Max Klaas E A, Zeeb Markus, Bienert Ralf, Balbach Jochen, Heinemann Udo
Max-Delbrück-Centrum für Molekulare Medizin 13125 Berlin, Germany.
J Mol Biol. 2006 Jul 14;360(3):702-14. doi: 10.1016/j.jmb.2006.05.044. Epub 2006 Jun 2.
Bacterial cold shock proteins (CSPs) are involved in cellular adaptation to cold stress. They bind to single-stranded nucleic acids with a KD value in the micro- to nanomolar range. Here we present the structure of the Bacillus subtilis CspB (Bs-CspB) in complex with hexathymidine (dT6) at a resolution of 1.78 A. Bs-CspB binds to dT6 with nanomolar affinity via an amphipathic interface on the protein surface. Individual binding subsites interact with single nucleobases through stacking interactions and hydrogen bonding. The sugar-phosphate backbone and the methyl groups of the thymine nucleobases remain solvent exposed and are not contacted by protein groups. Fluorescence titration experiments monitoring the binding of oligopyrimidines to Bs-CspB reveal binding preferences at individual subsites and allow the design of an optimised heptapyrimidine ligand, which is bound with sub-nanomolar affinity. This study reveals the stoichiometry and sequence determinants of the binding of single-stranded nucleic acids to a preformed site on Bs-CspB and thus provides the structural basis of the RNA chaperone and transcription antitermination activities of the CSP.
细菌冷休克蛋白（CSPs）参与细胞对冷应激的适应。它们以微摩尔至纳摩尔范围内的KD值与单链核酸结合。在此，我们展示了枯草芽孢杆菌CspB（Bs-CspB）与六聚胸苷（dT6）复合物的结构，分辨率为1.78 Å。Bs-CspB通过蛋白质表面的两性界面以纳摩尔亲和力与dT6结合。各个结合亚位点通过堆积相互作用和氢键与单个核碱基相互作用。胸腺嘧啶核苷酸碱基的糖-磷酸主链和甲基仍暴露于溶剂中，且不与蛋白质基团接触。监测寡嘧啶与Bs-CspB结合的荧光滴定实验揭示了各个亚位点的结合偏好，并允许设计一种优化的七嘧啶配体，该配体以亚纳摩尔亲和力结合。这项研究揭示了单链核酸与Bs-CspB上预先形成的位点结合的化学计量和序列决定因素，从而为CSP的RNA伴侣和转录抗终止活性提供了结构基础。
