|Abstract: ||摘要:膜融合蛋白質MacA，內膜蛋白MacB及外膜白TolC組成的轉運幫浦，能將巨環類藥物轉運至細胞外，為細菌抗藥性的重要因素。研究指出細菌失去macAB基因導致溶血素STII的分泌大幅減少，倘若是由MacAB負責STII的轉運，將使前者成為一個好的藥物標的。然而，沒有證據指出STII對MaA或MacB有直接的相互作用。以轉運幫浦為目標的藥物設計首先需要了解其作用機制，而動力學的研究有助了解幫浦如何組成、調控及其藥物轉運。我們的報告指出MacA能調控MacB的ATP水解速率，而為了解幫浦的調控機制，必須徹底探討MacB從結合ATP到釋放ADP及Pi其各步驟之酵素動力學，並測試溶血蛋白STII及TolC是否調控ATP水解速率。為了進一步了解作用機轉，首先必須了解幫浦組成及藥物轉運是否經由MacB結合ATP來起動? 本計劃於第一年將克隆及表達此三個蛋白質及溶血蛋白STII，利用恆溫滴定熱卡機及分析型超過速離心來測試STII對於膜融合蛋白MacA及內膜蛋白MacB的相互作用，以期確認STII的轉運途徑。第二年擬利用偏極化螢光技術，計算出紅黴素及STII對MacB之結合與解離常數。第三年，預計利用停止流技術來監測並分析ATP結合與ADP解離內膜蛋白MacB的速率常數及調控，並進一步地利用ITC來探討MacA, MacB及TolC的幫浦組成。|
Abstract:MacA, a membrane fusion protein (MFP), MacB, an inner membrane protein (IMP) and TolC, an outer membrane protein (OMP), form a tripartite pump to transport macrolide drugs across the inner and outer membranes, one of the factors to multidrug resistance in bacteria. A model has been proposed MacAB is also reponsible for secretion of hemolysin STII due to large decrease of extracellular secretion of STII in the macAB deletion mutant. If MacAB is the bona fide transporter for STII, which will make the former to be a good potential drug target. However, there is no direct evidence for the interaction of STII with MacA and/or MacB. The elucidation of dynamics of the assembly of the tripartite pump helps understand how it transports drugs and toxins out of the cell, a requisite for the rational design of inhibitors that block the pump or its assembly. Our previous study indicated a step after ATP hydrolysis is rate-limiting in the presence of MacA. In order to understand how ATP binding and hydrolysis is connected to the assembly of tripartite pumps and of the subsequent transport of drugs and toxins. There is clearly a need to rigorously define the kinetic steps governing the ATPase mechanism of a complete transporter and to determine how these are regulated during the assembly of a tripartite-pump. Furthermore, considering the translocation sequence is unclear, it will be important to determine whether the assembly of tripartite-pump-complex is dependent upon the conformational states of MacB (inward-facing and outward-facing) and TolC. In this manner we can determine the sequence of events that govern the assembly and function of the tripartite pump. In this proposal, for the first year, we plan to clone, overexpress and purify MaAB, TolC and STII proteins and use biophysical approaches, such as size exclusion chromatography, isothermal titration calorimetry (ITC) and analytical ultracentrifugation (AUC), to directly montor the possible interaction of STII to MacA or/and MacB. In the second year, we plan to determine the association and dissociation rate constants for the binding of macrolide antbiotic erythromycin and STII to MacB, by using fluorescence-polarization to monitor the bindings. In the last year, by using stopped-flow techniques, we plan to determinate the kinetic steps of MacB ATPase reaction-mechanism, and elucidate if, and how, each step is regulated by the binding of drugs, STII, MacA and TolC. By using ITC, we aim to determine the binding strengths of these interactions in the absence and presence of nucleotides to test our prediction that ATP stabilizes a conformation of MacB that interacts with TolC, either directly or via MacA.