STRUCTURE AND FUNCTION OF THE CLPXP ATP-DEPENDENT PROTEASE

CLPXP ATP 依赖性蛋白酶的结构和功能

• 批准号: 7721201
• 负责人: TANIA A BAKER
• 金额: $ 0.35万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-15 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7721201
• 关键词: Adaptor Signaling Protein; Amino Acids; Binding; Biochemical; Complex; Computer Retrieval of Information on Scientific Projects Database; Endopeptidases; Epitopes; Escherichia coli; Funding; Genes; Grant; Institution; Membrane Proteins; Peptide Hydrolases; Peptides; Positioning Attribute; Process; Proteins; Reporting; Research; Research Personnel; Resources; Ribosomes; Sigma Factor; Site; Source; Stretching; Structure; Techniques; United States National Institutes of Health; base; biological adaptation to stress; protein aminoacid sequence; protein function

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We had previously solved the structure of the AAA+ adaptor protein SspB in complex with one of its substrates, the ssrA tag. In E. coli, this tag targets incompletely synthesized proteins from stalled ribosomes for destruction by the ClpXP degradation machine, a process that is accelerated by SspB. Biochemical studies have revealed that other substrates are targeted to ClpXP by SspB, including the n-terminal fragment of RseA. RseA is a trans-membrane protein that functions as a master regulator of the extra-cytoplasmic stress response in E. coli. Targeting of n-terminal fragment for degradation by ClpXP is required for the release of the sigma factor (sigma E) and activation stress response genes. Using biochemical and biophysical techniques, a 31 amino acid stretch of the RseA sequence was identified as the SspB-binding determinant. In order to understand the structural basis for the SspB-RseA interaction, we crystallized the protein in complex with this 31 amino acid peptide. Energy-dependent proteases often rely on adaptor proteins to modulate substrate recognition. The SspB adaptor binds peptide sequences in the stress-response regulator RseA and in ssrA-tagged proteins and delivers these molecules to the AAA+ ClpXP protease for degradation. The structure of SspB bound to an ssrA peptide is known. Here, we reported the crystal structure of a complex between SspB and its recognition peptide in RseA in Nat. Struct. Mol. Biol. Notably, the RseA sequence is positioned in the peptide-binding groove of SspB in a direction opposite to the ssrA peptide, the two peptides share only one common interaction with the adaptor, and the RseA interaction site is substantially larger than the overlapping ssrA site. This marked diversity in SspB recognition of different target proteins indicates that it is capable of highly flexible and dynamic substrate delivery

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 我们之前已经解决了AAA+衔接蛋白SspB与其底物之一ssrA标签复合的结构。 在大肠在大肠杆菌中，该标签靶向来自停滞核糖体的不完全合成的蛋白质，以通过ClpXP降解机器进行破坏，这一过程由SspB加速。 生物化学研究表明，其他底物通过SspB靶向ClpXP，包括RseA的n-末端片段。 RseA是一种跨膜蛋白，在大肠杆菌的胞质外应激反应中起主要调节作用。杆菌ClpXP降解的N-末端片段的靶向对于σ因子（σ E）的释放和活化应激反应基因是必需的。 使用生物化学和生物物理技术，RseA序列的31个氨基酸的延伸被确定为SspB结合决定簇。 为了理解SspB-RseA相互作用的结构基础，我们将蛋白质与这31个氨基酸的肽复合结晶。 能量依赖性蛋白酶通常依赖于衔接蛋白来调节底物识别。SspB衔接子结合应激反应调节因子RseA和ssrA标记的蛋白质中的肽序列，并将这些分子递送至AAA+ ClpXP蛋白酶进行降解。与ssrA肽结合的SspB的结构是已知的。在这里，我们报道了SspB和它的识别肽在RseA在国家之间的复合物的晶体结构。值得注意的是，RseA序列以与ssrA肽相反的方向位于SspB的肽结合沟中，这两种肽仅共享一种与衔接子的共同相互作用，并且RseA相互作用位点基本上大于重叠的ssrA位点。SspB识别不同靶蛋白的显著多样性表明它能够高度灵活和动态地递送底物