CRYSTAL STRUCTURES OF B SUBTILIS SECA MUTANTS

枯草芽孢杆菌 SECA 突变体的晶体结构

• 批准号: 7726207
• 负责人: JOHN Francis HUNT
• 金额: $ 0.52万
• 依托单位: BROOKHAVEN SCIENCE ASSOC-BROOKHAVEN LAB
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-18 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7726207
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; ATP-Binding Cassette Transporters; Active Sites; Adenylyl Imidodiphosphate; Affinity; Binding; Cell membrane; Computer Retrieval of Information on Scientific Projects Database; Coupling; Data; Data Collection; Exhibits; F1-ATPase; Family; Funding; Glutamates; Glutamine; Grant; Hour; Hydrogen Bonding; Hydrolysis; Institution; Lead; Light; Mechanics; MgATP; Molecular Machines; Movement; Mutagenesis; Mutation; Nucleotides; Pattern; Photons; Process; Property; Protein translocation; Proteins; Publications; Reaction; Research; Research Personnel; Resolution; Resources; Screening procedure; Source; Structure; System; Testing; Time; United States National Institutes of Health; analog; beamline; electron density; improved; mutant; translocase

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. SecA is a bacterial ATPase involved in protein translocation through the cytoplasmic membrane. SecA uses the energy derived from cycles of ATP binding and hydrolysis to drive translocation of preproteins through the translocase SecYEG. However, the details of the mechanism of this process are unclear. A crystal structure of an ATP-bound state of SecA would provide the necessary structural information to decipher a mechanism for this mechanoenzyme. Because non-hydrolyzable analogs such as AMP-PNP have only remedial binding affinity in this system, we have employed active-site mutagenesis to obtain an ATP-bound form of SecA. SecA shares structural homology to the ABC transporter family as well as F1-ATPase, and thus may exhibit a similar mechanism in coupling ATP binding and hydrolysis to mechanical movements. Previously in ABC transporters it was observed that mutagenesis of the catalytic glutamate in the active site to glutamine rendered the protein inactive while maintaining its ability to bind ATP, thereby locking it into an ATP-bound state. Wild-type SecA from B. subtilis has an analogous glutamate in the SecA active site hypothesized to be the catalytic glutamate in the ATP hydrolysis reaction. The same mutant was made in BsSecA (E208Q) and tested for ATPase activity and binding efficiency. While reducing ATPase activity to that of background levels, the E208Q mutation also inhibits any nucleotide binding. This result led us to investigate why this mutation would create such a drastic change in nucleotide affinity. The crystal structure of BsSecA E208Q was solved at Brookhaven NSLS (X12B) to about 3.4¿¿¿ . However, the low resolution was not sufficient to decipher how and why the mutation caused a loss of nucleotide affinity. Improving the resolution of this structure would allow us to investigate changes in cooperative hydrogen bonding patterns and any stereochemical shifts involved in this phenomenon. A double mutant of BsSecA, E208Q R489K, is shown to restore nucleotide binding. Preliminary data collection of apo crystals of this double mutant diffracted to about 3.3¿¿¿ at beamlines such as X12B and X4A. These crystals were then tested at the Advanced Photon Source 24a-ID and the data were significantly improved to 3.0¿¿¿ . This resolution was sufficient to see well-defined electron density, and a similar resolution for the single mutant is needed to confidently assess the structure. Data collection of BsSecA E208Q at a beamline at the NSLS with a high flux beam such as X29 or X25 would greatly improve the crystal structure of this mutant and lead to an understanding of the SecA active site interactions. In addition, crystals of the double mutant containing MgATP were grown and are ready to be collected for diffraction. A structure of BsSecA bound to MgATP would shed light on the mechanistic properties of this molecular machine, providing a functional description of how SecA and related ATPases utilize ATP to convert nucleotide binding and hydrolysis to mechanical energy. High-resolution structures of these mutants are necessary to finalize data for publication. Six to eight hours of beamtime on X25 or X29 would provide sufficient time for minimal screening of crystals to find the best quality diffraction and also data collection of the best crystals.

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 SECA是一种细菌ATPase，参与蛋白质通过细胞膜的转运。SECA通过转位酶SecYEG利用来自于ATP结合和水解循环的能量来驱动前蛋白的易位。然而，这一过程的机制细节尚不清楚。SECA的ATP结合态的晶体结构将为破译这种机械酶的机制提供必要的结构信息。由于非水解性类似物，如AMP-PNP在该系统中只具有补救结合亲和力，我们采用活性部位诱变的方法获得了ATP结合形式的SecA。 SECA与ABC转运蛋白家族以及F1-ATPase在结构上具有同源性，因此在将ATP结合和水解与机械运动耦合方面可能表现出类似的机制。以前在ABC转运蛋白中观察到，活性部位的催化谷氨酸突变为谷氨酰胺，使蛋白质失去活性，同时保持其与ATP的结合能力，从而将其锁定在ATP结合的状态。枯草芽孢杆菌的野生型SecA在其活性部位含有类似的谷氨酸，推测其为催化ATP水解反应的谷氨酸。在BsSecA(E208Q)中获得了相同的突变体，并测定了ATPase活性和结合效率。在将ATPase活性降低到背景水平的同时，E208Q突变也抑制了任何核苷酸结合。这一结果促使我们调查为什么这种突变会导致核苷酸亲和力发生如此剧烈的变化。 BsSecA E208Q的晶体结构在Brookaven NSLS(X12B)上得到解析，约为3.4°。然而，低分辨率不足以破译这种突变是如何以及为什么导致核苷酸亲和力丧失的。提高这种结构的分辨率将使我们能够研究合作氢键模式的变化以及这一现象所涉及的任何立体化学位移。 BsSecA的一个双突变体E208Q R489K可以恢复核苷酸结合。初步收集了这个双突变体的载脂蛋白晶体的数据，在X12B和X4A等光束线上衍射率约为3.3°。这些晶体随后在先进的光子源24a-ID上进行了测试，数据显著提高到3.0。这一分辨率足以看到明确定义的电子密度，而单个突变体需要类似的分辨率才能自信地评估结构。在NSLS的光束线上用高通量束流(如X29或X25)收集BsSecA E208Q的数据将极大地改善该突变体的晶体结构，并有助于了解SecA活性中心的相互作用。此外，还生长了含有镁-三磷酸腺苷的双突变体晶体，并准备收集用于衍射。与镁三磷酸腺苷结合的BsSecA的结构将揭示这一分子机器的机械性质，为SecA和相关的ATPase如何利用ATP将核苷酸结合和水解转化为机械能提供功能描述。 这些突变体的高分辨率结构对于最终确定要发表的数据是必要的。X25或X29上6到8小时的光束时间将为最小限度地筛选晶体提供足够的时间，以找到最高质量的衍射，并收集最好的晶体的数据。