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

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    Contribution of Active Site Residues to Substrate Hydrolysis by USP2: Insights into Catalysis by Ubiquitin Specific Proteases
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    ‡ Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024, People's Republic of China
    § Biotechnology Research Institute, National Research Council of Canada, Montréal, Québec, Canada H4P 2R2
    *Phone: (514) 496-6317. Fax: (514) 496-5143. E-mail: [email protected] (R.M.); [email protected] (Y.L.).
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    Biochemistry

    Cite this: Biochemistry 2011, 50, 21, 4775–4785
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    https://doi.org/10.1021/bi101958h
    Published May 4, 2011
    Copyright © 2011 American Chemical Society
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    Abstract

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    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.

    Copyright © 2011 American Chemical Society

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    Details of protein purification and characterization, molecular dynamics simulations, and additional structural analysis. This material is available free of charge via the Internet at http://pubs.acs.org.

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    Cited By

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    Biochemistry

    Cite this: Biochemistry 2011, 50, 21, 4775–4785
    Click to copy citationCitation copied!
    https://doi.org/10.1021/bi101958h
    Published May 4, 2011
    Copyright © 2011 American Chemical Society
    Request reuse permissions

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