Velamakanni Saroj, Yao Yao, Gutmann Daniel A P, van Veen Hendrik W
Department of Pharmacology, University of Cambridge CB2 1PD, Cambridge, UK.
Biochemistry. 2008 Sep 2;47(35):9300-8. doi: 10.1021/bi8006737. Epub 2008 Aug 9.
Sav1866 is an ATP-binding cassette (ABC) protein from the pathogen Staphylococcus aureus and is a homologue of bacterial and human multidrug ABC transporters. Recently, the three-dimensional crystal structure of Sav1866 was determined at 3.0 A resolution [Dawson, R. J., and Locher, K. P. (2006) Nature 443, 180-185]. Although this structure is frequently used to homology model human and microbial ABC multidrug transporters by computational methods, the ability of Sav1866 to transport multiple drugs has not been described. We obtained functional expression of Sav1866 in the drug-sensitive, Gram-positive bacterium Lactococcus lactis Delta lmrA Delta lmrCD lacking major endogenous multidrug transporters. Sav1866 displayed a Hoechst 33342, verapamil, tetraphenylphosphonium, and vinblastine-stimulated ATPase activity. In growing cells, Sav1866 expression conferred resistance to Hoechst 33342. In transport assays in intact cells, Sav1866 catalyzed the translocation of amphiphilic cationic ethidium. Additionally, Sav1866 mediated the active transport of Hoechst 33342 in membrane vesicles and proteoliposomes containing purified and functionally reconstituted protein. Sav1866-mediated resistance and transport were inhibited by the human ABCB1 and ABCC1 modulator verapamil. This work represents the first demonstration of multidrug transport by Sav1866 and suggests that Sav1866 can serve as a well-defined model for studies on the molecular bases of drug-protein interactions in ABC transporters. Our methods for the overexpression, purification, and functional reconstitution of Sav1866 are described in detail.
Sav1866是一种来自病原体金黄色葡萄球菌的ATP结合盒（ABC）蛋白，是细菌和人类多药ABC转运蛋白的同源物。最近，Sav1866的三维晶体结构在3.0埃分辨率下被确定[道森，R.J.，和洛赫，K.P.（2006年）《自然》443，180 - 185]。尽管该结构常被用于通过计算方法对人类和微生物ABC多药转运蛋白进行同源建模，但Sav1866转运多种药物的能力尚未见报道。我们在缺乏主要内源性多药转运蛋白的药物敏感革兰氏阳性细菌乳酸乳球菌ΔlmrAΔlmrCD中实现了Sav1866的功能性表达。Sav1866表现出对Hoechst 33342、维拉帕米、四苯基鏻和长春碱刺激的ATP酶活性。在生长的细胞中，Sav1866的表达赋予了对Hoechst 33342的抗性。在完整细胞的转运试验中，Sav1866催化两亲性阳离子乙锭的转运。此外，Sav1866在含有纯化且功能重构蛋白的膜囊泡和蛋白脂质体中介导了Hoechst 33342的主动转运。Sav1866介导的抗性和转运受到人类ABCB1和ABCC1调节剂维拉帕米的抑制。这项工作首次证明了Sav1866的多药转运能力，并表明Sav1866可作为研究ABC转运蛋白中药物 - 蛋白质相互作用分子基础的一个明确模型。我们详细描述了Sav1866的过表达、纯化和功能重构方法。
