title: Specificity profiling of dual specificity phosphatase vaccinia VH1-related (VHR) reveals two distinct substrate binding modes.
abstract: Vaccinia VH1-related (VHR) is a dual specificity phosphatase that consists of only a single catalytic domain. Although several protein substrates have been identified for VHR, the elements that control the in vivo substrate specificity of this enzyme remain unclear. In this work, the in vitro substrate specificity of VHR was systematically profiled by screening combinatorial peptide libraries. VHR exhibits more stringent substrate specificity than classical protein-tyrosine phosphatases and recognizes two distinct classes of Tyr(P) peptides. The class I substrates are similar to the Tyr(P) motifs derived from the VHR protein substrates, having sequences of (D/E/ϕ)(D/S/N/T/E)(P/I/M/S/A/V)pY(G/A/S/Q) or (D/E/ϕ)(T/S)(D/E)pY(G/A/S/Q) (where ϕ is a hydrophobic amino acid and pY is phosphotyrosine). The class II substrates have the consensus sequence of (V/A)P(I/L/M/V/F)X1-6pY (where X is any amino acid) with V/A preferably at the N terminus of the peptide. Site-directed mutagenesis and molecular modeling studies suggest that the class II peptides bind to VHR in an opposite orientation relative to the canonical binding mode of the class I substrates. In this alternative binding mode, the Tyr(P) side chain binds to the active site pocket, but the N terminus of the peptide interacts with the carboxylate side chain of Asp(164), which normally interacts with the Tyr(P) + 3 residue of a class I substrate. Proteins containing the class II motifs are efficient VHR substrates in vitro, suggesting that VHR may act on a novel class of yet unidentified Tyr(P) proteins in vivo.
authors: Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA.
authors: Rinrada
authors: R
authors: Luechapanichkul
authors: Xianwen
authors: X
authors: Chen
authors: Hashem A
authors: HA
authors: Taha
authors: Shubham
authors: S
authors: Vyas
authors: Xiaoyan
authors: X
authors: Guan
authors: Michael A
authors: MA
authors: Freitas
authors: Christopher M
authors: CM
authors: Hadad
authors: Dehua
authors: D
authors: Pei
mesh_terms: D020816:Amino Acid Motifs; D054640:Dual Specificity Phosphatase 3 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D011485:Protein Binding; D013379:Substrate Specificity / Q000502:physiology
chemical_list: D019151:Peptide Library; C517025:DUSP3 protein, human; D054640:Dual Specificity Phosphatase 3