Protein Hydraulics: Water Mediated Cooperativity of Substrate Binding in PKA

Abstract

Catalytic subunits of protein kinases share a common fold centered around several precisely arranged key motifs. Multiple amino acids mutations within such conserved regions have been identified to either abrogate kinase catalytic function or switch it to permanently active state, in any case leading to severe disorders. The analysis of crystallographic structures representing diverse kinases reveals the presence of at least several water molecules whose positions within the protein core are equally well preserved as amino acid types in many functionally important locations. It remains unknown whether those water molecules play any important role, and whether their removal - disturbing local interaction patterns to no smaller degree than amino acid mutations - can affect kinase stability and function. We present the results of long computer simulations of PKA catalytic subunit targeted at the analysis of water structure, kinetics and affinity to buried hydration sites. In addition to already available structural information our simulations give insights into hydration pockets filled by disordered, highly mobile water molecules, not fully resolved in X-ray structures. We show that such regions are vital for the existence of flexible or partially disordered protein segments such as the activation and peptide binding loops in kinases. We further demonstrate that communication between the ATP and protein binding sites involves an isolated water molecule embedded between the DFG and YRD regions. Modification of its hydrogen bonding pattern induced by ATP binding affects the configuration of the DFG+1 residue. This in turn shifts the conformational equilibrium of the P+1 peptide positioning loop, promoting more frequent occurrence of peptide binding conformation. The above findings complement well NMR-based data on changes in local kinase mobility upon substrate binding, and provide mechanistic explanation for experimentally observed binding cooperativity of the two substrates.