Regulation of protein-protein binding by coupling between phosphorylation and intrinsic disorder: analysis of human protein complexes

Abstract

Phosphorylation offers a dynamic way to regulate protein activity, subcellular localization, and stability. The majority of signaling pathways involve an extensive set of protein-protein interactions, and phosphorylation is widely used to regulate protein-protein binding by affecting the stability, kinetics and specificity of interactions. Previously it was found that phosphorylation sites tend to be located on protein-protein binding interfaces and may orthosterically modulate the strength of interactions. Here we studied the effect of phosphorylation on protein binding in relation to intrinsic disorder for different types of human protein complexes with known structure of the binding interface. Our results suggest that the processes of phosphorylation, binding and disorder-order transitions are coupled to each other, with about one quarter of all disordered interface Ser/Thr/Tyr sites being phosphorylated. Namely, residue site disorder and interfacial states significantly affect the phosphorylation of serine and to a lesser extent of threonine. Tyrosine phosphorylation might not be directly associated with binding through disorder, and is often observed in ordered interface regions which are not predicted to be disordered in the unbound state. We analyze possible mechanisms of how phosphorylation might regulate protein-protein binding via intrinsic disorder, and specifically focus on how phosphorylation could prevent disorder-order transitions upon binding.

Figure 1. Cluster tree showing the association between phosphorylation, disorder, interface and homo/ heterooligomeric states

The clustering was done with the Manhattan distance used as a distance measure and weighted pair-group clustering algorithm.

Figure 2. Dependence of fraction of phosphorylation sites on different factors

Fraction of phosphorylated sites on solvent accessible regions is calculated for each level of joint variable: D=1, I=1: Disordered Interface; D=1, I=0: Disordered nonInterface; D=0, I=1: Ordered Interface and D=0, I=0: Ordered nonInterface. Error bars indicate 95% confidential interval. Homo and heterooligomers are shown in red and blue, respectively.

Figure 3. Examples of phosphorylation sites on disordered interface regions

Phosphorylation site are shown in red stick models. A: p21/PCNA complex (PDBID: 1AXC, chain A, B). p21 (colored in yellow) forms an ordered conformation upon binding to PCNA (green). B: RhoGDI-Rac1 complex (PDBID: 1HH4, chain B, E). RhoGDI has a C-terminal domain (shown in cyan) and a flexible N-terminal region (yellow) which become ordered upon binding to Rac1 (pink).