Huang Yuanpeng Janet, Swapna G V T, Rajan P K, Ke Haiping, Xia Bing, Shukla Kamal, Inouye Masayori, Montelione Gaetano T
Department of Molecular Biology and Biochemistry, Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, NJ 08854, USA.
J Mol Biol. 2003 Mar 21;327(2):521-36. doi: 10.1016/s0022-2836(03)00061-5.
Ribosome-binding factor A (RbfA) from Escherichia coli is a cold-shock adaptation protein. It is essential for efficient processing of 16S rRNA and is suspected to interact with the 5'-terminal helix (helix I) of 16S 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 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 15N R(1), R(2) relaxation rate and heteronuclear 15N-1H 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 II KH domains suggest previously unsuspected evolutionary relationships between these cold-shock associated proteins.
来自大肠杆菌的核糖体结合因子A（RbfA）是一种冷休克适应蛋白。它对于16S rRNA的高效加工至关重要，并且被怀疑与16S rRNA的5'-末端螺旋（螺旋I）相互作用。RbfA是在大多数细菌生物体中发现的一大类小蛋白家族的成员，这使其成为结构蛋白质组学的重要靶点。在此，我们描述了RbfADelta25的三维结构，这是一种从全长RbfA的羧基末端去除了25个残基的108个残基构建体，通过异核核磁共振方法在pH 5.0的溶液中测定。结构测定主要使用自动方法来确定共振归属、解释核Overhauser效应（NOE）光谱（NOESY）以及生成结构。RbfADelta25具有α+β折叠，包含三个螺旋和三条β链，即α1-β1-β2-α2-α3-β3。该结构具有II型KH结构域折叠拓扑，与保守的KH序列家族蛋白相关，其β-α-α-β亚基的特征是在转角处具有序列特征GxxG的螺旋-转角-螺旋基序。在RbfA中，这个β-α-α-β亚基的特征是螺旋-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。RbfADelta25周围的静电场分布是双极的；分子的一侧强烈带负电，而相对的面具有强正静电场。通过15N R(1)、R(2)弛豫率和异核15N-1H NOE测量，在高度保守的残基Ser39附近的β-凸起附近确定了RbfADelta25的一个“动态热点”。对这些静电场和内部动力学分布的分析，以及折叠和序列保守性的进化意义表明，RbfA确实是一种核酸结合蛋白，并在对应于α3-环-β3螺旋-环-链结构的保守多肽片段Ser76-Asp100内或其周围确定了一个潜在的RNA结合位点。虽然RbfADelta25的结构与大肠杆菌Era GTPase的KH结构域最相似，但其静电场分布与来自嗜热栖热菌的NusA蛋白的KH1结构域最相似，NusA也是一种与冷休克相关的RNA结合蛋白。RbfA和NusA在同一个大肠杆菌操纵子中受到调控。RbfA、NusA和其他细菌II型KH结构域之间的结构和功能相似性表明，这些与冷休克相关的蛋白之间存在以前未被怀疑的进化关系。
