Li Hongyuan, Byeon In Ja L, Ju Yong, Tsai Ming Daw
Department of Biochemistry and Chemistry, Campus Chemical Instrument Center, The Ohio State University, Columbus, OH 43210, USA.
J Mol Biol. 2004 Jan 2;335(1):371-81. doi: 10.1016/j.jmb.2003.10.032.
Recent studies by use of short phosphopeptides showed that forkhead-associated (FHA) domains recognize pTXX(D/I/L) motifs. Solution structures and crystal structures of several different FHA domains and their complexes with short phosphopeptides have been reported by several groups. We now report the solution structure of the FHA domain of human Ki67, a large nuclear protein associated with the cell-cycle. Using fragments of its binding partner hNIFK, we show that Ki67-hNIFK binding involves ca 44 residues without a pTXX(D/I/L) motif. The pThr site of hNIFK recognized by Ki67 FHA is pThr234-Pro235, a motif also recognized by the proline isomerase Pin1. Heteronuclear single quantum coherence (HSQC) NMR was then used to map out the binding surface, and structural analyses were used to identify key binding residues of Ki67 FHA. The results represent the first structural characterization of the complex of an FHA domain with a biologically relevant target protein fragment. Detailed analyses of the results led us to propose that three major factors control the interaction of FHA with its target protein: the pT residue, +1 to +3 residues, and an extended binding surface, and that variation in the three factors is the likely cause of the great diversity in the function and specificity of FHA domains from different sources.
最近使用短磷酸肽的研究表明，叉头相关（FHA）结构域识别pTXX（D/I/L）基序。多个研究小组已经报道了几种不同FHA结构域及其与短磷酸肽复合物的溶液结构和晶体结构。我们现在报告人Ki67的FHA结构域的溶液结构，Ki67是一种与细胞周期相关的大型核蛋白。使用其结合伴侣hNIFK的片段，我们发现Ki67-hNIFK结合涉及约44个残基，且没有pTXX（D/I/L）基序。Ki67 FHA识别的hNIFK的磷酸苏氨酸位点是pThr234-Pro235，这也是脯氨酸异构酶Pin1识别的基序。然后使用异核单量子相干（HSQC）核磁共振来绘制结合表面，并通过结构分析来确定Ki67 FHA的关键结合残基。这些结果代表了FHA结构域与具有生物学相关性的靶蛋白片段复合物的首次结构表征。对结果的详细分析使我们提出，三个主要因素控制FHA与其靶蛋白的相互作用：磷酸苏氨酸残基、+1至+3残基以及扩展的结合表面，并且这三个因素的变化可能是不同来源FHA结构域功能和特异性存在巨大差异的原因。
