A dissection of specific and non-specific protein-protein interfaces

We compare the geometric and physical-chemical properties of interfaces involved in specific and non-specific protein-protein interactions in crystal structures reported in the Protein Data Bank. Specific interactions are illustrated by 70 protein-protein complexes and by subunit contacts in 122 homodimeric proteins; non-specific interactions are illustrated by 188 pairs of monomeric proteins making crystal-packing contacts selected to bury more than 800 A2 of protein surface. A majority of these pairs have 2-fold symmetry and form "crystal dimers" that cannot be distinguished from real dimers on the basis of the interface size or symmetry. The chemical and amino acid compositions of the large crystal-packing interfaces resemble the protein solvent-accessible surface. These interfaces are less hydrophobic than in homodimers and contain much fewer fully buried atoms. We develop a residue propensity score and a hydrophobic interaction score to assess preferences seen in the chemical and amino acid compositions of the different types of interfaces, and we derive indexes to evaluate the atomic packing, which we find to be less compact at non-specific than at specific interfaces. We test the capacity of these parameters to identify homodimeric proteins in crystal structures, and show that a simple combination of the non-polar interface area and the fraction of buried interface atoms assigns the quaternary structure of 88% of the homodimers and 77% of the monomers in our data set correctly. These success rates increase to 93-95% when the residue propensity score of the interfaces is taken into consideration.     

The Subunit Interfaces of Weakly Associated Homodimeric Proteins

We analyzed subunit interfaces in 315 homodimers with an X-ray structure in the Protein Data Bank, validated by checking the literature for data that indicate that the proteins are dimeric in solution and that, in the case of the “weak” dimers, the homodimer is in equilibrium with the monomer. The interfaces of the 42 weak dimers, which are smaller by a factor of 2.4 on average than in the remainder of the set, are comparable in size with antibody-antigen or protease-inhibitor interfaces. Nevertheless, they are more hydrophobic than in the average transient protein-protein complex and similar in amino acid composition to the other homodimer interfaces. The mean numbers of interface hydrogen bonds and hydration water molecules per unit area are also similar in homodimers and transient complexes. Parameters related to the atomic packing suggest that many of the weak dimer interfaces are loosely packed, and we suggest that this contributes to their low stability. To evaluate the evolutionary selection pressure on interface residues, we calculated the Shannon entropy of homologous amino acid sequences at 60% sequence identity. In 93% of the homodimers, the interface residues are better conserved than the residues on the protein surface. The weak dimers display the same high degree of interface conservation as other homodimers, but their homologs may be heterodimers as well as homodimers. Their interfaces may be good models in terms of their size, composition, and evolutionary conservation for the labile subunit contacts that allow protein assemblies to share and exchange components, allosteric proteins to undergo quaternary structure transitions, and molecular machines to operate in the cell.     

Interaction geometry involving planar groups in protein-protein interfaces

The geometry of interactions of planar residues is nonrandom in protein tertiary structures and gives rise to conventional, as well as nonconventional (X--H...pi, X--H...O, where X = C, N, or O) hydrogen bonds. Whether a similar geometry is maintained when the interaction is across the protein-protein interface is addressed here. The relative geometries of interactions involving planar residues, and the percentage of contacts giving rise to different types of hydrogen bonds are quite similar in protein structures and the biological interfaces formed by protein chains in homodimers and protein-protein heterocomplexes--thus pointing to the similarity of chemical interactions that occurs during protein folding and binding. However, the percentage is considerably smaller in the nonspecific and nonphysiological interfaces that are formed in crystal lattices of monomeric proteins. The C--H...O interaction linking the aromatic and the peptide groups is quite common in protein structures as well as the three types of interfaces. However, as the interfaces formed by crystal contacts are depleted in aromatic residues, the weaker hydrogen bond interactions would contribute less toward their stability.     

