Cross-talk between energy supply and drug translocation in the ABC transporter Pdr5

多药耐药(MDR)是公共卫生中的一个主要问题，特别是对多种疗法产生抗药性的微生物的持续上升。在微生物用来抵抗治疗药物的武器库中，一个主要威胁是使用多药转运体，它可以驱逐许多结构上无关的药物。其中一些多特异性转运蛋白以ATP为能源，它们属于一个非常大的超家族，即ATP结合盒(ABC)蛋白。这个ABC家族的所有成员，主要是膜蛋白，都拥有参与ATP结合和水解的保守序列基序，最初认为它们是按照保守的能量转导机制工作的。然而，由于ABC转运蛋白功能的多功能性和许多不同的拓扑结构，能量的使用方式和类型可能会有很大的不同，可能会出现具有独特特征的转运蛋白亚类。例如，即使所有的ABC转运蛋白都有两个ATP结合位点，它们要么是对称的，两个位点都能水解ATP，要么是不对称的，只有一个简并的位点，不能或不能水解ATP。例如，Pdr5是一个全长的酵母MDR ABC转运蛋白，也是最极端的ABC转运蛋白，它包含一个简并的ATP结合位点，因为参与ATP结合的所有保守基序都显示出催化相关氨基酸的交换。在能量供应方面，Pdr5似乎根据运输的药物而调整其核苷酸依赖性。此外，最近用Pdr5获得的结果有力地支持了该转运体共同运输药物和质子，这可能也与其他多药ABC转运体相关。MDR ABC转运体中的这些奇点需要对其分子机制进行详细的研究，并特别关注其能量需求。因此，我们将解决以下核心问题：(I)Pdr5运输周期中质子的共同运输机制，(Ii)某些核苷酸的特异性也取决于运输底物，(Iii)继续我们对底物和抑制剂的结构分析，以了解是什么使底物成为底物，是什么使抑制剂成为抑制剂，以及(Vi)确定Pdr5运输周期中的潜在动力学和能量学。两个团队的互补专业知识以及他们在酵母转运蛋白以及电生理学和单分子荧光技术方面的知识将是解决这些紧迫问题和揭开Pdr5催化机制的宝贵财富。

Multidrug resistance (MDR) is a major concern in public health and in particular with the relentless rise of microbes that become resistant to multiple therapies. Among the arsenal used by microorganisms to resist to therapeutic drugs, a major threat is the use of multidrug transporters that expel many structurally unrelated drugs. Some of these polyspecific transporters use ATP as the energy source and they belong to a very large superfamily, the ATP-Binding Cassette (ABC) proteins. All members of this ABC family, primarily membrane proteins, possess conserved sequence motifs involved in ATP binding and hydrolysis and it was originally suggested that they work according to a conserved ‘unified’ mechanism of energy transduction. However, due to the versatility of function and many different topologies of ABC transporters, how and which energy is used may vary considerably, and subclasses of transporters with singular features are likely to emerge. For instance, even if all ABC transporters possess two ATP-binding sites, they are either symmetric, both sites being able to hydrolyse ATP, or asymmetric with one degenerate site, unable, or poorly able, to hydrolyse ATP. For example, Pdr5 is a full-length yeast MDR ABC transporter and the most extreme example of an ABC transporter containing a degenerated ATP binding site as all conserved motifs involved in ATP binding show exchange of the catalytical relevant amino acids. With respect to energy supply, Pdr5 seems to adapt its nucleotide dependency according to the drug transported. Moreover, recent results obtained with Pdr5 strongly support that this transporter co-transport drugs and protons and this might be relevant for other multidrug ABC transporters as well. These singularities among MDR ABC transporters warrant a detailed investigation of their molecular mechanisms with a particular focus on their energy requirement. We will therefore address central questions regarding (i) the mechanism of co-transport of protons during the transport cycle of Pdr5, (ii) specificity for certain nucleotides also in dependence of the transport substrate, (iii) continue our structural analysis of substrates and inhibitors to understand what make a substrate a substrate and an inhibitor to an inhibitor and (vi) determine the underlying dynamics and energetics during the transport cycle of Pdr5. The complementary expertise of the two teams and their knowledge of this yeast transporter as well as in electrophysiology and single molecule fluorescence techniques will be an asset to tackle these burning questions and unravel the catalytic mechanisms of Pdr5.