Biophysical mechanisms of ABC-F proteins

ABC-F蛋白的生物物理机制

• 批准号: 9160450
• 负责人: JOHN Francis HUNT
• 金额: $ 41.85万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9160450
• 关键词: ATP phosphohydrolase; ATP-Binding Cassette Transporters; ATPase Domain; Antibiotic Resistance; Antibiotics; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Biology; Biophysical Process; Classification; Complex; Disease; Electron Microscopy; Escherichia coli; Escherichia coli Proteins; Family; Family member; Fluorescence Spectroscopy; Foundations; Genes; Genetic Translation; Genetic study; Genome; Genomics; Goals; Hibernation; Human; Kinetics; Life; Mediating; Messenger RNA; Methods; Microbial Antibiotic Resistance; Modeling; Molecular; Molecular Conformation; Monitor; Names; Peptides; Process; Protein Biosynthesis; Protein Family; Proteins; Publications; Publishing; Regulation; Research; Ribosomes; Role; Specificity; Stereotyping; Structure; Testing; Thermodynamics; Transfer RNA; Translating; Translations; Transmembrane Domain; Variant; Work; Yeasts; base; biophysical techniques; cryogenics; deep sequencing; gene function; genome sequencing; genome-wide; human disease; insight; microbial; microscopic imaging; novel; paralogous gene; particle; peptidyl-tRNA; programs; reconstruction; research study; single molecule; structural biology; tool; transcriptome sequencing; translation factor; ATP phosphohydrolase; ATP-Binding Cassette Transporters; ATPase Domain; Antibiotic Resistance; Antibiotics; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Biology; Biophysical Process; Classification; Complex; Disease; Electron Microscopy; Escherichia coli; Escherichia coli Proteins; Family; Family member; Fluorescence Spectroscopy; Foundations; Genes; Genetic Translation; Genetic study; Genome; Genomics; Goals; Hibernation; Human; Kinetics; Life; Mediating; Messenger RNA; Methods; Microbial Antibiotic Resistance; Modeling; Molecular; Molecular Conformation; Monitor; Names; Peptides; Process; Protein Biosynthesis; Protein Family; Proteins; Publications; Publishing; Regulation; Research; Ribosomes; Role; Specificity; Stereotyping; Structure; Testing; Thermodynamics; Transfer RNA; Translating; Translations; Transmembrane Domain; Variant; Work; Yeasts; base; biophysical techniques; cryogenics; deep sequencing; gene function; genome sequencing; genome-wide; human disease; insight; microbial; microscopic imaging; novel; paralogous gene; particle; peptidyl-tRNA; programs; reconstruction; research study; single molecule; structural biology; tool; transcriptome sequencing; translation factor

Genome sequencing has revolutionized biology by providing unprecedented insight into the molecular basis of life. However, it has also established new research challenges. One critical challenge is the elucidation of the function of uncharacterized protein families, because at least half of the proteins encoded in any genome lack reliably described biochemical functions. We have attempted to develop systematic approaches to tackle this problem by combining the tools of structural biology with those of genomics. Using this approach, we recently elucidated the biochemical function of one of four E. coli proteins belonging to the “ABC-F” sequence family, which has multiple representatives encoded in all eukaryotic and almost all eubacterial genomes. No other ABC-F protein had previously had its function characterized in detail, even though published studies using genome-scale profiling methods have identified the three human paralogs as contributing to a variety of different diseases. Furthermore, several of the ~30 eubacterial ABC-F paralog groups have been implicated in mediating microbial antibiotic resistance. Our recently published studies of the E. coli YjjK protein, which we renamed EttA (Energy-dependent Translational Throttle A), demonstrated that this ABC-F family member is a regulatory translation factor that mediates hibernation of ribosome initiation complexes dependent on ADP/ATP ratio. We determined a cryogenic electron microscopy (cryo-EM) structure of EttA trapped in an ATP-bound state bound to a 70S ribosome, which established that its unprecedented translational control activity is mediated by binding to a novel factor-binding site between the exit (E) and peptidyl-tRNA-binding (P) tRNA- binding sites on the ribosome. These studies, together with previous research from the Hunt lab on the mechanochemistry of homologous ATPases in the ABC Transporter superfamily, suggested that EttA uses a novel molecular mechanism to sense ADP/ATP ratio, which is a critical monitor of cellular energy status. The research proposed in this application will harness a wide variety of biochemical, biophysical, and structural methods to critically evaluate our hypothesis explaining this novel activity while also characterizing the range of biochemical functions performed by the four ABC-F paralogs expressed by E. coli. These studies will provide a foundation to understand the biochemical functions of the microbial ABC-F paralogs that confer antibiotic resistance and the three human ABC-F paralogs, which is critical for understanding their roles in disease.

基因组测序通过为分子基础提供前所未有的洞察力彻底改变了生物学 生活。但是，它也建立了新的研究挑战。一个关键挑战是阐明 未表征的蛋白质家族的功能，因为至少一半的蛋白质在任何基因组中编码 缺乏可靠描述的生化功能。我们试图开发系统的解决方法 通过将结构生物学的工具与基因组学的工具相结合，这一问题。使用这种方法，我们 最近阐明了属于“ ABC-F”序列的四种大肠杆菌蛋白之一的生化功能 家族在所有真核和几乎所有真菌基因组中都有多种代表。不 尽管已发表的研究 使用基因组规模的分析方法已经确定了三个人类旁系同源物，这有助于多种 不同的疾病。此外，在〜30个大细菌的ABC-F旁系同源组中，有几个暗示 介导微生物抗生素耐药性。我们最近发表的关于大肠杆菌YJJK蛋白的研​​究，我们 重命名为ETTA（能量依赖性的翻译节气门A），证明了ABC-F家族成员是一个 介导核糖体倡议复合物的冬眠的调节翻译因子取决于ADP/ATP 比率。我们确定了被困在ATP结合的ETTA的低温电子显微镜（Cryo-EM）结构 状态绑定到70年代的核糖体，该状态确定其前所未有的翻译控制活动是 通过与出口（E）和肽基-TRNA结合（P）tRNA-之间的新因子结合位点结合而介导 核糖体上的结合位点。这些研究，以及亨特实验室先前的研究 ABC转运蛋白超家族中同源ATPases的机械化学表明ETTA使用 了解ADP/ATP比率的新型分子机制，这是细胞能量状态的关键监视器。这 该应用中提出的研究将利用各种生化，生物物理和结构 批判性评估我们的假设的方法，解释了这一新型活动，同时还表征了 大肠杆菌表达的四个ABC-F旁系同源物执行的生化功能。这些研究将提供 了解会议抗生素的微生物ABC-F旁系同源物的生化功能的基础 抗性和三个人类ABC-F旁系同源物，这对于理解其在疾病中的作用至关重要。