Molecular basis for substrate discrimination by transporter protein MexY of the MexXY-OprM efflux pump in Pseudomonas aeruginosa

铜绿假单胞菌中 MexXY-OprM 外排泵转运蛋白 MexY 区分底物的分子基础

• 批准号: 10655590
• 负责人: Logan Kavanaugh
• 金额: $ 4.77万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10655590
• 关键词: Adaptor Signaling Protein; Affinity; Aminoglycoside resistance; Aminoglycosides; Antibiotic Resistance; Antibiotics; Bacteria; Binding; Biological Assay; Calorimetry; Carrier Proteins; Cells; Centers for Disease Control and Prevention (U.S.); Charge; Childbirth; Clinical; Complement; Complex; Computer Models; Coupled; Cryoelectron Microscopy; Dangerousness; Development; Discrimination; Distal; Drug Efflux; Escherichia coli; Family; Health; In Vitro; Infection; Knock-out; Lactams; Lead; Membrane; Membrane Proteins; Membrane Transport Proteins; Minimum Inhibitory Concentration measurement; Modeling; Molecular; Mutagenesis; Operative Surgical Procedures; Pathway interactions; Pharmaceutical Preparations; Pneumonia; Procedures; Process; Property; Proteins; Pseudomonas aeruginosa; Public Health; Publishing; Pump; Reporting; Research; Research Personnel; Resistance; Resolution; Sepsis; Shapes; Site-Directed Mutagenesis; Structure; Substrate Specificity; System; Testing; Therapeutic; Titrations; Training; Variant; Vestibule; X-Ray Crystallography; antibiotic efflux; beta-Lactams; cancer care; career; combat; design; efflux pump; in silico; in vivo; inhibitor; insight; mutant; novel; novel strategies; pathogen; pathogenic bacteria; periplasm; preference; protein folding; resistance mechanism; skills; uptake; Adaptor Signaling Protein; Affinity; Aminoglycoside resistance; Aminoglycosides; Antibiotic Resistance; Antibiotics; Bacteria; Binding; Biological Assay; Calorimetry; Carrier Proteins; Cells; Centers for Disease Control and Prevention (U.S.); Charge; Childbirth; Clinical; Complement; Complex; Computer Models; Coupled; Cryoelectron Microscopy; Dangerousness; Development; Discrimination; Distal; Drug Efflux; Escherichia coli; Family; Health; In Vitro; Infection; Knock-out; Lactams; Lead; Membrane; Membrane Proteins; Membrane Transport Proteins; Minimum Inhibitory Concentration measurement; Modeling; Molecular; Mutagenesis; Operative Surgical Procedures; Pathway interactions; Pharmaceutical Preparations; Pneumonia; Procedures; Process; Property; Proteins; Pseudomonas aeruginosa; Public Health; Publishing; Pump; Reporting; Research; Research Personnel; Resistance; Resolution; Sepsis; Shapes; Site-Directed Mutagenesis; Structure; Substrate Specificity; System; Testing; Therapeutic; Titrations; Training; Variant; Vestibule; X-Ray Crystallography; antibiotic efflux; beta-Lactams; cancer care; career; combat; design; efflux pump; in silico; in vivo; inhibitor; insight; mutant; novel; novel strategies; pathogen; pathogenic bacteria; periplasm; preference; protein folding; resistance mechanism; skills; uptake

