Enmon J L, de Beer T, Overduin M
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309, USA.
Biochemistry. 2000 Apr 18;39(15):4309-19. doi: 10.1021/bi9927383.
Eps15 homology (EH) domains interact with proteins involved in endocytosis and signal transduction. EH domains bind to Asn-Pro-Phe (NPF) consensus motifs of target proteins. A few EH domains, such as the third EH domain (EH(3)) of human Eps15, prefer to bind Phe-Trp (FW) sequences. The structure of EH(3) has been solved by nuclear magnetic resonance (NMR) spectroscopy and is the first of an FW- and NPF-binding EH domain. Both FW and NPF sequences bind in the same hydrophobic pocket as shown by heteronuclear chemical shift mapping. EH(3) contains the dual EF-hand fold characteristic of the EH domain family, but it binds calcium with high affinity in the first EF-hand rather than the usual coordination in the second EF-hand. Point mutations were designed based on differences in the EH(3) and the second EH domain (EH(2)) of human Eps15 that alter the affinity of the domains for FW or NPF motif peptides. Peptides that mimic binding sites in the potential EH(3) targets Rab, synaptojanin, and the cation-dependent mannose 6-phosphate receptor were used to explore wild-type and mutant affinities. Characterization of the structure and binding properties of an FW- and NPF-binding EH domain and comparison to an NPF-specific EH domain provide important insights into the mechanisms of EH domain ligand recognition.
Eps15同源（EH）结构域与参与内吞作用和信号转导的蛋白质相互作用。EH结构域与靶蛋白的天冬酰胺-脯氨酸-苯丙氨酸（NPF）共有基序结合。一些EH结构域，如人Eps15的第三个EH结构域（EH(3)），更倾向于结合苯丙氨酸-色氨酸（FW）序列。EH(3)的结构已通过核磁共振（NMR）光谱解析，它是第一个结合FW和NPF的EH结构域。如异核化学位移图谱所示，FW和NPF序列都结合在同一个疏水口袋中。EH(3)包含EH结构域家族特有的双EF手型折叠，但它在第一个EF手中以高亲和力结合钙，而不是在第二个EF手中进行通常的配位。基于人Eps15的EH(3)和第二个EH结构域（EH(2)）的差异设计了点突变，这些差异改变了结构域对FW或NPF基序肽的亲和力。模拟潜在EH(3)靶标Rab、突触素和阳离子依赖性甘露糖6-磷酸受体中结合位点的肽被用于探究野生型和突变体的亲和力。对结合FW和NPF的EH结构域的结构和结合特性进行表征，并与NPF特异性EH结构域进行比较，为深入了解EH结构域配体识别机制提供了重要线索。