Surface, subunit interfaces and interior of oligomeric proteins

The solvent-accessible surface area (As) of 23 oligomeric proteins is calculated using atomic co-ordinates from high-resolution and well-refined crystal structures. As is correlated with the protein molecular weight, and a power law predicts its value to within 5% on average. The accessible surface of the average oligomer is similar to that of monomeric proteins in its hydropathy and amino acid composition. The distribution of the 20 amino acid types between the protein surface and its interior is also the same as in monomers. Interfaces, i.e. surfaces involved in subunit contacts, differ from the rest of the subunit surface. They are enriched in hydrophobic side-chains, yet they contain a number of charged groups, especially from Arg residues, which are the most abundant residues at interfaces except for Leu. Buried Arg residues are involved in H-bonds between subunits. We counted H-bonds at interfaces and found that several have none, others have one H-bond per 200 A2 of interface area on average (1 A = 0.1 nm). A majority of interface H-bonds involve charged donor or acceptor groups, which should make their contribution to the free energy of dissociation significant, even when they are few. The smaller interfaces cover about 700 A2 of the subunit surface. The larger ones cover 3000 to 10,000 A2, up to 40% of the subunit surface area in catalase. The lower value corresponds to an estimate of the accessible surface area loss required for stabilizing subunit association through the hydrophobic effect alone. Oligomers with small interfaces have globular subunits with accessible surface areas similar to those of monomeric proteins. We suggest that these oligomers assemble from preformed monomers with little change in conformation. In oligomers with large interfaces, isolated subunits should be unstable given their excessively large accessible surface, and assembly is expected to require major structural changes.     

Extent and nature of contacts between protein molecules in crystal lattices and between subunits of protein oligomers

A survey was compiled of several characteristics of the intersubunit contacts in 58 oligomeric proteins, and of the intermolecular contacts in the lattice for 223 protein crystal structures. The total number of atoms in contact and the secondary structure elements involved are similar in the two types of interfaces. Crystal contact patches are frequently smaller than patches involved in oligomer interfaces. Crystal contacts result from more numerous interactions by polar residues, compared with a tendency toward nonpolar amino acids at oligomer interfaces. Arginine is the only amino acid prominent in both types of interfaces. Potentials of mean force for residue–residue contacts at both crystal and oligomer interfaces were derived from comparison of the number of observed residue–residue interactions with the number expected by mass action. They show that hydrophobic interactions at oligomer interfaces favor aromatic amino acids and methionine over aliphatic amino acids; and that crystal contacts form in such a way as to avoid inclusion of hydrophobic interactions. They also suggest that complex salt bridges with certain amino acid compositions might be important in oligomer formation. For a protein that is recalcitrant to crystallization, substitution of lysine residues with arginine or glutamine is a recommended strategy. Proteins 28:494–514, 1997. © 1997 Wiley-Liss, Inc.     

Structural characterisation and functional significance of transient protein-protein interactions

Protein-protein complexes that dissociate and associate readily, often depending on the physiological condition or environment, play an important role in many biological processes. In order to characterise these "transient" protein-protein interactions, two sets of complexes were collected and analysed. The first set consists of 16 experimentally validated "weak" transient homodimers, which are known to exist as monomers and dimers at physiological concentration, with dissociation constants in the micromolar range. A set of 23 functionally validated transient (i.e. intracellular signalling) heterodimers comprise the second set. This set includes complexes that are more stable, with nanomolar binding affinities, and require a molecular trigger to form and break the interaction. In comparison to more stable homodimeric complexes, the weak homodimers demonstrate smaller contact areas between protomers and the interfaces are more planar and polar on average. The physicochemical and geometrical properties of these weak homodimers more closely resemble those of non-obligate hetero-oligomeric complexes, whose components can exist either as monomers or as complexes in vivo. In contrast to the weak transient dimers, "strong" transient dimers often undergo large conformational changes upon association/dissociation and are characterised with larger, less planar and sometimes more hydrophobic interfaces. From sequence alignments we find that the interface residues of the weak transient homodimers are generally more conserved than surface residues, consistent with being constrained to maintain the protein-protein interaction during evolution. Protein families that include members with different oligomeric states or structures are identified, and found to exhibit a lower sequence conservation at the interface. The results are discussed in terms of the physiological function and evolution of protein-protein interactions.     

