describe aspects of the atomic structure of the recognition sites seen in protein-protein complexes of known structure. The protein-protein complexes whose structures we analyse here are non-covalent assemblies of proteins that fold separately to carry out independent functions before they associate, as opposed to permanent macromolecular assemblies, such as oligomeric proteins, virus capsids or muscle fibres. Though the two types of complexes have features in common, they also have many that differ, and are best treated separately.
General analyses of structural aspects of protein-protein interaction have been carried out Chothia and Janin 1975, Argos 1988, Janin and Chothia 1990, Janin 1995, Janin 1996, Jones and Thornton 1995, Jones and Thornton 1996, Jones and Thornton 1997, Tsai et al 1996, Chothia 1997. Recently, however, there has been a large increase in the number of known three-dimensional structures that contain protein-protein recognition sites. These structures cover a much broader range of activities than earlier ones, which were almost exclusively protease-inhibitor and antibody-antigen complexes. They allow us to determine the extent to which rules based on the few structures that were first available, can be generalised. We also find features that were missed in previous work.
The aspects of structure with which we are concerned here are those related to the stabilization of protein association: the size and chemical character of the protein surface that is buried at interfaces; the packing density of atoms that make contacts across the interface, which expresses complementarity; and polar interactions through hydrogen bonds and interface water molecules. Each of these aspects can be described at the level of the individual atom.
From the Protein Databank (PDB; Bernstein et al., 1977), we selected 72 non-redundant files that contain X-ray structures of protein-protein complexes determined at a resolution of 3.1 Å or better. When more than one complex was present in the asymmetric unit, only one copy was retained. The atomic co-ordinates of another three complexes were given to us by their authors.
Table 1 lists the 75 structures: 24 are complexes of proteases with protein inhibitors, 19 are complexes of antibody Fab or Fv fragments with cognate protein antigens, and 32 complexes of other kinds. Among the latter, there are nine complexes between enzymes and protein inhibitors or substrates, and 11 that involve GTP/GDP-binding proteins (G-proteins) or other components of the cellular cycle or signal transduction pathways. Although nearly all these complexes are binary in the sense that they have only two protein components, a few also contain cofactors or other non-protein components.
Section snippets
The interface areas of protein-protein recognition sites
Our basic tool for measuring the extent of a protein-protein recognition site is the interface area: the area of the accessible surface on both partners that becomes inaccessible to solvent due to protein-protein contacts. This area is the sum of the solvent-accessible surface areas (ASA) of the isolated components less that of the complex.
Solvent accessible surface areas were determined here using the Lee & Richards (1971) algorithm. The computer program is based on that originally produced by
Conformation changes
For many of the complexes listed in Table 1, atomic structures are known not just for the complex but also for one or both of the free components. Comparison of the free and complexed structures can show the changes that take place upon association. With one exception, the proteases, inhibitors, antigens and antibodies that form complexes with standard size interfaces undergo only small changes in conformation. These include shifts in surface loops or movements of short segments of polypeptide
Classes of atoms at protein-protein recognition sites
In describing the atomic structure of recognition sites it is useful to distinguish between three classes of atoms (Figure 2). These are (i) interface atoms (types A, B, C): all atoms that lose solvent-accessible surface on the formation of the complex. Interface atoms on different components are within the sum of the Van der Waals radii plus the diameter of the water probe (2.8 Å). Not all interface atoms make actual Van der Waals contact across the interface. Those which do will be called:
The chemical character of the interfaces
Chemical groups at the protein surface may be allotted to one of three types: non-polar (all groups containing aliphatic and aromatic carbons); neutral polar groups (all groups containing non-carbon atoms, except those carrying a net electric charge), and charged groups. Their relative contribution to the interface area is listed in Table 6 (see page 2186). The average composition of the solvent-accessible surface of small globular proteins is 57 % non-polar, 24 % neutral polar and 19 % charged
The character of amino acids at interfaces
Table 4 describes the contribution of each of the 20 amino acid types to the interfaces and to the protein surface that remains accessible to solvent in the complexes. The two surfaces have amino acid compositions that are significantly different. Interfaces are much richer in aromatic residues His, Tyr, Phe and Trp than the average protein surface (21 % versus 8 %), and somewhat richer in aliphatic residues Leu, Ile, Val and Met (17 % versus 11 %). They are depleted in the charged residues
Polar interactions: hydrogen bonds and interface water molecules
Polar interactions at interfaces were determined using the program HBPLUS (McDonald et al., 1994) and standard geometrical parameters. The number of such bonds, Nhb, in each complex is listed in Table 1. The average interface contains ten hydrogen bonds, but Nhb can be as low as 1 between cytochrome c and cytochrome peroxidase (2pcc) or as high as 34 between Gtβγ and phosducin (2trc). Standard-size interfaces in protease-inhibitor and antibody-antigen complexes interfaces contain 9 (±5)
Atomic packing at interfaces
The volumes occupied by atoms inside proteins or buried in their interfaces can be determined by constructing Voronoi polyhedra around their atoms, and calculating their volume. The packing density at interfaces was determined by calculating the volume of the Voronoi polyhedron of each atom buried in the interface, summing the values to give a total volume V, and comparing V to a reference value Vo. To derive Vo, we used the mean volumes that the atomic groups of residues occupy in protein
Diversity of interfaces
The analysis of the atomic structure presented in the previous sections shows that recognition sites have some features that all, or almost all, share and other features that vary. To illustrate these general points at a more detailed level we describe and discuss here the recognition sites in two complexes that have standard size interfaces and two complexes with large interfaces. All four structures were determined at high resolution (2 Å or better). The recognition sites in these complexes
Discussion
Early analyses of protein-protein recognition sites were based on a small number of structures, nearly exclusively protease-inhibitor and antigen-lysozyme complexes Chothia and Janin 1975, Janin and Chothia 1990. They revealed a remarkable homogeneity in the size of the interfaces measured by the interface area, and suggested that regions of the protein surface that form recognition sites do not differ greatly in their chemical character from the rest of the solvent-accessible surface. The
Conclusion
The examples of antibody-antigen, protease-inhibitors and many other complexes show that a standard-size interface is sufficient for both stability and specificity. These standard recognition sites form stable protein-protein complexes irrespective of the size of the proteins, and no large conformational change is involved in their formation. On average, these interfaces bury 1600 Å2, have a chemical character that is close to the average protein surface, and involve nine hydrogen bonds and the
Acknowledgements
L.L. thanks IBM for an IBM Cooperative Fellowship and The Fondazione Cassa di Risparmio and the Compagnia di San Paolo of Torino for support. J.J. acknowledges financial support from EMBL-European Bioinformatics Institute (Hinxton, Cambridge, UK) and Université Paris-Sud (Orsay, France) during a sabbatical leave. We thank Dr S. Wodak for discussion, Dr N. Strynadka, S. Smerdon, M. Knossow, D. Fleury, N. Chinardet and H.K. Song for providing co-ordinates prior to deposition.
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Edited by A. R. Fersht