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Critical Role of the Secondary Binding Pocket in Modulating the Enzymatic Activity of DUSP5 toward Phosphorylated ERKs. Biochemistry 2016 Nov 08;55(44):6187-6195

Date

10/16/2016

Pubmed ID

27739308

DOI

10.1021/acs.biochem.6b00498

Abstract

DUSP5 is an inducible nuclear dual-specificity phosphatase that specifically interacts with and deactivates extracellular signal-regulated kinases ERK1 and ERK2, which are responsible for cell proliferation, differentiation, and survival. The phosphatase domain (PD) of DUSP5 has unique structural features absent from other nuclear DUSPs, such as the presence of a secondary anion-binding site in the proximity of the reaction center and a glutamic acid E264 positioned next to the catalytic cysteine C263, as well as a remote intramolecular disulfide linkage. The overall 400 ns molecular dynamics simulations indicate that the secondary binding site of DUSP5 PD acts as an allosteric regulator of the phosphatase activity of DUSP5. Our studies have identified E264 as a critical constituent of the dual binding pocket, which regulates the catalytic activity of DUSP5 by forming a salt bridge with arginine R269. Molecular dynamics studies showed that initial occupation of the secondary binding pocket leads to the breakage of the salt bridge, which then allows the occupation of the active site. Indeed, biochemical analysis using the pERK assay on mutant E264Q demonstrated that mutation of glutamic acid E264 leads to an increase in the DUSP5 catalytic activity. The role of the secondary binding site in assembling the DUSP5-pERK pre-reactive complex was further demonstrated by molecular dynamics simulations that showed that the remote C197-C219 disulfide linkage controls the structure of the secondary binding pocket based on its redox state (i.e., disulfide/dithiol) and, in turn, the enzymatic activity of DUSP5.

Author List

Talipov MR, Nayak J, Lepley M, Bongard RD, Sem DS, Ramchandran R, Rathore R

Author

Ramani Ramchandran PhD Professor in the Pediatrics department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Binding Sites
Dual-Specificity Phosphatases
Extracellular Signal-Regulated MAP Kinases
Humans
Molecular Dynamics Simulation
Mutagenesis, Site-Directed
Phosphorylation
jenkins-FCD Prod-484 8aa07fc50b7f6d102f3dda2f4c7056ff84294d1d