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

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.

基因组测序通过提供对分子基础的前所未有的洞察而彻底改变了生物学 的生活。然而，它也提出了新的研究挑战。一项关键挑战是阐明 未表征的蛋白质家族的功能，因为任何基因组中至少有一半的蛋白质编码 缺乏可靠描述的生化功能。我们试图制定系统的方法来解决 通过将结构生物学工具与基因组学工具相结合来解决这个问题。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，在所有真核生物和几乎所有真细菌基因组中都有编码的多个代表。不 其他 ABC-F 蛋白之前已对其功能进行了详细描述，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这三种人类旁系同源物对多种 不同的疾病。此外，大约 30 个真细菌 ABC-F 旁系同源群中的几个与 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 更名为 EttA (Energy dependent Translational Throttle A)，表明该 ABC-F 家族成员是 依赖于 ADP/ATP 介导核糖体起始复合物冬眠的调节翻译因子 比率。我们确定了捕获在 ATP 结合物中的 EttA 的低温电子显微镜 (cryo-EM) 结构 与 70S 核糖体结合的状态，这证实了其前所未有的翻译控制活性 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介导 核糖体上的结合位点。这些研究以及亨特实验室之前的研究 ABC 转运蛋白超家族中同源 ATP 酶的机械化学表明，EttA 使用 检测 ADP/ATP 比率的新分子机制，这是细胞能量状态的关键监测器。这 本申请中提出的研究将利用广泛的生物化学、生物物理和结构 批判性地评估我们的假设的方法，该假设解释了这种新颖的活动，同时也描述了 由大肠杆菌表达的四种 ABC-F 旁系同源物执行的生化功能。这些研究将提供 为了解赋予抗生素的微生物 ABC-F 旁系同源物的生化功能奠定基础 抗性和三种人类 ABC-F 旁系同源物，这对于了解它们在疾病中的作用至关重要。