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.

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

“…The critical residues in the PD domain are the P loop residues in DUSP5 namely 262 HCEAGISRS 270 . E264Q mutation has been shown to increase DUSP5′s activity toward pERK in a manner similar to H262F. This data would suggest that H262 and E264 residues are acting via a similar mechanism in the DUSP5-ERK assembly and catalysis reaction.…”

“…The active site of DUSP5 includes the phosphatase signature motif HCXXXXXRS that is essential for the catalytic activity of DUSP5 and known as the catalytic P-loop. − Compared to other DUSPs, DUSP5 has a uniquely positioned glutamic acid (E264), adjacent to the enzymatically nucleophilic cysteine (or catalytic cysteine) at the 263rd amino acid position. It also possesses other unique features such as an additional substrate-binding site or secondary site, which we have hypothesized to influence the accessibility to the active site. , In our proposed “lock-and-key” mechanism, E264 forms a salt bridge (ionic interaction) with R269 residue thus preventing access to catalytic C263. On occupation of the secondary site with an anion residue (phosphate group), the ionic interaction is broken, thus facilitating entry of a second anion (phosphate group) into the catalytic pocket thus initiating catalysis by C263 nucleophilic attack.…”

“…Our laboratory has made a systematic attempt to decipher the underlying mechanism through which DUSP5 dephosphorylates pERK1/2 . The active site of DUSP5 includes the phosphatase signature motif HCXXXXXRS that is essential for the catalytic activity of DUSP5 and known as the catalytic P-loop. − Compared to other DUSPs, DUSP5 has a uniquely positioned glutamic acid (E264), adjacent to the enzymatically nucleophilic cysteine (or catalytic cysteine) at the 263rd amino acid position.…”

Discovery and characterization of halogenated xanthene inhibitors of DUSP5 as potential photodynamic therapeutics

“…Initially, sulfates had been proposed to occupy the binding pockets that are presumably occupied by the phosphate groups on the substrate, pERK 14 . For the ERK2 substrate, the pThr-Glu-pTyr tripeptide region of the ERK2 activation loop presumably occupies this region in the DUSP5 PD 7 . In general, inhibitors are expected to occupy one or both of these basic binding pockets, which are located 7.2 Å apart (Fig.…”

“…Selectivity of DUSP5 toward pERK2 is provided by the structural composition of DUSP5 that consists of two globular domains-ERK-binding domain (EBD) and phosphatase domain (PD)-connected by a ~40 amino acid-long linker, which also contributes to the selectivity toward pERK 6 . Additionally, our previous molecular dynamics simulation work 7 indicates a secondary binding site as a critical regulator of the phosphatase activity of DUSP5.…”