Availability of bound and unbound structures of proteins provides an opportunity to address various questions regarding structural alterations occurring due to protein-protein interactions. Our study underlines that macromolecular liganded forms of proteins undergo larger structural alterations in terms of change in local conformation (captured using PBs) as well as atomic positions (captured using RMSD) compared to unliganded proteins (Figure
). These changes are much larger than those observed due to random fluctuations characteristic of intrinsic flexibility or experimental artifacts (see Additional file 3
: Figure S1).
Non-obligatory complexes occupy a niche position as key regulators of cellular homeostasis. Their specific and timely association and dissociation are crucial for bringing about required biological function. Spatial and temporal regulation of the interacting proteins is one of the ways of avoiding unsuitable complexation [85
]. The other mechanism could be the use of different conformations of binding sites, which provide favourable or unfavourable binding-competence to the partner.
Transformation of binding site structure into the active form can serve as a switch to ensure correct binding at the appropriate time. Our analysis of structural alterations provides credence to this view. Surprisingly, pre-made interfaces, which are structurally invariant upon binding, shows distribution of %PB changes similar to that observed for induced-fit interfaces. This indicates that there is some extent of conformational change in all interfaces; only the nature and magnitude varies (Figure
B). Additionally, interface of the partner of pre-made interface is usually observed to undergo significant structural changes (Figure
A). In essence, there are no ‘completely pre-made’ interfaces in non-obligatory complexes. Crucially, significant structural changes are observed at backbone level in most of the interfaces used in this study. It is well known that side-chains undergo large structural changes upon protein-protein complexation [86
]. Considered together, these results support the view that structural conformations by themselves can serve as a good mechanism to implement the required tight regulation. Lower magnitude of structural changes is generally observed to optimize complex formation, whereas larger magnitude of structural changes is observed to remove steric clashes.
We also observe a substantial proportion of instances with significant conformational changes in non-interacting regions away from the interface (Figure
). Identification of these cases is facilitated by the ability of PBs to capture subtle structural variations. Observation of structural changes away from interface changes has been reported previously [87
]. They could be due to various factors:
1. Flexible regions are dynamic and can take up several distinct conformations, which can have specific functional relevance [45
]. Several studies have revealed that flexibility is localized to certain regions of protein structure and such dynamic sites are usually involved in both small and large molecular interaction [90
] and enzymatic catalysis. In our study, the interface regions of TolB - Pal complex (Figure
A) and Complement C3 - Epstein-Barr virus receptor C2 complex (Figure
B) are shown by normal mode analysis to be intrinsically mobile.
2. Studies show that thermodynamic entropy redistribution is a common outcome of protein-protein interaction, irrespective of the net change in entropy after complexation [92
]. Loss of entropy at interacting sites is many times accompanied by gain of entropy in other regions of surface. ‘Entropy-entropy compensation’ may be due to significant intermolecular motion between the interacting molecules, which recovers about half of the entropy lost due to rotational and translational components [92
]. This compensatory mechanism has been postulated to be the mechanism responsible for high-specificity binding of multiple ligands at the same region of a protein.
3. The region may be functionally relevant, for e.g. a ligand/macromolecule binding site, whose conformation is regulated by an allosteric mechanism. Since binding sites are observed to be a combination of flexible and rigid sites [90
], the signal based on protein-protein complexation may alter the stability and facilitate conformational change at the functionally relevant distant region. The complex of Ran GTPase with its cognate guanine nucleotide exchange factor probably utilizes this mechanism since complexation helps in altering the accessibility to the ligand on Rho protein (Figure
4. Crystallization is known to induce substantially altered conformations [44
]. In our study, we ensure that this bias is accounted for (Table
) and that the conformational changes observed are not due to such effects.
5. Trivial factors, such as missing residues near the region of interest (or) the region being near termini, could contribute to such changes [93
]. Since we ruled out complexes exhibiting such changes (Table
), the changes observed have other biological origin.
In-depth analysis of several complexes using rigorous coarse-grained NMA and literature survey indicates that a fair proportion of structural changes upon protein-protein complexation are allosteric (Figure
). Such communication is largely enriched in signalling proteins, which seems plausible considering the complex regulation of signal transduction pathways achieved using the interplay of several modular elements [12
]. The lesser frequency of occurrence of such changes in enzyme-inhibitor and antibody-antigen complexes is expected. In the case of the former, their interaction is usually the result of an allosteric modulation and in the latter, a very high-affinity complex is formed, which needs to be cleared.
The classical view of allostery is as a mechanism of effector binding causing functionally relevant conformational changes at a distant site [10
]. The salient features of the models involves two key attributes: the presence of two conformational states of the protein, one stabilised in the unbound state and the other favoured upon binding of the allosteric effector, and induction of structural change at the target site leading to functional modulation. However, studies in the last two decades have thrown new light on this phenomenon. The observations of allosteric modulation in the absence of conformational change [94
] and the introduction of allosteric perturbation in non-allosteric proteins [95
] have raised the viewpoint that all dynamic proteins are possibly allosteric [96
]. These studies indicate that proteins in their unbound states exist in several conformational sub-states, characterized by different population densities [97
]. Allosteric perturbation results in change in the relative populations of these conformers [98
]. Such studies resulted in a paradigm shift in the understanding of allostery from a structure-centric to a thermodynamics-centric phenomenon [98
]. Although newer studies on allostery indicate that change in dynamics also enables allosteric communication in many cases [94
], in this study we have confined ourselves to the study of allostery in the classical sense, as communicated by structural changes. Surprisingly, allosteric communication established only via structural changes appears to be established in almost half of the complexes upon protein binding. Consideration of dynamics along with structural changes would most probably lead to uncovering of many more protein-induced allosteric changes. Therefore, our study suggests that protein-protein binding in the case of signalling complexes, is often likely to result in downstream effects. The smaller of the two proteins in a complex, usually comprising of an unaltered interface upon protein-protein complexation, appears to be the effector molecule in most cases. The binding event generally causes changes at the interface and concomitant structural changes at the target site.
Signalling proteins are key drug targets and the usage of allosteric modulators as drugs is gaining acceptance [100
]. In such a scenario, the understanding that most protein-protein interactions in signalling proteins are allosteric provides impetus for the design of allosteric modulators as drugs. Allosteric regulators provide certain advantages over traditional drugs, which are usually competitive inhibitors. Binding of an allosteric drug at a distant site provides reduced side-effects, saturability, modulation in the presence of true agonist etc. [84
]. We hope that knowledge of possible allosterically modified sites identified in the signalling complexes studied in our analysis (see Additional file 10
: Table S3 & Additional file 11
: Table S4) serves as a starting point for combating disease manifestations.