Mechanism of SecA-dependent protein translocation

SecA 依赖性蛋白易位机制

• 批准号: 8685679
• 负责人: DONALD B. OLIVER
• 金额: $ 37.41万
• 依托单位: WESLEYAN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8685679
• 关键词: ATP phosphohydrolase; Anti-Bacterial Agents; Antibiotics; Area; Bacteria; Bacterial Proteins; Binding; Binding Sites; Biochemical; Biological; Biological Products; Chimera organism; Communicable Diseases; Complex; Discrimination; Engineering; Escherichia coli; Evaluation; Future; Genetic; Goals; Heart; Human; In Vitro; Knowledge; Location; Maps; Membrane; Modeling; Molecular; Pathway interactions; Peptide Signal Sequences; Pharmaceutical Preparations; Protein translocation; Proteins; Research; Resistance; Series; Site; Structure; System; Testing; Virulence Factors; antimicrobial drug; base; dimer; improved; in vivo; membrane activity; monomer; novel; pathogen; protein transport; public health relevance

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to elucidate the molecular details of preprotein targeting to and translocation across biological membranes utilizing Escherichia coli as a facile genetic and biochemical system suitable for structural analysis. The project will specifically focus on a central component of this system, SecA ATPase, which binds both preproteins and the SecYEG channel complex, and whose translocation ATPase activity and membrane insertion and retraction cycle are at the heart of the protein translocation mechanism. Three specific aims are proposed. (1) To resolve the controversy surrounding the location and topology of the SecA signal peptide-binding site, a series of chimeras with E. coli SecA attached to two different signal peptides will be constructed and tested for signal peptide binding and transport function. Functionally-optimized chimeras will be re-engineered by substitution with B. subtilis or T. maritima SecA, retested and crystallized in order to determine their x- ray structures. These studies should define the molecular basis of signal peptide binding to SecA, and they should also help to reveal how the signal peptide is inserted into the SecYEG channel. (2) To reconcile the SecA membrane insertion-retraction model with recent structural studies of the SecYEG complex that limit channel size as well as to characterize the integral membrane structural state of SecA at SecYEG, an in vivo and in vitro site-specific photocrosslinking approach will be utilized to map SecY-interaction sites with this form of SecA deep within the translocon complex during normal or translocation-arrested conditions. These studies should help to define the poorly understood structural state of integral membrane SecA and allow for a re-evaluation of this dominant model. (3) To resolve the controversy as to whether SecA functions as a monomer or dimer and define any relevant dimer state at the active translocon, an in vivo and in vitro site-specific photocrosslinking approach wil be undertaken to identify and characterize SecYEG-bound SecA dimer during normal or translocation- arrested conditions. Use of photocrosslinkable "signature" residues unique to each SecA dimer interface will allow discrimination between the different SecA dimer states. These studies should clarify the oligomeric state(s) of SecA at the active translocon and allow more definitive models of SecA action to be proposed and tested. Overall, these studies will allow a better understanding of Sec-dependent protein transport at the molecular level in three critical unresolved areas of SecA structure and function, and they should be of broad significance to ultimately engineer this pathway for secretion of biopharmaceuticals and develop novel anti-bacterial agents to its conserved components in human pathogens.

描述（由申请人提供）：拟议研究的长期目标是阐明前蛋白靶向生物膜和跨生物膜易位的分子细节，利用大肠杆菌作为适合结构分析的简易遗传和生化系统。该项目将特别关注该系统的核心组成部分SecA ATP酶，它结合前蛋白和SecYEG通道复合物，其易位ATP酶活性和膜插入和收缩周期是蛋白质易位机制的核心。提出了三个具体目标。(1)为了解决围绕SecA信号肽结合位点的位置和拓扑结构的争议，我们用E。将构建连接到两种不同信号肽的coli SecA，并测试信号肽结合和转运功能。功能优化的嵌合体将通过用B取代来重新工程化。subtilis或T. maritima SecA，重新测试和结晶， 以确定它们的X射线结构。这些研究应该定义信号肽与SecA结合的分子基础，它们也应该有助于揭示信号肽如何插入SecYEG通道。(2)为了使SecA膜插入-回缩模型与SecYEG复合物的最近结构研究相一致，该结构研究限制了通道大小，并且为了表征SecYEG处SecA的整体膜结构状态，将利用体内和体外位点特异性光交联方法来映射SecY相互作用位点，其中在正常或易位停滞条件下，该形式的SecA深入易位子复合物内。这些研究应有助于确定了解不多的结构状态的整体膜SecA，并允许重新评估这一主导模式。(3)为了解决关于SecA是作为单体还是二聚体起作用的争议，并定义活性易位子处的任何相关二聚体状态，将采用体内和体外位点特异性光交联方法来鉴定和表征正常或易位停滞条件下SecYEG结合的SecA二聚体。使用对每个SecA二聚体界面独特的可光交联的“特征”残基将允许区分不同的SecA二聚体状态。这些研究应该澄清SecA在活性易位子上的寡聚状态，并允许提出和测试SecA作用的更明确的模型。总的来说，这些研究将允许在分子水平上更好地理解SecA结构和功能的三个关键未解决领域中的Sec-dependent蛋白质转运，并且它们应该对最终设计用于生物药物分泌的这一途径以及开发针对其在人类病原体中的保守组分的新型抗菌剂具有广泛意义。

项目成果

SecA functions in vivo as a discrete anti-parallel dimer to promote protein transport.

SECA在体内发挥离散的抗平行二聚体，可促进蛋白质转运。

• DOI: 10.1111/mmi.13567
• 发表时间: 2017-02
• 期刊: Molecular microbiology
• 影响因子: 3.6
• 作者: Banerjee T;Lindenthal C;Oliver D
• 通讯作者: Oliver D

Alignment of the protein substrate hairpin along the SecA two-helix finger primes protein transport in Escherichia coli.

蛋白质底物发夹沿着 SecA 双螺旋指的排列启动了大肠杆菌中的蛋白质运输。

• DOI: 10.1073/pnas.1702201114
• 发表时间: 2017
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Zhang,Qi;Lahiri,Sudipta;Banerjee,Tithi;Sun,Zhongmou;Oliver,Donald;Mukerji,Ishita
• 通讯作者: Mukerji,Ishita

Substrate Proteins Take Shape at an Improved Bacterial Translocon.

底物蛋白在改进的细菌转运中成形。

• DOI: 10.1128/jb.00618-18
• 发表时间: 2019
• 期刊: Journal of bacteriology
• 影响因子: 3.2
• 作者: Oliver,Donald