PROJECT SUMMARY/ ABSTRACT Antibiotic resistance is a major global public health threat, with approximately three million resistant infections reported each year in the US alone. Many distinct mechanisms of antibiotic resistance have been observed in bacteria, but pervasive among Gram-negative pathogens is the ability to actively efflux drugs out of the cell. Efflux pumps in the Resistance-Nodulation-Division (RND) family contribute extensively to intrinsic, clinical antibiotic resistance. RND pumps are tripartite complexes composed of an inner membrane transporter protein, a periplasmic adaptor protein, and an outer membrane factor protein. Many Gram-negative pathogens encode multiple RND systems; for example, the serious threat pathogen Pseudomonas aeruginosa contains four RND efflux systems that contribute to antibiotic resistance. Here, two of these pumps, MexAB-OprM and MexXY- OprM, will serve as an ideal model to define the basis of substrate selectivity due to their overlapping but distinct preferences for b-lactams and aminoglycosides, respectively. Although preferred substrates are known, the molecular determinants behind substrate recognition are not currently understood. Guided by the published structure of MexAB-OprM, our lab generated a model for MexXY-OprM. Using this structural framework for comparison of the transporter proteins MexB and MexY, specific regions and residues within them were identified that could underpin substrate selectivity. In particular, the distal binding pocket (DBP) is predicted to be critical for substrate selection and translocation within the transporter protein. Based on these findings, I hypothesize that critical residues within the distal binding pocket (DBP) of MexY define its physicochemical properties (shape, charge, distribution, and volume) that control aminoglycoside substrate recognition and translocation. In this project, I will test this hypothesis and elucidate the molecular basis of substrate selectivity and translocation through the transporter MexY of the P. aeruginosa RND efflux pump MexXY-OprM. In Aim 1, I will determine the preferred aminoglycoside entry channel(s) from the cell periplasm into the transporter MexY using mutagenesis coupled with in vivo functional assays in both lab and pan-aminoglycoside resistant clinical isolates and high-resolution cryogenic electron microscopy structural studies. In Aim 2, I will define the residues within the DBP of MexY that control selectivity for substrates over non-substrates (e.g. aminoglycosides over b-lactams) by using in vitro binding affinity and high-resolution X-ray crystallographic structural studies, complemented with in vivo functional assays. Understanding what defines uptake, binding, and selection for substrates versus non-substrates by RND transporters can provide critical insight into antibiotic resistance mechanisms and influence the redesign of current therapeutics or design of novel efflux pump inhibitors. Because 11 of the 14 bacterial pathogens currently identified by the Centers for Disease Control and Prevention as “urgent” or “serious” contain at least one RND efflux pump, these alternative therapeutic strategies are urgently needed to combat the growing threat of antibiotic resistance.

项目摘要/摘要 抗生素耐药性是全球主要的公共卫生威胁，大约有300万种抵抗感染 仅在美国，每年都有报道。在 细菌，但革兰氏阴性病原体中普遍存在的是能够从细胞中积极排出药物的能力。 抗性 - 突出区（RND）家族中的外排泵对内在的，临床的贡献很大 抗生素抗性。 RND泵是由内膜转运蛋白组成的三方复合物， 外围衔接蛋白和外膜因子蛋白。许多革兰氏阴性病原体编码 多个RND系统；例如，严重的威胁病原体铜绿假单胞菌包含四个 有助于抗生素耐药性的外排系统。在这里，其中两个泵，mexab-oprm和mexxy- OPRM将作为定义底物选择性基础的理想模型，因为它们的重叠但不同 B-内酰胺和氨基糖苷的偏好。尽管已知首选底物，但 目前尚不了解底物识别背后的分子决定。在已出版的指导下 MEXAB-OPRM的结构，我们的实验室生成了Mexxy-Oprm的模型。使用这个结构框架进行 鉴定了转运蛋白MEXB和MEXY，特定区域和保留的比较 这可能是基材的选择性。特别是，预计明显的结合口袋（DBP）至关重要 用于底物选择和转运蛋白。根据这些发现，我假设 Mexy的盘状结合口袋（DBP）中的关键残差定义了其物理 控制氨基糖苷底物识别的属性（形状，电荷，分布和体积） 和易位。在这个项目中，我将检验这一假设并阐明底物的分子基础 铜绿假单胞菌RND泵泵Mexxy-oprm的转运蛋白MEXY的选择性和运输。 在AIM 1中，我将确定从细胞张到细胞到达的首选氨基糖苷进入通道 使用诱变与实验室和泛氨基糖苷的体内功能测定相结合的转运蛋白mexy 耐药性临床分离株和高分辨率低温电子显微镜结构研究。在AIM 2中，我会 定义MEXY DBP内的残差，该残差控制非材料的底物的选择性（例如 通过使用体外结合亲和力和高分辨率X射线晶体学 结构研究，由体内功能测定完成。了解什么定义了摄取，约束力， RND转运蛋白对底物与非材料的选择可以提供对抗生素的关键见解 电阻机制并影响当前治疗剂或新型外排泵的设计的重新设计 抑制剂。因为疾病控制中心目前确定的14个细菌病原体中有11个 预防“紧急”或“严重”至少包含一个RND外排泵，这些替代性治疗策略 迫切需要应对日益增长的抗生素耐药性威胁。