Major groove recognition by three-stranded β-sheets:: Affinity determinants and conserved structural features

被引:17
作者
Connolly, KM
Ilangovan, U
Wojciak, JM
Iwahara, M
Clubb, RT
机构
[1] Univ Calif Los Angeles, Dept Chem, Los Angeles, CA 90095 USA
[2] Univ Calif Los Angeles, DOE Lab Struct Biol & Genet, Los Angeles, CA 90095 USA
关键词
protein-DNA complex; binding affinity; mutagenesis; fluorescence spectroscopy; three-stranded beta-sheet; Tn916; transposon;
D O I
10.1006/jmbi.2000.3888
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We present the results of a rational mutagenesis and binding-affinity study of the three-stranded beta-sheet-DNA interface in the complex formed by the amino-terminal DNA-binding domain of the Tn916 integrase protein and its cognate binding site. The relative importance of interfacial contacts present in its NMR-derived solution structure have been tested through mutagenesis, fluorescence anisotropy, and intrinsic quenching DNA-binding assays. We find that seven protein-DNA hydrogen bonds (two base-specific and five to phosphate groups) significantly contribute to the level of affinity. These interactions span the entire DNA-binding surface on the protein, but primarily originate from residues in only two strands of the sheet and loop L2. Interestingly, we show that highly populated, precisely defined intermolecular hydrogen bonds in the ensemble of conformers are invariably important for DNA-binding, implying that NMR-derived solution structures provide direct insight into the energetics of recognition. Unusual three-stranded beta-sheet-DNA interfaces have recently been discovered in three unrelated protein-DNA complexes. A comparative analysis of these structures reveals similar sheet positioning, the presence of two invariant interfacial contacts to the phosphodiester backbone, and two semi-conserved base-specific hydrogen bonds. Two of these conserved contacts significantly contribute to the affinity of the integrase-DNA complex, suggesting that the three-stranded beta-sheet DNA-binding motif exhibits conserved principles of recognition. (C) 2000 Academic Press.
引用
收藏
页码:841 / 856
页数:16
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