Specificity Profiling of Dual Specificity Phosphatase Vaccinia VH1-related (VHR) Reveals Two Distinct Substrate Binding Modes
Specificity Profiling of Dual Specificity Phosphatase Vaccinia VH1-related (VHR) Reveals Two Distinct Substrate Binding Modes
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DOI：
10.1074/jbc.m112.449611
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发表时间：
2013-03-01
影响因子：
4.8
通讯作者：
Pei, Dehua
中科院分区：
文献类型：
--
作者：
Luechapanichkul, Rinrada;Chen, Xianwen;Pei, Dehua
痘苗病毒VH1相关蛋白(VHR)是一种只由单一催化结构域组成的双特异性磷酸酶。虽然已经确定了VHR的几种蛋白质底物，但控制这种酶在体内的底物特异性的元件仍然不清楚。在这项工作中，通过筛选组合多肽文库，系统地描述了VHR的体外底物特异性。VHR表现出比经典的蛋白酪氨酸磷酸酶更严格的底物特异性，并识别两类不同的Tyr(P)肽。I类底物类似于来源于VHR蛋白底物的Tyr(P)基序，具有(D/E/Phi)(D/S/N/T/E)(P/I/M/S/A/V)Py(G/A/S/Q)或(D/E/Phi)(T/S)(D/E)Py(G/A/S/Q)(其中Phi是疏水氨基酸，Py是磷酸酪氨酸)的序列。II类底物具有(V/A)P(I/L/M/V/F)X(1-6)Py(其中X是任何氨基酸)的共同序列，其中V/A优选在肽的N端。定点突变和分子模拟研究表明，与I类底物的典型结合模式相比，II类肽与VHR以相反的方向结合。在这种可供选择的结合模式中，Tyr(P)侧链与活性部位口袋结合，但肽的N端与Asp(164)的羧基侧链相互作用，后者通常与I类底物的Tyr(P)+3残基相互作用。含有II类基序的蛋白质在体外是有效的VHR底物，提示VHR在体内可能作用于一类新的尚未鉴定的Tyr(P)蛋白。
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/phi)(D/S/N/T/E)(P/I/M/S/A/V) pY(G/A/S/Q) or (D/E/phi)(T/S)(D/E) pY(G/A/S/Q) (where phi 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.