title: Critical Role of the Secondary Binding Pocket in Modulating the Enzymatic Activity of DUSP5 toward Phosphorylated ERKs.
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.
authors.affiliation: Department of Chemistry, Marquette University , Wehr Chemistry Building, P.O. Box 1881, 535 North 14th Street, Milwaukee, Wisconsin 53201, United States.
authors.forename: Marat R
authors.initials: MR
authors.lastname: Talipov
authors.affiliation: Department of Pediatrics, Department of Obstetrics and Gynecology, Children's Research Institute (CRI) Developmental Vascular Biology Program, and Translational and Biomedical Research Center, Medical College of Wisconsin , 8701 Watertown Plank Road, P.O. Box 26509, Milwaukee, Wisconsin 53226, United States.
authors.forename: Jaladhi
authors.initials: J
authors.lastname: Nayak
authors.affiliation: Department of Pediatrics, Department of Obstetrics and Gynecology, Children's Research Institute (CRI) Developmental Vascular Biology Program, and Translational and Biomedical Research Center, Medical College of Wisconsin , 8701 Watertown Plank Road, P.O. Box 26509, Milwaukee, Wisconsin 53226, United States.
authors.forename: Michael
authors.initials: M
authors.lastname: Lepley
authors.affiliation: Center for Structure-based Drug Design and Development, Department of Pharmaceutical Sciences, Concordia University of Wisconsin , Mequon, Wisconsin 53097, United States.
authors.forename: Robert D
authors.initials: RD
authors.lastname: Bongard
authors.affiliation: Center for Structure-based Drug Design and Development, Department of Pharmaceutical Sciences, Concordia University of Wisconsin , Mequon, Wisconsin 53097, United States.
authors.forename: Daniel S
authors.initials: DS
authors.lastname: Sem
authors.affiliation: Department of Pediatrics, Department of Obstetrics and Gynecology, Children's Research Institute (CRI) Developmental Vascular Biology Program, and Translational and Biomedical Research Center, Medical College of Wisconsin , 8701 Watertown Plank Road, P.O. Box 26509, Milwaukee, Wisconsin 53226, United States.
authors.forename: Ramani
authors.initials: R
authors.lastname: Ramchandran
authors.affiliation: Department of Chemistry, Marquette University , Wehr Chemistry Building, P.O. Box 1881, 535 North 14th Street, Milwaukee, Wisconsin 53201, United States.
authors.forename: Rajendra
authors.initials: R
authors.lastname: Rathore
mesh_terms: D001665:Binding Sites; D054637:Dual-Specificity Phosphatases / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D006801:Humans; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation
chemical_list: D048049:Extracellular Signal-Regulated MAP Kinases; C442320:DUSP5 protein, human; D054637:Dual-Specificity Phosphatases