Analysis of coupling between substrate binding and ATP hydrolysis in canonical homo- and heterodimeric amino acid ABC import systems

在原核生物中，介导溶质摄取的典型ABC转运系统由一个胞质外溶质结合蛋白、两个成孔亚基和一个atp酶二聚体组成。我们目前对I型进口机制的了解主要基于大肠杆菌/沙门氏菌麦芽糖转运蛋白（MalE-FGK2）的结构、生化和生物物理数据。直到最近，这也是唯一一个在跨膜结构域内鉴定出（单一）底物结合位点的系统。具有两个底物分子的同型二聚体氨基酸转运体（artii - q2n2）的新型晶体结构提出了一些关于I型进口物的一般机制的重要问题：(I)在同型二聚体转运体中是否需要两个底物分子来触发ATP水解？（ii）只有同型二聚体氨基酸转运体含有两个底物结合位点，还是异型二聚体系统也是如此？（iii）异二聚体转运体能否转化为同二聚体转运体？我们建议在下一个和最后一个资助期解决这些问题，再次使用鼠伤寒沙门氏菌的异质二聚体组氨酸转运体HisJ/LAO-QMP2和嗜脂嗜热地杆菌的同源二聚体精氨酸转运体ArtJ-(MP)2作为模型系统。目前将继续研究结合蛋白HisJ和HisQMP2转运体在EPR（DEER）光谱研究的转运周期中的相互作用。此外，我们将应用位点定向诱变结合atp酶和转运试验来研究灭活Art(MP)2转运体中两个底物结合位点之一的功能后果。作为补充，我们将分析在HisQMP2转运体中引入第二个结合位点（HisQ）的功能后果。最后，通过随机诱变，我们希望分离HisJ/HisQM中允许同型二聚体变体（HisM2P2和HisQ2P2）运输底物偶联ATP水解的抑制突变。我们相信，这些研究的结果（其可行性已被初步实验证明）将扩大我们对ABC转运蛋白机制的认识。

Canonical ABC transport systems mediating the uptake of solutes in prokaryotes are composed of an extracytoplasmic solute binding protein, two pore-forming subunits and an ATPase dimer. Our current knowledge on the mechanism of Type I importers is largely based on structural, biochemical and biophysical data of the maltose transporter (MalE-FGK2) of E. coli/Salmonella. Until recently, this was also the only system for which a (single) substrate binding site had been identified within the transmembrane domains. Novel crystal structures of a homo-dimeric amino acid transporter (ArtI-Q2N2) in complex with two substrate molecules raise some important questions concerning the general mechanism of Type I importers: (i) are two substrate molecules required to trigger ATP hydrolysis in a homo-dimeric transporter? (ii) do only homo-dimeric amino acid transporters contain two substrate binding sites or does this hold also for hetero-dimeric systems? and (iii) can a hetero-dimeric transporter be converted into a homo-dimeric system? We propose to address these questions in the next and final funding period as follows, using again the well-characterized hetero-dimeric histidine transporter, HisJ/LAO-QMP2, of S. Typhimurium and the homo-dimeric arginine transporter, ArtJ-(MP)2 of Geobacillus stearothermophilus as model systems. Current investigations on the interactions of the binding protein HisJ and the HisQMP2 transporter during the transport cycle studied by EPR(DEER) spectroscopy will be continued. Furthermore, we will apply site-directed mutagenesis combined with ATPase and transport assays to study the functional consequences of inactivating one of two substrate binding sites in the Art(MP)2 transporter. Complementary, we will analyse the functional consequences of introducing a second binding site (in HisQ) in the HisQMP2 transporter in addition to that proposed for HisM. Finally, by random mutagenesis, we hope to isolate suppressor mutations in HisJ/HisQM that allow homo-dimeric variants (HisM2P2 and HisQ2P2) to transport substrate coupled to ATP hydrolysis. We are confident that the results of these investigations the feasibility of which has been proven by initial experiments, will expand our knowledge on the mechanism of ABC transporters.