Lubelski Jacek, van Merkerk Ronald, Konings Wil N, Driessen Arnold J M
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, NL-9751 Haren, The Netherlands.
Biochemistry. 2006 Jan 17;45(2):648-56. doi: 10.1021/bi051276s.
LmrCD is a lactococcal, heterodimeric multidrug transporter, which belongs to the ABC superfamily. It consists of two half-transporters, LmrC and LmrD, that are necessary and sufficient for drug extrusion and ATP hydrolysis. LmrCD is asymmetric in terms of the conservation of the functional motifs of the nucleotide-binding domains (NBDs). Important residues of the nucleotide-binding site of LmrC and the C loop of LmrD are not conserved. To investigate the functional importance of the LmrC and LmrD subunits, the putative catalytic base residue adjacent to the Walker B motif of both NBDs were substituted for the respective carboxamides. Our data demonstrate that Glu587 of LmrD is essential for both drug transport and ATPase activity of the LmrCD heterodimer, whereas mutation of Asp495 of LmrC has a less severe effect on the activity of the complex. Structural and/or functional asymmetry is further demonstrated by differential labeling of both subunits by 8-azido-[alpha-32P]ATP, which, at 4 degrees C, occurs predominantly at LmrC, while aluminiumfluoride (AlF(x))-induced trapping of the hydrolyzed nucleotide at 30 degrees C results in an almost exclusive labeling of LmrD. It is concluded that the LmrCD heterodimer contains two structurally and functionally distinct NBDs.
LmrCD是一种来自乳球菌的异源二聚体多药转运蛋白，属于ABC超家族。它由两个半转运蛋白LmrC和LmrD组成，这两个半转运蛋白对于药物外排和ATP水解是必需且足够的。LmrCD在核苷酸结合结构域（NBDs）功能基序的保守性方面是不对称的。LmrC核苷酸结合位点的重要残基和LmrD的C环并不保守。为了研究LmrC和LmrD亚基的功能重要性，将两个NBDs中与沃克B基序相邻的假定催化碱基残基替换为各自的羧酰胺。我们的数据表明，LmrD的Glu587对于LmrCD异源二聚体的药物转运和ATP酶活性都是必不可少的，而LmrC的Asp495突变对复合物活性的影响较小。通过用8-叠氮基-[α-32P]ATP对两个亚基进行差异标记进一步证明了结构和/或功能的不对称性，在4℃时，这种标记主要发生在LmrC上，而在30℃时，氟化铝（AlF(x)）诱导的水解核苷酸捕获导致几乎只标记LmrD。得出的结论是，LmrCD异源二聚体包含两个结构和功能不同的NBDs。
