Xu Yongbin, Jo Inseong, Wang Lulu, Chen Jinli, Fan Shengdi, Dong Yuesheng, Quan Chunshan, Ha Nam-Chul
Department of Bioengineering, College of Life Science, Dalian Minzu University, Dalian 116600, Liaoning, China; Key Laboratory of Biotechnology and Bioresources Utilization (Dalian Minzu University), Ministry of Education, China.
Department of Agricultural Biotechnology, Research Institute for Agriculture and Life Sciences, Seoul National University, Gwanak-gu, Seoul 08826, Republic of Korea.
Biochem Biophys Res Commun. 2017 Nov 4;493(1):152-157. doi: 10.1016/j.bbrc.2017.09.059. Epub 2017 Sep 14.
Membrane fusion proteins (MFPs) play an essential role in the action of the drug efflux pumps and protein secretion systems in bacteria. The sporulation delaying protein (SDP) efflux pump YknWXYZ has been identified in diverse Bacillus species. The MFP YknX requires the ATP-binding cassette (ABC) transporter YknYZ and the Yip1 family protein YknW to form a functional complex. To date, the crystal structure, molecular function and mechanism of action of YknX remain unknown. In this study, to characterize the structural and biochemical roles of YknX in the functional assembly of YknWXYZ from B. amyloliquefaciens, we successfully obtained crystals of the YknX protein that diffracted X-rays to a resolution of 4.4 Å. We calculated an experimentally phased map using single-wavelength anomalous diffraction (SAD), revealing that YknX forms a hexameric assembly similar to that of MacA from Gram-negative bacteria. The hexameric assembly of YknX exhibited a funnel-like structure with a central channel and a conical mouth. Functional studies in vitro suggest that YknX can bind directly to peptidoglycan. Our study provides an improved understanding of the assembly of the YknWXYZ efflux pump and the role of YknX in the complex.
膜融合蛋白（MFPs）在细菌的药物外排泵和蛋白质分泌系统的作用中起着至关重要的作用。已在多种芽孢杆菌属物种中鉴定出延迟芽孢形成蛋白（SDP）外排泵YknWXYZ。膜融合蛋白YknX需要ATP结合盒（ABC）转运蛋白YknYZ和Yip1家族蛋白YknW形成功能复合物。迄今为止，YknX的晶体结构、分子功能和作用机制仍不清楚。在本研究中，为了表征YknX在解淀粉芽孢杆菌YknWXYZ功能组装中的结构和生化作用，我们成功获得了YknX蛋白的晶体，其X射线衍射分辨率为4.4 Å。我们使用单波长反常衍射（SAD）计算了一个实验相位图，揭示YknX形成了一种类似于革兰氏阴性菌MacA的六聚体组装。YknX的六聚体组装呈现出一种具有中央通道和锥形口的漏斗状结构。体外功能研究表明，YknX可以直接结合肽聚糖。我们的研究增进了对YknWXYZ外排泵组装以及YknX在复合物中作用的理解。
