Structural Analysis of Multidrug Transport

多药物转运的结构分析

• 批准号: 7104908
• 负责人: MARK E. GIRVIN
• 金额: $ 31.93万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7104908
• 关键词: Staphylococcus aureus; bacterial proteins; conformation; glutamates; inhibitor /antagonist; micelles; multidrug resistance; nuclear magnetic resonance spectroscopy; pharmacokinetics; protein kinase A; protein structure function; protonation

DESCRIPTION (provided by applicant): Bacteria have developed several methods to resist the lethal effects of antibiotics. The broadest spectrum resistance results from the action of Multidrug resistant pumps (MDRs), which extrude a range of compounds of quite diverse chemical structure. The Small Multidrug Resistance pumps (SMRs) are 100- 110 residue dimeric proton-drug antiporters that contain the full multidrug transport machinery, stripped to its barest essentials. Hence they are ideal transporters for a comprehensive structural and functional understanding of drug transport and inhibition in a medically important MDR. We propose to determine the structures of the conformations making up the functional cycle of an SMR, and identify the binding determinants for multiple drugs and inhibitors using solution NMR by: 1) Determining the structure of a dimeric SMR in lysolipid micelles in its protonated state, 2) Measuring the affinities of multiple drugs and inhibitors above and below the pKa of the glutamate essential for transport, 3) Identifying the binding determinants for inhibitors and transportable drugs, and 4) Determining the conformational changes induced by substrate and inhibitor binding, and by deprotonation of the critical glutamate. In addition to the MDR pumps, membrane proteins are responsible for transmembrane signaling, energy transduction, and ion and metabolite transport. These proteins are important in infectious disease, genetic disorders, and cancer. Despite their importance, and the need for structure to understand their function, relatively few such structures are available. For transporters and receptors, ligand binding and transport involve multiple conformations and dynamic changes. Solution NMR is an ideal method for examining these processes. Firmly establishing NMR methods to study larger helical membrane proteins - here a dimer with total of eight transmembrane helices, will have as long-term an impact as the specific findings for an SMR.

描述（由申请人提供）：细菌已经开发出多种方法来抵抗抗生素的致命作用。最广泛的耐药性来自于多重耐药泵 (MDR) 的作用，该泵可挤出一系列具有相当不同化学结构的化合物。小型多药耐药泵 (SMR) 是 100-110 个残留二聚体质子药物反向转运蛋白，包含完整的多药转运机制，仅保留最基本的要素。因此，它们是全面了解医学上重要的 MDR 中药物转运和抑制的结构和功能的理想转运蛋白。我们建议确定构成 SMR 功能循环的构象结构，并使用溶液 NMR 鉴定多种药物和抑制剂的结合决定因素：1) 确定质子化状态的溶血脂胶束中二聚体 SMR 的结构， 2) 测量多种药物和抑制剂的亲和力（高于和低于运输必需的谷氨酸的 pKa）， 3) 识别抑制剂和可运输药物的结合决定因素， 4) 确定由底物和抑制剂结合以及关键谷氨酸去质子化诱导的构象变化。 除了 MDR 泵之外，膜蛋白还负责跨膜信号传导、能量转导以及离子和代谢物运输。这些蛋白质在传染病、遗传性疾病和癌症中很重要。尽管它们很重要，并且需要结构来理解它们的功能，但可用的此类结构相对较少。对于转运蛋白和受体，配体结合和转运涉及多种构象和动态变化。溶液核磁共振是检查这些过程的理想方法。牢固建立 NMR 方法来研究较大的螺旋膜蛋白（这里是总共有 8 个跨膜螺旋的二聚体），将产生与 SMR 的具体发现一样的长期影响。