Peptide segments in protein-protein interfaces

An important component of functional genomics involves the understanding of protein association. The interfaces resulting from protein-protein interactions - (i) specific, as represented by the homodimeric quaternary structures and the complexes formed by two independently occurring protein components, and (ii) non-specific, as observed in the crystal lattice of monomeric proteins - have been analysed on the basis of the length and the number of peptide segments. In 1000 A2 of the interface area, contributed by a polypeptide chain, there would be 3.4 segments in homodimers, 5.6 in complexes and 6.3 in crystal contacts. Concomitantly, the segments are the longest (with 8.7 interface residues) in homodimers. Core segments (likely to contribute more towards binding) are more in number in homodimers (1.7) than in crystal contacts (0.5), and this number can be used as one of the parameters to distinguish between the two types of interfaces. Dominant segments involved in specific interactions, along with their secondary structural features, are enumerated.     

Effect of N-terminal residues on the structural stability of recombinant horse L-chain apoferritin in an acidic environment

The denaturation of recombinant horse L-chain apoferritin (rLF), which is composed of 24 L-chain subunits, in acidic solution was studied. Using two rLF mutants, lacking four (Fer4) or eight (Fer8) N-terminal amino acid residues, the effect of N-terminal residues on the protein's stability was investigated. Of the two mutants and wild-type rLF, the tertiary and secondary structures of Fer8 were found to be most sensitive to an acidic environment. The Fer8 protein dissociated easily into subunit dimers at or below pH 2.0. Comparing the crystal structures of the mutant proteins, deletion of the N-terminal residues was found to result in fewer inter- and intra-subunit hydrogen bonds. The loss of these bonds is assumed to be responsible for lower endurance against acidic denaturation in N-terminus-deleted mutants. These results indicated that the inter- and intra-subunit hydrogen bonds of N-terminal residues affect the denaturation, especially oligomer formation of apoferritin subunits and will be of use in designing ferritin-based nanodevices.     

A dissection of the protein-protein interfaces in icosahedral virus capsids

We selected 49 icosahedral virus capsids whose crystal structures are reported in the Protein Data Bank. They belong to the T=1, T=3, pseudo T=3 and other lattice types. We identified in them 779 unique interfaces between pairs of subunits, all repeated by icosahedral symmetry. We analyzed the geometric and physical chemical properties of these interfaces and compared with interfaces in protein-protein complexes and homodimeric proteins, and with crystal packing contacts. The capsids contain one to 16 subunits implicated in three to 66 unique interfaces. Each subunit loses 40-60% of its accessible surface in contacts with an average of 8.5 neighbors. Many of the interfaces are very large with a buried surface area (BSA) that can exceed 10,000 A(2), yet 39% are small with a BSA<800 A(2) comparable to crystal packing contacts. Pairwise capsid interfaces overlap, so that one-third of the residues are part of more than one interface. Those with a BSA>800 A(2) resemble homodimer interfaces in their chemical composition. Relative to the protein surface, they are non-polar, enriched in aliphatic residues and depleted of charged residues, but not of neutral polar residues. They contain one H-bond per about 200 A(2) BSA. Small capsid interfaces (BSA<800 A(2)) are only slightly more polar. They have a similar amino acid composition, but they bury fewer atoms and contain fewer H-bonds for their size. Geometric parameters that estimate the quality of the atomic packing suggest that the small capsid interfaces are loosely packed like crystal packing contacts, whereas the larger interfaces are close-packed as in protein-protein complexes and homodimers. We discuss implications of these findings on the mechanism of capsid assembly, assuming that the larger interfaces form first to yield stable oligomeric species (capsomeres), and that medium-size interfaces allow the stepwise addition of capsomeres to build larger intermediates.     

Distribution of amino acid residues and residue-residue contacts in molecular chaperones.

The amino acid distribution and residue-residue contacts in molecular chaperones are different when compared to normal globular proteins. The study of molecular chaperones reveals a different surrounding environment to exist for the residues Cys, Trp, and His which may play an important role in determining the chaperone structures. Unlike globular proteins, it has been observed that a one-to-one correspondence between the amino acid distribution in a sequence and the structures of molecular chaperones. The preference of amino acid residues surrounding all 20 types of residues in secondary structures and their accessible surface areas have been analysed.     

