Solution NMR structure of ribosome-binding factor A (RbfA), a cold-shock adaptation protein from Escherichia coli
Solution NMR structure of ribosome-binding factor A (RbfA), a cold-shock adaptation protein from Escherichia coli
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DOI：
10.1016/s0022-2836(03)00061-5
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发表时间：
2003-03-21
影响因子：
5.6
通讯作者：
Montelione, GT
中科院分区：
文献类型：
--
作者：
Huang, YPJ;Swapna, GVT;Montelione, GT
核糖体结合因子A（Ribosome-binding factor A，RbfA）是大肠杆菌的一种冷休克适应蛋白。它对于16 S rRNA的有效加工至关重要，并且被怀疑与16 S rRNA的5-末端螺旋（螺旋1）相互作用。RbfA是在大多数细菌生物体中发现的小蛋白大家族的成员，使其成为结构蛋白质组学的重要靶标。在这里，我们描述的三维结构的RbfADelta 25，一个2 - 108个残基的构建体，从全长RbfA的羧基末端除去25个残基，在溶液中确定在pH 5.0的异相NMR方法。使用用于确定共振归属、解释、核奥弗豪泽效应（NOE）光谱（NOESY）光谱和结构生成的大部分自动化方法进行结构测定。- RbfADelta 25具有包含三个螺旋和三个β链的α + β折叠，α 1-β 1-β 2-α 2-α 3-β 3。该结构具有II型KH-结构域折叠拓扑结构，与保守的KH序列家族蛋白相关，其β α α亚基的特征在于螺旋-转角-螺旋基序，在转角处具有序列签名GxxG。在RbfA中，该β α α亚基的特征在于螺旋-扭结-螺旋基序，其中GxxG序列被保守的AxG序列取代，包括在位置75处形成螺旋间扭结的高度保守的Ala残基。RbfADelta 25周围的静电场分布是双极的;分子的一侧是强负的，而相对面具有强正静电场。通过N-15 R-1、R-2弛豫速率和异源N-15-H-1 NOE测量，在高度保守的残基Ser 39处的β凸起附近鉴定了RbfAD elta 25的“动态热点”。分析这些分布的静电场和内部动力学，再加上进化意义的折叠和序列保守性，表明RbfA确实是一种核酸结合蛋白，并确定一个潜在的RNA结合位点或周围的保守的多肽片段Ser 76-Asp 100对应的α 3环β 3螺旋环链结构。而RbfADelta 25的结构与E. coli Era GTexpress 1的静电场分布与来自海栖热袍菌的NusA蛋白的KH 1结构域最相似，后者是另一种冷休克相关的RNA结合蛋白。RbfA和NusA在同一个E.大肠杆菌操纵子。RbfA、NusA和其他细菌11型KH结构域之间的结构和功能相似性表明这些冷休克相关蛋白之间存在先前未被怀疑的进化关系。(C)2003爱思唯尔科技有限公司版权所有。
Ribosome-binding factor A (RbfA) from Escherichia coli is a cold-shock adaptation protein. It is essential for efficient processing of 16 S rRNA and is suspected to interact with the 5-terminal helix (helix 1) of 16 S rRNA. RbfA is a member of a large family of small proteins found in most bacterial organisms, making it an important target for structural proteomics. Here, we describe the three-dimensional structure of RbfADelta25, a 2 108 residue construct with 25 residues removed from the carboxyl terminus of full-length RbfA, determined in solution at pH 5.0 by heteronuclear NMR methods. The structure determination was carried out using largely automated methods for determining resonance assignments, interpreting, nuclear Overhauser effect (NOE) spectroscopy (NOESY) spectra, and structure generation. - RbfADelta25 has an alpha + beta fold containing three helices and three beta-strands, alpha1-beta1-beta2-alpha2-alpha3-beta3. The structure has type-II KH-domain fold topology, related to conserved KH sequence family proteins whose betaalphaalphabeta subunits are characterized by a helix-turn-helix motif with sequence signature GxxG at the turn. In RbfA, this betaalphaalphabeta subunit is characterized by a helix-kink-helix motif in which the GxxG sequence is replaced by a conserved AxG sequence, including a strongly conserved, Ala residue at position 75 forming an interhelical kink. The electrostatic field distribution about RbfADelta25 is bipolar; one side of the molecule is, strongly negative and the opposite face has a strong positive electrostatic field. A "dynamic hot spot" of RbfADelta25 has been identified in the vicinity of a beta-bulge at strongly conserved residue Ser39 by N-15 R-1, R-2 relaxation rate and heteronuclear N-15-H-1 NOE measurements. Analyses of these distributions of electrostatic field and internal dynamics, together with evolutionary implications of fold and sequence conservation, suggest that RbfA is indeed a nucleic acid-binding protein, and identify a potential RNA-binding site in or around the conserved polypeptide segment Ser76-Asp100 corresponding to the alpha3-loop-beta3 helix-loop-strand structure. While the structure of RbfADelta25 is most similar to that of the KH domain of the E. coli Era GTPase, its electrostatic field distribution is most similar to the KH1 domain of the NusA protein from Thermotoga maritima, another cold-shock associated RNA-binding protein. Both RbfA and NusA are regulated in the same E. coli operon. Structural and functional similarities between RbfA, NusA, and other bacterial type 11 KH domains suggest previously unsuspected evolutionary relationships between these cold-shock associated proteins. (C) 2003 Elsevier Science Ltd. All rights reserved.