Functional coupling between the α4 - α5 loop and allosteric site on MKP5 is a critical determinant of catalysis

Dual-specificity phosphatases (DUSPs) that inactivate mitogen-activated protein kinases (MAPKs) are called MAPK phosphatases (MKPs). The stress-responsive MKP, MKP5, dephosphorylates p38 MAPK and c-Jun NH ₂ -terminal kinase (JNK). Previously, we identified an allosteric compound that binds to the site within the phosphatase domain of MKP5 that is critical for the binding and dephosphorylation of both p38 MAPK and JNK. The allosteric site, comprised of the α4-α5 loop, is an essential region for transmitting MAPK binding to the catalytic site. Here, we examine the contribution of additional structural rearrangements that occur upon allosteric site engagement. We show that binding of an inhibitor that engages the MKP5 allosteric site induces conformational changes in the α4-α5 loop. Mutants of residues in this loop inhibited enzymatic activity, and some mutants exhibited changes in dynamics, indicating that this loop has a significant structural and dynamic role in controlling MKP5 catalysis. Enzymatic and NMR studies supported the interpretation that conformational changes and dynamics in the α4-α5 loop are required for enzymatic function. Additionally, alanine mutants of R442 (α4-α5 loop), the catalytic base D377 (β4-α2 loop), and Q409A and S413A (β5-α3 loop) disrupted catalytic activity. These results highlight the α4-α5, β4-α2, and β5-α3 loops as structurally, dynamically, and functionally interconnected for communication between the allosteric and enzymatic sites.