On the specificity of protein-protein interactions in the context of disorder

Abstract

With the increased focus on intrinsically disordered proteins (IDPs) and their large interactomes, the question about their specificity - or more so on their multispecificity - arise. Here we recapitulate how specificity and multispecificity are quantified and address through examples if IDPs in this respect differ from globular proteins. The conclusion is that quantitatively, globular proteins and IDPs are similar when it comes to specificity. However, compared with globular proteins, IDPs have larger interactome sizes, a phenomenon that is further enabled by their flexibility, repetitive binding motifs and propensity to adapt to different binding partners. For IDPs, this adaptability, interactome size and a higher degree of multivalency opens for new interaction mechanisms such as facilitated exchange through trimer formation and ultra-sensitivity via threshold effects and ensemble redistribution. IDPs and their interactions, thus, do not compromise the definition of specificity. Instead, it is the sheer size of their interactomes that complicates its calculation. More importantly, it is this size that challenges how we conceptually envision, interpret and speak about their specificity.

Keywords: intrinsically disordered proteins; multispecificity; multivalency; protein–protein interactions; specificity; structural biology.

Figure 1.. Specificity, complementarity, and valency.

(A) Specific binding as opposed to non-specific binding implicating complementary contact between ligand and receptor, as illustrated here by a π-cation interaction between a tryptophan and a lysine, and a hydrogen bond between the indole NH and a carbonyl backbone. The graph at the top illustrates that total binding to a receptor on a cell typically is the sum of specific binding to the receptor as well as non-specific, non-saturable binding e.g. to the membrane. (B) Non-specific binding in terms of lack of discrimination between any phosphorylated residue (top) or lack of complementary matching and increased dynamics in the binding site (bottom). (C) Multispecificity of the protein (in red) binding to several different binding partners (black) using the same or different sites along the chain. This is in contrast to mono-specific where only one ligand can bind the protein. (D) Multivalent interactions in the form of hetero-multivalent binding where different partners (black) binds to the protein (left) and homo-multivalent binding, where the similar ligand binding sites of the protein (in red) binds to similar partner proteins (in black, here globular proteins). In the latter case one may have homotypic interactions, e.g. IDP–IDP interactions or heterotypic between IDPs and folded domains.

Figure 2.. Part of the interactome of p53 and illustration of the role of concentration in specificity.

(A) domain structure of p53 with its TAD2 domain highlighted in red. (B) Variations in bound-state structures of the p53–TAD2 domain (in red) in complex with different folded domains (in grey) from RPA (the N-terminal DBD domain (DBD-F); PDB 2B3G [49]), TFIIH (The TFB1 domain; PDB 2GS0 [50]), CBP (the TAZ2 domain; PDB 2MZD [51]), HMGB1 (the A-box; PDB 2LY4 [52]) and p62 (The PB1 domain; PDB 2RUK [148]). The N-terminus of p53 is to the left in each figure. (C) Association constants K_a for p53 TAD2 from [49–53]. (D) Cellular concentrations taken from PaxDB [55] in two different cell lines HEK293 and HeLa (Geiger) converted from ppm to µM [58]. (E) Specificity of p53 given by the specificity factor α for different ligands of the p53–TAD2 in two different cell lines. HEK293 cells in black and HeLa cells in red.

Figure 3.. Multivalency and multispecificity is also a property of globular domains.

The PCNA trimer (in grey) is shown in the central part with three Cdt1 ligands bound (in red). The canonical PIP box motif is shown below. With a few possible exceptions, all ligands have the motif within an intrinsically disordered region and bind to the same site on PCNA forming a 3₁₀ turn and with varying structures and dynamics in the flanking regions. Here, seven crystal structures of PCNA and small ligand peptides are shown with their PIP box motif indicated below. The bound ligands and the corresponding PDB codes are: Left panel: DNA polymerase iota (2ZVM), RNase H2B (3P87), and p15 (6GWS); central panel: Cdt1 (6QCG); right panel: DNA pol delta (1U76), DNA pol eta (2ZVK), and DNA pol kappa (2ZVL).

Figure 4.. Dynamic complexes enable facilitated exchange.

Dynamics in the complex facilitated by the disordered chains allows for temporary simultaneous binding of two ligands (red and grey) to a partner, here illustrated by another IDP (in black). Once two (or more) ligands share the partner, being it through charges, ions, or a mean-field, this lowers the affinity of both ligands. In this case, the dissociation rate constants for the ligands will be concentration dependent. Depending on the life time of the trimers (or higher order oligomers) compared with the chain reconfiguration times of the IDPs, either ligand may leave the complex as indicated.