Botuyan Maria Victoria E, Nominé Yves, Yu Xiaochun, Juranic Nenad, Macura Slobodan, Chen Junjie, Mer Georges
Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation, 200 First Street S.W., Rochester, MN 55905 USA.
Structure. 2004 Jul;12(7):1137-46. doi: 10.1016/j.str.2004.06.002.
BRCT tandem domains, found in many proteins involved in DNA damage checkpoint and DNA repair pathways, were recently shown to be phosphopeptide binding motifs. Using solution nuclear magnetic resonance (NMR) spectroscopy and mutational analysis, we have characterized the interaction of BRCA1-BRCT domains with a phosphoserine-containing peptide derived from the DNA repair helicase BACH1. We show that a phenylalanine in the +3 position from the phosphoserine of BACH1 is bound to a conserved hydrophobic pocket formed between the two BRCT domains and that recognition of the phosphate group is mediated by lysine and serine side chains from the amino-terminal BRCT domain. Mutations that prevent phosphopeptide binding abolish BRCA1 function in DNA damage-induced checkpoint control. Our NMR data also reveal a dynamic interaction between BRCA1-BRCT and BACH1, where the bound phosphopeptide exists as an equilibrium of two conformations and where BRCA1-BRCT undergoes a transition to a more rigid conformation upon peptide binding.
BRCT串联结构域存在于许多参与DNA损伤检查点和DNA修复途径的蛋白质中，最近被证明是磷酸肽结合基序。利用溶液核磁共振（NMR）光谱和突变分析，我们对BRCA1-BRCT结构域与源自DNA修复解旋酶BACH1的含磷酸丝氨酸肽的相互作用进行了表征。我们发现，BACH1磷酸丝氨酸+3位置的苯丙氨酸与两个BRCT结构域之间形成的保守疏水口袋结合，并且磷酸基团的识别由氨基末端BRCT结构域的赖氨酸和丝氨酸侧链介导。阻止磷酸肽结合的突变会消除BRCA1在DNA损伤诱导的检查点控制中的功能。我们的NMR数据还揭示了BRCA1-BRCT与BACH1之间的动态相互作用，其中结合的磷酸肽以两种构象的平衡形式存在，并且BRCA1-BRCT在肽结合后会转变为更刚性的构象。
