Yum Soohwan, Xu Yongbin, Piao Shunfu, Sim Se-Hoon, Kim Hong-Man, Jo Wol-Soon, Kim Kyung-Jin, Kweon Hee-Seok, Jeong Min-Ho, Jeon Hyesung, Lee Kangseok, Ha Nam-Chul
College of Pharmacy and Research Institute for Drug Development, Pusan National University, Busan 609-735, Korea.
J Mol Biol. 2009 Apr 17;387(5):1286-97. doi: 10.1016/j.jmb.2009.02.048. Epub 2009 Feb 28.
In Gram-negative bacteria, type I protein secretion systems and tripartite drug efflux pumps have a periplasmic membrane fusion protein (MFP) as an essential component. MFPs bridge the outer membrane factor and an inner membrane transporter, although the oligomeric state of MFPs remains unclear. The most characterized MFP AcrA connects the outer membrane factor TolC and the resistance-nodulation-division-type efflux transporter AcrB, which is a major multidrug efflux pump in Escherichia coli. MacA is the periplasmic MFP in the MacAB-TolC pump, where MacB was characterized as a macrolide-specific ATP-binding-cassette-type efflux transporter. Here, we report the crystal structure of E. coli MacA and the experimentally phased map of Actinobacillus actinomycetemcomitans MacA, which reveal a domain orientation of MacA different from that of AcrA. Notably, a hexameric assembly of MacA was found in both crystals, exhibiting a funnel-like structure with a central channel and a conical mouth. The hexameric MacA assembly was further confirmed by electron microscopy and functional studies in vitro and in vivo. The hexameric structure of MacA provides insight into the oligomeric state in the functional complex of the drug efflux pump and type I secretion system.
在革兰氏阴性菌中，I型蛋白质分泌系统和三联体药物外排泵都有一个周质膜融合蛋白（MFP）作为必需组分。MFP连接外膜因子和内膜转运蛋白，尽管MFP的寡聚状态仍不清楚。研究最深入的MFP AcrA连接外膜因子TolC和抗药-固氮-细胞分裂型外排转运蛋白AcrB，AcrB是大肠杆菌中的一种主要多药外排泵。MacA是MacAB-TolC泵中的周质MFP，其中MacB被鉴定为一种大环内酯特异性ATP结合盒式外排转运蛋白。在此，我们报道了大肠杆菌MacA的晶体结构以及伴放线放线杆菌MacA的实验相位图谱，它们揭示了MacA与AcrA不同的结构域取向。值得注意的是, 在两种晶体中均发现了MacA的六聚体组装体，呈现出具有中央通道和锥形口的漏斗状结构。通过电子显微镜以及体外和体内功能研究进一步证实了MacA的六聚体组装体。MacA的六聚体结构为深入了解药物外排泵和I型分泌系统功能复合物中的寡聚状态提供了线索。
