Work

Specificity Profiling of Dual Specificity Phosphatase Vaccinia VH1-related (VHR) Reveals Two Distinct Substrate Binding Modes*

Year: 2013

Type: article

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) X 1–6 pY (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 . Background: Factors that determine the in vivo substrate specificity of dual specificity phosphatases are currently unknown. Results: Specificity profiling of VHR through peptide library screening identified two distinct classes of peptide substrates, which bind to VHR in opposite orientations. Conclusion: VHR may act on a previously unrecognized class of protein substrates. Significance: The results should help identify new VHR substrates and elucidate its biological function.

Source: Journal of Biological Chemistry

DOI: 10.1074/jbc.m112.449611