Role of Conserved Histidine and Serine in the HCXXXXXRS Motif of Human Dual-Specificity Phosphatase 5

• Published in: Journal of chemical information and modeling Vol. 59; no. 4; pp. 1563 - 1574
• Main Authors: Gupta, Ankan, Brahmbhatt, Jaladhi, Syrlybaeva, Raulia, Bodnar, Catherine, Bodnar, Natalia, Bongard, Robert, Pokkuluri, Phani Raj, Sem, Daniel S, Ramchandran, Ramani, Rathore, Rajendra, Talipov, Marat R
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 22.04.2019
• Subjects: Amino acids; Anomalies; Catalysis; Catalytic activity; Computation; Computer simulation; Enzyme activity; Glutathione; Histidine; Homeostasis; Kinases; Mathematical models; Molecular docking; Mutation; Phosphatase; Physiology; Predictions; Proteins; Residues; Substrates; Tyrosine
• ISSN: 1549-9596; 1549-960X
• DOI: 10.1021/acs.jcim.8b00919

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.