Critton David A, Tortajada Antoni, Stetson Geoffrey, Peti Wolfgang, Page Rebecca
Department of Molecular Biology, Brown University, Providence, Rhode Island 02912, USA.
Biochemistry. 2008 Dec 16;47(50):13336-45. doi: 10.1021/bi801724n.
Hematopoietic tyrosine phosphatase (HePTP) is one of three members of the kinase interaction motif (KIM) phosphatase family which also includes STEP and PCPTP1. The KIM-PTPs are characterized by a 15 residue sequence, the KIM, which confers specific high-affinity binding to their only known substrates, the MAP kinases Erk and p38, an interaction which is critical for their ability to regulate processes such as T cell differentiation (HePTP) and neuronal signaling (STEP). The KIM-PTPs are also characterized by a unique set of residues in their PTP substrate binding loops, where 4 of the 13 residues are differentially conserved among the KIM-PTPs as compared to more than 30 other class I PTPs. One of these residues, T106 in HePTP, is either an aspartate or asparagine in nearly every other PTP. Using multiple techniques, we investigate the role of these KIM-PTP specific residues in order to elucidate the molecular basis of substrate recognition by HePTP. First, we used NMR spectroscopy to show that Erk2-derived peptides interact specifically with HePTP at the active site. Next, to reveal the molecular details of this interaction, we solved the high-resolution three-dimensional structures of two distinct HePTP-Erk2 peptide complexes. Strikingly, we were only able to obtain crystals of these transient complexes using a KIM-PTP specific substrate-trapping mutant, in which the KIM-PTP specific residue T106 was mutated to an aspartic acid (T106D). The introduced aspartate side chain facilitates the coordination of the bound peptides, thereby stabilizing the active dephosphorylation complex. These structures establish the essential role of HePTP T106 in restricting HePTP specificity to only those substrates which are able to interact with KIM-PTPs via the KIM (e.g., Erk2, p38). Finally, we describe how this interaction of the KIM is sufficient for overcoming the otherwise weak interaction at the active site of KIM-PTPs.
造血酪氨酸磷酸酶（HePTP）是激酶相互作用基序（KIM）磷酸酶家族的三个成员之一，该家族还包括STEP和PCPTP1。KIM磷酸酶的特征在于有一个15个残基的序列，即KIM，它赋予与它们唯一已知的底物——丝裂原活化蛋白激酶（MAP激酶）Erk和p38特异性的高亲和力结合，这种相互作用对于它们调节诸如T细胞分化（HePTP）和神经元信号传导（STEP）等过程的能力至关重要。KIM磷酸酶的另一个特征是其PTP底物结合环中有一组独特的残基，与其他30多种I类PTP相比，13个残基中的4个在KIM磷酸酶中差异保守。这些残基之一，HePTP中的T106，在几乎所有其他PTP中要么是天冬氨酸要么是天冬酰胺。我们使用多种技术研究这些KIM磷酸酶特异性残基的作用，以阐明HePTP识别底物的分子基础。首先，我们使用核磁共振光谱表明源自Erk2的肽在活性位点与HePTP特异性相互作用。接下来，为了揭示这种相互作用的分子细节，我们解析了两种不同的HePTP-Erk2肽复合物的高分辨率三维结构。令人惊讶的是，我们仅使用KIM磷酸酶特异性底物捕获突变体获得了这些瞬时复合物的晶体，其中KIM磷酸酶特异性残基T106被突变为天冬氨酸（T106D）。引入的天冬氨酸侧链促进了结合肽的配位，从而稳定了活性去磷酸化复合物。这些结构确立了HePTP T106在将HePTP特异性仅限制于那些能够通过KIM与KIM磷酸酶相互作用的底物（例如，Erk2、p38）中的重要作用。最后，我们描述了KIM的这种相互作用如何足以克服KIM磷酸酶活性位点处原本较弱的相互作用。