Dissecting subunit interfaces in homodimeric proteins

The subunit interfaces of 122 homodimers of known three-dimensional structure are analyzed and dissected into sets of surface patches by clustering atoms at the interface; 70 interfaces are single-patch, the others have up to six patches, often contributed by different structural domains. The average interface buries 1,940 A2 of the surface of each monomer, contains one or two patches burying 600-1,600 A2, is 65% nonpolar and includes 18 hydrogen bonds. However, the range of size and of hydrophobicity is wide among the 122 interfaces. Each interface has a core made of residues with atoms buried in the dimer, surrounded by a rim of residues with atoms that remain accessible to solvent. The core, which constitutes 77% of the interface on average, has an amino acid composition that resembles the protein interior except for the presence of arginine residues, whereas the rim is more like the protein surface. These properties of the interfaces in homodimers, which are permanent assemblies, are compared to those of protein-protein complexes where the components associate after they have independently folded. On average, subunit interfaces in homodimers are twice larger than in complexes, and much less polar due to the large fraction belonging to the core, although the amino acid compositions of the cores are similar in the two types of interfaces.     

Extent of structural asymmetry in homodimeric proteins: prevalence and relevance

Most homodimeric proteins have symmetric structure. Although symmetry is known to confer structural and functional advantage, asymmetric organization is also observed. Using a non-redundant dataset of 223 high-resolution crystal structures of biologically relevant homodimers, we address questions on the prevalence and significance of asymmetry. We used two measures to quantify global and interface asymmetry, and assess the correlation of several molecular and structural parameters with asymmetry. We have identified rare cases (11/223) of biologically relevant homodimers with pronounced global asymmetry. Asymmetry serves as a means to bring about 2:1 binding between the homodimer and another molecule; it also enables cellular signalling arising from asymmetric macromolecular ligands such as DNA. Analysis of these cases reveals two possible mechanisms by which possible infinite array formation is prevented. In case of homodimers associating via non-topologically equivalent surfaces in their tertiary structures, ligand-dependent mechanisms are used. For stable dimers binding via large surfaces, ligand-dependent structural change regulates polymerisation/depolymerisation; for unstable dimers binding via smaller surfaces that are not evolutionarily well conserved, dimerisation occurs only in the presence of the ligand. In case of homodimers associating via interaction surfaces with parts of the surfaces topologically equivalent in the tertiary structures, steric hindrance serves as the preventive mechanism of infinite array. We also find that homodimers exhibiting grossly symmetric organization rarely exhibit either perfect local symmetry or high local asymmetry. Binding of small ligands at the interface does not cause any significant variation in interface asymmetry. However, identification of biologically relevant interface asymmetry in grossly symmetric homodimers is confounded by the presence of similar small magnitude changes caused due to artefacts of crystallisation. Our study provides new insights regarding accommodation of asymmetry in homodimers.     

Evolution in the structure and function of carboxyl proteases

Summary A model for the structure and function of extracellular carboxyl (acid) proteases can be established from three amino acid sequences and four crystal structures of these enzymes. The carboxyl proteases from gastric and fungal origins are very homologous in both primary and tertiary structures. The molecules consist of about 320 residues organized with a secondary structure which is primarily comprised of -strands and very similar tertiary structures. An apparent binding cleft, which can accommodate a substrate with about eight amino acid residues, contains near its midpoint the active center residues Asp-215, Asp-32, and Ser-35. These three residues are hydrogen bonded to each other.An intracellular carboxyl protease, cathepsin D, is very homologous to the extracellular enzymes in N-terminal amino acid sequence and primary structure location of active center residues. The tertiary structure of cathepsin D is probably similar, as well. However, cathepsin D contains a unique hydrophobic tail made up of about 100 residues added on the C-terminal side. Cathepsin D precursor is over 100,000 daltons in molecular weights, as contrasted to the gastric carboxyl protease zymogens, which are about 40,000 daltons.     

