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Role of Conserved Histidine and Serine in the HCXXXXXRS Motif of Human Dual-Specificity Phosphatase 5. J Chem Inf Model 2019 04 22;59(4):1563-1574

Date

03/06/2019

Pubmed ID

30835471

DOI

10.1021/acs.jcim.8b00919

Abstract

BACKGROUND: The mitogen-activated protein kinase (MAPK) pathway is functionally generic and critical in maintaining physiological homeostasis and normal tissue development. This pathway is under tight regulation, which is in part mediated by dual-specific phosphatases (DUSPs), which dephosphorylate serine, threonine, and tyrosine residues of the ERK family of proteins. DUSP5 is of high clinical interest because of mutations we identified in this protein in patients with vascular anomalies. Unlike other DUSPs, DUSP5 has unique specificity toward substrate pERK1/2. Using molecular docking and simulation strategies, we previously showed that DUSP5 has two pockets, which are utilized in a specific fashion to facilitate specificity toward catalysis of its substrate pERK1/2. Remarkably, most DUSPs share high similarity in their catalytic sites. Studying the catalytic domain of DUSP5 and identifying amino acid residues that are important for dephosphorylating pERK1/2 could be critical in developing small molecules for therapies targeting DUSP5.

RESULTS: In this study, we utilized computational modeling to identify and predict the importance of two conserved amino acid residues, H262 and S270, in the DUSP5 catalytic site. Modeling studies predicted that catalytic activity of DUSP5 would be altered if these critical conserved residues were mutated. We next generated independent Glutathione-S-Transferase (GST)-tagged full-length DUSP5 mutant proteins carrying specific mutations H262F and S270A in the phosphatase domain. Biochemical analysis was performed on these purified proteins, and consistent with our computational prediction, we observed altered enzyme activity kinetic profiles for both mutants with a synthetic small molecule substrate (pNPP) and the physiological relevant substrate (pERK) when compared to wild type GST-DUSP5 protein.

CONCLUSION: Our molecular modeling and biochemical studies combined demonstrate that enzymatic activity of phosphatases can be manipulated by mutating specific conserved amino acid residues in the catalytic site (phosphatase domain). This strategy could facilitate generation of small molecules that will serve as agonists/antagonists of DUSP5 activity.

Author List

Gupta A, Brahmbhatt J, Syrlybaeva R, Bodnar C, Bodnar N, Bongard R, Pokkuluri PR, Sem DS, Ramchandran R, Rathore R, Talipov MR

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

Amino Acid Motifs
Amino Acid Sequence
Catalytic Domain
Conserved Sequence
Dual-Specificity Phosphatases
Extracellular Signal-Regulated MAP Kinases
Histidine
Humans
Serine
jenkins-FCD Prod-484 8aa07fc50b7f6d102f3dda2f4c7056ff84294d1d