Department of Biochemistry and Molecular Biology and Eppley Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska 68198-5870, USA.
J Biol Chem. 2010 Mar 19;285(12):8687-94. doi: 10.1074/jbc.M109.045666. Epub 2010 Jan 27.
Epidermal growth factor receptor tyrosine kinase substrate 15 (Eps15) homology (EH)-domain proteins can be divided into two classes: those with an N-terminal EH-domain(s), and the C-terminal Eps15 homology domain-containing proteins (EHDs). Whereas many N-terminal EH-domain proteins regulate internalization events, the best characterized C-terminal EHD, EHD1, regulates endocytic recycling. Because EH-domains interact with the tripeptide Asn-Pro-Phe (NPF), it is of critical importance to elucidate the molecular mechanisms that allow EHD1 and its paralogs to interact selectively with a subset of the hundreds of NPF-containing proteins expressed in mammalian cells. Here, we capitalize on our findings that C-terminal EH-domains possess highly positively charged interaction surfaces and that many NPF-containing proteins that interact with C-terminal (but not N-terminal) EH-domains are followed by acidic residues. Using the recently identified EHD1 interaction partner molecule interacting with CasL (MICAL)-Like 1 (MICAL-L1) as a model, we have demonstrated that only the first of its two NPF motifs is required for EHD1 binding. Because only this first NPF is followed by acidic residues, we have utilized glutathione S-transferase pulldowns, two-hybrid analysis, and NMR to demonstrate that the flanking acidic residues "fine tune" the binding affinity to EHD1. Indeed, our NMR solution structure of the EHD1 EH-domain in complex with the MICAL-L1 NPFEEEEED peptide indicates that the first two flanking Glu residues lie in a position favorable to form salt bridges with Lys residues within the EH-domain. Our data provide a novel explanation for the selective interaction of C-terminal EH-domains with specific NPF-containing proteins and allow for the prediction of new interaction partners with C-terminal EHDs.
表皮生长因子受体酪氨酸激酶底物 15（Eps15）同源（EH）结构域蛋白可分为两类：一类具有 N 端 EH 结构域，另一类具有 C 端 Eps15 同源结构域的蛋白（EHDs）。虽然许多 N 端 EH 结构域蛋白调节内化事件，但研究最深入的 C 端 EHD，EHD1，调节内体再循环。由于 EH 结构域与三肽 Asn-Pro-Phe（NPF）相互作用，因此阐明允许 EHD1 和其同源物与哺乳动物细胞中表达的数百种含有 NPF 的蛋白中的特定子集选择性相互作用的分子机制至关重要。在这里，我们利用我们的发现，即 C 端 EH 结构域具有高度带正电荷的相互作用表面，并且与 C 端（而非 N 端）EH 结构域相互作用的许多含有 NPF 的蛋白紧随其后是酸性残基。利用最近鉴定的 EHD1 相互作用伙伴分子 CasL（微管相关蛋白轻链激酶样 1）（MICAL-L1）作为模型，我们已经证明，只有其两个 NPF 基序中的第一个基序是与 EHD1 结合所必需的。由于只有第一个 NPF 随后是酸性残基，因此我们利用谷胱甘肽 S-转移酶 pull-downs、双杂交分析和 NMR 证明了侧翼酸性残基“微调”与 EHD1 的结合亲和力。事实上，我们的 EHD1 EH 结构域与 MICAL-L1 NPFEEEEED 肽复合物的 NMR 溶液结构表明，前两个侧翼 Glu 残基位于与 EH 结构域内的 Lys 残基形成盐桥的有利位置。我们的数据为 C 端 EH 结构域与特定含有 NPF 的蛋白的选择性相互作用提供了新的解释，并允许预测具有 C 端 EHD 的新相互作用伙伴。