Crystal Structures of α-Crystallin Domain Dimers of αB-Crystallin and Hsp20

Small heat shock proteins (sHsps) are a family of large and dynamic oligomers highly expressed in long-lived cells of muscle, lens and brain. Several family members are upregulated during stress, and some are strongly cytoprotective. Their polydispersity has hindered high-resolution structure analyses, particularly for vertebrate sHsps. Here, crystal structures of excised α-crystallin domain from rat Hsp20 and that from human αB-crystallin show that they form homodimers with a shared groove at the interface by extending a β sheet. However, the two dimers differ in the register of their interfaces. The dimers have empty pockets that in large assemblies will likely be filled by hydrophobic sequence motifs from partner chains. In the Hsp20 dimer, the shared groove is partially filled by peptide in polyproline II conformation. Structural homology with other sHsp crystal structures indicates that in full-length chains the groove is likely filled by an N-terminal extension. Inside the groove is a symmetry-related functionally important arginine that is mutated, or its equivalent, in family members in a range of neuromuscular diseases and cataract. Analyses of residues within the groove of the αB-crystallin interface show that it has a high density of positive charges. The disease mutant R120G α-crystallin domain dimer was found to be more stable at acidic pH, suggesting that the mutation affects the normal dynamics of sHsp assembly. The structures provide a starting point for modelling higher assembly by defining the spatial locations of grooves and pockets in a basic dimeric assembly unit. The structures provide a high-resolution view of a candidate functional state of an sHsp that could bind non-native client proteins or specific components from cytoprotective pathways. The empty pockets and groove provide a starting model for designing drugs to inhibit those sHsps that have a negative effect on cancer treatment.     

Structural role of the tyrosine residues of cytochrome c.

The tertiary structures of horse, tuna, Neurospora crassa, horse [Hse65,Leu67]- and horse [Hse65,Leu74]-cytochromes c were studied with high-resolution 1H n.m.r. spectroscopy. The amino acid sequences of these proteins differ at position 46, which is occupied by phenylalanine in the horse proteins but by tyrosine in the remaining two, and at positions 67, 74 and 97, which are all occupied by tyrosine residues in horse and tuna cytochrome c but in the other proteins are substituted by phenylalanine or leucine, though there is only one such substitution per protein. The various aromatic-amino-acid substitutions do not seriously affect the protein structure.     

Members of TALE and WUS subfamilies of homeodomain proteins with potentially important functions in development form dimers within each subfamily in rice

Transacting factors often form homo- and heterodimers and regulate various targets, the type of regulation depending on the dimeric combination. The WUS and TALE subfamilies are two atypical homeodomains in plants. A homeodomain mediates sequence-specific binding to its target DNA and usually consists of 60 amino acid residues, whereas atypical homeodomains have extra amino acid residues in the well-conserved region. The genes OsWUS and OsPRS, which encode atypical homeodomain proteins from the WUS subfamily, and OsBEL and OSH15, which encode those from the TALE subfamily, were isolated from rice and tested for their interactions by yeast two-hybrid analysis. OsWUS and OsPRS formed homodimers and formed heterodimers with each other but did not form dimers with the TALE family homeodomain proteins OSH15 or OsBEL. Likewise, OSH15 and OsBEL formed homodimers and heterodimers but did not form dimers with the WUS family homeodomain proteins OsWUS and OsPRS. These findings suggest that the combinations of dimers are well correlated with the classification of these proteins on the basis of sequence similarity. RT-PCR analysis revealed that expression of OsWUS and OsPRS was detected in the same organs, namely floral buds, roots, and suspension cells. Therefore, it is possible that the proteins encoded by both of these genes function as homo- and heterodimers in planta. These results suggest that, during the evolution of these subfamilies, various combinations of dimers within proteins encoded by paralogous genes were formed and generated independent regulatory networks that enabled complex patterns of plant development.     

