SOLUTION NMR STRUCTURE OF THE MAJOR COLD SHOCK PROTEIN (CSPA) FROM ESCHERICHIA-COLI - IDENTIFICATION OF A BINDING EPITOPE FOR DNA
SOLUTION NMR STRUCTURE OF THE MAJOR COLD SHOCK PROTEIN (CSPA) FROM ESCHERICHIA-COLI - IDENTIFICATION OF A BINDING EPITOPE FOR DNA
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DOI：
10.1073/pnas.91.11.5114
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发表时间：
1994-05-24
影响因子：
11.1
通讯作者：
MONTELIONE, GT
中科院分区：
文献类型：
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作者：
NEWKIRK, K;FENG, WQ;MONTELIONE, GT
利用最近开发的三维三共振核磁共振实验，确定了大肠杆菌主要冷休克蛋白（CspA）的序列特异性H-1和N-15共振配位。通过这些分配，从核磁共振数据分析中确定了五个反平行的β -链。链1-4具有经典的3-2-3 -4 - Creek关键β -sheet拓扑结构，并且在位置Lys(10)-Trp（11）和Gly(65)-Asn（66）处存在两个β -凸起。采用分子动力学模拟退火方法，利用核磁共振数据生成了CspA的三维结构。CspA的整体链褶为β -桶状结构，疏水核心排列紧密。采用二维同位素编辑脉冲场梯度N-15-H-1异核单量子相干光谱对含和不含24碱基寡脱氧核糖核苷酸5′-AACGGTTTGACGTACAGACCATTA-3′的N-15-H-1指纹图谱进行了表征。蛋白质- dna复合物的形成扰乱了大部分位于CspA分子单面的酰胺共振的一个子集。CspA分子表面的这一部分包括两个假定的rna结合序列蛾，它们构成了一个不寻常的8个表面芳香侧链簇：Trp（11）、Phe（12）、Phe（18）、Phe（20）、Phe（31）、His（33）、Phe（34）和Tyr（42）。这些表面芳香基团以及位于CspA同一面上的残基Lys（16）、Ser（44）和Lys（60）在CspA同系物家族中是高度保守的。这些同位素编辑的脉冲场梯度核磁共振数据提供了CspA上dna结合表位的低分辨率映射。
Sequence-specific H-1 and N-15 resonance assignments have been determined for the major cold shock protein (CspA) from Escherichia coli with recently developed three-dimensional triple-resonance NMR experiments. By use of these assignments, five antiparallel beta-strands were identified from analysis of NMR data. Strands 1-4 have a classical 3-2-1-4 Creek key beta-sheet topology and there are two beta-bulges, at positions Lys(10)-Trp(11) and Gly(65)-Asn(66). Three-dimensional structures of CspA were generated from NMR data by using simulated annealing with molecular dynamics. The overall chain fold of CspA is a beta-barrel structure, with a tightly packed hydrophobic core. Two-dimensional isotope-edited pulsed-field gradient N-15-H-1 heteronuclear single-quantum coherence spectroscopy was used to characterize the N-15-H-1 fingerprint spectrum with and without a 24-base oligodeoxyribonucleotide, 5'-AACGGTTTGACGTACAGACCATTA-3'. Protein-DNA complex formation perturbs a subset of the amide resonances that are located mostly on one face of the CspA molecule. This portion of the CspA molecular surface includes two putative RNA-binding sequence moths which contribute to an unusual cluster of eight surface aromatic side chains: Trp(11), Phe(12), Phe(18), Phe(20), Phe(31) His(33), Phe(34) and Tyr(42). These surface aromatic groups, and also residues Lys(16) Ser(44), and Lys(60) located on this same face of CspA, are highly conserved in the family of CspA homologues. These isotope-edited pulsed-field gradient NMR data provide a low-resolution mapping of a DNA-binding epitope on CspA.