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。 SecA是一种细菌ATP酶，参与蛋白质通过细胞质膜的转运。SecA使用来自ATP结合和水解循环的能量来驱动前蛋白通过移位酶SecYEG的移位。 然而，这一过程的机制细节尚不清楚。 SecA的ATP结合状态的晶体结构将提供必要的结构信息来解释这种机械酶的机制。 由于非水解类似物，如AMP-PNP在这个系统中只有补救性的结合亲和力，我们采用了活性位点诱变，以获得ATP结合形式的SecA。 SecA与ABC转运蛋白家族以及F1-ATP酶具有结构同源性，因此在ATP结合和水解与机械运动的偶联中可能表现出类似的机制。 以前在ABC转运蛋白中观察到，活性位点中的催化谷氨酸突变为谷氨酰胺使蛋白失活，同时保持其结合ATP的能力，从而将其锁定在ATP结合状态。 来自B的野生型SecA。枯草芽孢杆菌在SecA活性位点具有类似的谷氨酸，假设其是ATP水解反应中的催化谷氨酸。 在BsSecA（E208 Q）中制备相同的突变体，并测试ATP酶活性和结合效率。 在将ATP酶活性降低至背景水平的同时，E208 Q突变还抑制任何核苷酸结合。 这一结果使我们研究了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 E208 Q的晶体结构在Brookhaven NSLS（X12 B）解析为约3.4。 然而，低分辨率不足以解释突变如何以及为什么导致核苷酸亲和力丧失。 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和这种现象中涉及的任何立体化学位移。 显示BsSecA的双突变体E208 Q R489 K恢复核苷酸结合。 这种双突变体的载脂蛋白晶体在X12 B和X4 A等光束线处衍射至约3.3的初步数据收集。 这些晶体随后在Advanced Photon Source 24 a-ID上进行了测试，数据显著改善至3.0。 该分辨率足以看到明确的电子密度，并且需要单个突变体的类似分辨率来自信地评估结构。 在NSLS的光束线处用高通量光束如X29或X25收集BsSecA E208 Q的数据将极大地改善该突变体的晶体结构，并导致对SecA活性位点相互作用的理解。 此外，生长含有MgATP的双突变体的晶体，并准备收集用于衍射。 BsSecA结合MgATP的结构将揭示这种分子机器的机械特性，提供SecA和相关ATP酶如何利用ATP将核苷酸结合和水解转化为机械能的功能描述。 这些突变体的高分辨率结构是必要的，以最终确定数据出版。 在X25或X29上的六到八小时的光束时间将提供足够的时间对晶体进行最小的筛选，以找到最佳质量的衍射和最佳晶体的数据收集。