Contribution of Active Site Residues to Substrate Hydrolysis by USP2: Insights into Catalysis by Ubiquitin Specific Proteases

Abstract

The ubiquitin-specific protease (USP) structural class represents the largest and most diverse family of deubiquitinating enzymes (DUBs). Many USPs assume important biological roles and emerge as potential targets for therapeutic intervention. A clear understanding of USP catalytic mechanism requires a functional evaluation of the proposed key active site residues. Crystallographic data of ubiquitin aldehyde adducts of USP catalytic cores provided structural details on the catalytic triad residues, namely the conserved Cys and His, and a variable putative third residue, and inferred indirect structural roles for two other conserved residues (Asn and Asp), in stabilizing via a bridging water molecule the oxyanion of the tetrahedral intermediate (TI). We have expressed the catalytic domain of USP2 and probed by site-directed mutagenesis the role of these active site residues in the hydrolysis of peptide and isopeptide substrates, including a synthetic K48-linked diubiquitin substrate for which a label-free, mass spectrometry based assay has been developed to monitor cleavage. Hydrolysis of ubiquitin-AMC, a model substrate, was not affected by the mutations. Molecular dynamics simulations of USP2, free and complexed with the TI of a bona fide isopeptide substrate, were carried out. We found that Asn271 is structurally poised to directly stabilize the oxyanion developed in the acylation step, while being structurally supported by the adjacent absolutely conserved Asp575. Mutagenesis data functionally confirmed this structural role independent of the nature (isopeptide vs peptide) of the bond being cleaved. We also found that Asn574, structurally located as the third member of the catalytic triad, does not fulfill this role functionally. A dual supporting role is inferred from double-point mutation and structural data for the absolutely conserved residue Asp575, in oxyanion hole formation, and in maintaining the correct alignment and protonation of His557 for catalytic competency.

A Role for Intersubunit Interactions in Maintaining SAGA Deubiquitinating Module Structure and Activity

“…The activating S144N and S149N substitutions in Ubp8 both lie near the asparagine residue, N141 that has been proposed to stabilize the oxyanion intermediate in USP isopeptidases (Hu et al, 2002; W. Zhang et al, 2011).…”

“…The enzymatic subunit, Ubp8, contains a C-terminal catalytic domain belonging to the U biquitin S pecific P rotease (USP) family of deubiquitinating enzymes (DUBs) (Reyes-Turcu and Wilkinson, 2010; Henry et al, 2003) and an N-terminal ZnF-UBP domain (Henry et al, 2003; Ingvarsdottir et al, 2005; Reyes-Turcu et al, 2006) that is required for incorporation into the SAGA complex (Ingvarsdottir et al, 2005). USP enzymes are papain-like cysteine proteases that contain a conserved Cys-His catalytic dyad as well as conserved oxyanion hole residues that are required for a conserved general mechanism of deubiquitination (Zhang et al, 2011; Komander et al, 2009; Hu et al, 2002). Although Ubp8 contains a canonical USP domain, this subunit is inactive unless it is in complex with the other three DUBm subunits (Kohler et al, 2008; Lee et al, 2009).…”

Oroxylin A Inhibits Hemolysis via Hindering the Self-Assembly of α-Hemolysin Heptameric Transmembrane Pore

“…It has been known for years, and was restated recently [23], that free energy calculations from MD simulations can prove to be a powerful tool for exploring the process of ligand-protein binding when used in combination with mutagenesis experiments [24]–[27]. In addition, MM-GBSA calculations were performed on a series of derivatives of TIBO (a substituted tetrahydroimidazole benzodiazepine thione) to explore their potential as inhibitors of HIV-1 reverse transcriptase [28].…”

Resistance of Francisella Novicida to Fosmidomycin Associated with Mutations in the Glycerol-3-Phosphate Transporte

“…Spontaneous resistance to Fos has been observed with E. coli (Kanimoto and Greenwood, 1987). Known mechanisms of resistance to Fos include overexpression of the target enzymes DXR or IspD (Zhang et al, 2011), overexpression of efflux pumps ( fsr ) to remove the drug from the bacterial cell (Messiaen et al, 2011), and mutation or down-regulation of the GlpT transporter (Fujisaki et al, 1996; Sakamoto et al, 2003; Brown and Parish, 2008; Messiaen et al, 2011). …”

“…This result also suggests that the spontaneous resistance is not due to mutations in DXR, the intracellular bacterial target of Fos, FR900098, and compound 1. It has recently been shown that Fos also targets the enzyme following DXR in the MEP pathway, 2-C-methylerythritol-4-phoshpate cytidyltransferase (or IspD) in E. coli and P. falciparum (Zhang et al, 2011). This is a promising discovery, as there is a lower frequency of resistance to antibiotics that have multiple targets (Silver, 2011).…”