Crystal structure of tobacco necrosis virus at 2.25 A resolution

The crystal structure of tobacco necrosis virus (TNV) has been determined by real-space averaging with 5-fold non-crystallographic symmetry, and refined to R=25.3 % for diffraction data to 2.25 A resolution. A total of 180 subunits form a T=3 virus shell with a diameter of about 280 A and a small protrusion at the 5-fold axis. In 276 amino acid residues, the respective amino terminal 86, 87 and 56 residues of the A, B and C subunits are disordered. No density for the RNA was found. The subunits have a "jelly roll" beta-barrel structure, as have the structures of the subunits of other spherical viruses. The tertiary and quaternary structures of TNV are, in particular, similar to those of southern bean mosaic virus, although they are classified in different groups. Invisible residues 1 to 56 with a high level of basic residues are considered to be located inside the particle. Sequence comparison of the coat proteins of several TNV strains showed that the sequences of the disordered segment diverge considerably as compared with those of the ordered segment, consistent with a small tertiary structural constraint being imposed on the N-terminal segment. Basic residues are localized on the subunit interfaces or inner surface of the capsid. Positive charges of the basic residues facing the interior, as well as those of the N-terminal segment, may neutralize the negative charge of the RNA inside. Five calcium ions per icosahedral asymmetric unit are located at the subunit interfaces; three are close to the exterior surface, the other two away from it. The environments of the first three are similar, and those of the other two sites are similar. These calcium ions are assumed to be responsible for the stabilization/transition of the quaternary structure of the shell. Three peptide segments ordered only in the C subunits are clustered around each 3-fold (quasi-6-fold) axis forming a beta-annulus, and may lead to quasi-equivalent interactions for the organization of the T=3 shell.     

Structure-function relationship of [2Fe2S] ferredoxins and design of a model molecule

[2Fe2S] ferredoxins isolated from various plants and algae comprise 93–99 amino acid residues and resemble each other not only in sequences, but also in physiological functions. One of them isolated from Spirulina platensis was subjected to X-ray analysis and its three dimensional structure is now known. [2Fe2S] ferredoxins of a different type are found in halobacteria and comprise 128 amino acid residues. Both types of the [2Fe2S] ferredoxins exhibit low redox potentials. By comparing the amino acid sequences of 28 [2Fe2S] ferredoxins and the tertiary structure of S. platensis ferredoxin we predicted a common three-dimensional structure to the [2Fe2S] ferredoxins and proposed a molecular surface area to be interacting with FNR. An artificial small molecule composed of 20 amino acid residues is designed on the basis of the tertiary structure of S. platensis ferredoxin. The amino acid sequence was predicted to be ProTyrSerCysArgAlaGlyAlaCysSerThrCysAlaGly ProLeuLeuThr CysVal which should have a [2Fe2S] cluster with a low redox potential     

Oligomeric protein structure networks: insights into protein-protein interactions

Background  

Protein-protein association is essential for a variety of cellular processes and hence a large number of investigations are being carried out to understand the principles of protein-protein interactions. In this study, oligomeric protein structures are viewed from a network perspective to obtain new insights into protein association. Structure graphs of proteins have been constructed from a non-redundant set of protein oligomer crystal structures by considering amino acid residues as nodes and the edges are based on the strength of the non-covalent interactions between the residues. The analysis of such networks has been carried out in terms of amino acid clusters and hubs (highly connected residues) with special emphasis to protein interfaces.     

Identification of amino acids involved in protein structural uniqueness: implication for de novo protein design

Structural uniqueness is characteristic of native proteins and is essential to express their biological functions. The major factors that bring about the uniqueness are specific interactions between hydrophobic residues and their unique packing in the protein core. To find the origin of the uniqueness in their amino acid sequences, we analyzed the distribution of the side chain rotational isomers (rotamers) of hydrophobic amino acids in protein tertiary structures and derived deltaS(contact), the conformational-entropy changes of side chains by residue-residue contacts in each secondary structure. The deltaS(contact) values indicate distinct tendencies of the residue pairs to restrict side chain conformation by inter-residue contacts. Of the hydrophobic residues in alpha-helices, aliphatic residues (Leu, Val, Ile) strongly restrict the side chain conformations of each other. In beta-sheets, Met is most strongly restricted by contact with Ile, whereas Leu, Val and Ile are less affected by other residues in contact than those in alpha-helices. In designed and native protein variants, deltaS(contact) was found to correlate with the folding-unfolding cooperativity. Thus, it can be used as a specificity parameter for designing artificial proteins with a unique structure.