STRUCTURE OF TYROSINE AND DUAL-SPECIFICITY PHOSPHATASES

描述：蛋白酪氨酸磷酸酶（PTP）， 结构相似的双特异性蛋白磷酸酶（dsPTP）， 在调节细胞功能如细胞生长中的关键作用， 细胞分裂和免疫细胞激活。 几种晶体结构具有 揭示了底物结合的催化机制和模式的细节 和抑制，但很少有人知道PTPs如何靶向特定的 基质，或者它们是如何被调节的。 这一问题的解决办法是 通过X射线晶体学确定结合到 功能相关的配体。 在未来5年内，申请人 建议继续研究dsPTP催化机制，通过解决一个 与钒酸盐络合的VHR的过渡态模拟物（Aim 1）和 使新的磷酸半胱氨酸中间体在 催化缺陷型突变体（Aim 2）。 它们还将使 具有磷酸肽和小分子底物失活VHR突变体（目的 3和4）。 晶体已获得的催化领域的LAR， PTP受体参与细胞粘附和细胞骨架重组。 将收集衍射数据并通过分子解析结构 替换（目标5）。 最近的结构表明，受体PTP在体内 可二聚化以调节PTP活性。 其次，肽底物将 与LAR结构域共结晶，以比较其与 最近解决的PTPa（目标6）。 第二个，来自LAR的无活性PTP结构域 将表达并纯化与细胞膜相关蛋白结合的 用于结晶（目标7）。 致病性耶尔森氏菌分泌一种 高活性PTP进入宿主巨噬细胞，抑制吞噬作用， 抑制有效的免疫反应 像LAR一样，这些证据表明， 耶尔森氏菌PTP中的非催化结构域将酶靶向细胞骨架 proteins. 申请人已经纯化了该氨基末端结构域， 结晶实验（目标8）。

DESCRIPTION: Protein tyrosine phosphatases (PTPs), and the structurally-similar dual-specificity protein phosphatases (dsPTP), play critical roles in the regulation of cellular functions such as cell growth, cell division, and immune cell activation. Several crystal structures have revealed details of the catalytic mechanism and mode of substrate binding and inhibition, but little is known about how PTPs target specific substrates, or how they are regulated. This question is addressed by determining structures by X-ray crystallography of PTPs bound to functionally-relevant ligands. During the next 5-year period, the applicant proposes to continue studies of the dsPTP catalytic mechanism by solving a transition state mimic of VHR complexed with vanadate (Aim 1) and visualizing the novel phosphocysteine intermediate in a catalytically-deficient mutant (Aim 2). They will also co-crystallize an inactive VHR mutant with a phosphopeptide and small molecule substrate (Aims 3 & 4). Crystals have been obtained of the catalytic domain of LAR, a receptor PTP involved in cell adhesion and cytoskeletal reorganization. Diffraction data will be collected and the structure solved by molecular replacement (Aim 5). Recent structures suggest that receptor PTPs in vivo may dimerize to regulate PTP activity. Secondly, a peptide substrate will be co-crystallized with the LAR domain to compare its specificity with the recently-solved PTPa (Aim 6). The second, inactive PTP domain from LAR which binds cytoskeletal-associated proteins, will be expressed and purified for crystallization (Aim 7). Pathogenic Yersinia bacteria secrete a highly-active PTP into host macrophages which inhibits phagocytosis and suppresses an effective immune response. Like LAR, these is evidence that a non-catalytic domain in the Yersinia PTP targets the enzyme to cytoskeletal proteins. The applicants have purified this amino-terminal domain for crystallization experiments (Aim 8).