Rath Arianna, Melnyk Roman A, Deber Charles M
Division of Structural Biology & Biochemistry, Research Institute, Hospital for Sick Children, Toronto, Ontario M5G 1X8.
J Biol Chem. 2006 Jun 2;281(22):15546-53. doi: 10.1074/jbc.M600434200. Epub 2006 Mar 30.
Clinically significant bacterial resistance to drugs and cytotoxic compounds can be conferred by the energy-dependent efflux of toxicants, catalyzed by proteins embedded in the bacterial cell membrane. One such group of proteins, the small multidrug resistance family, are drug/proton antiporters that must oligomerize to function, a process that requires the assembly of at least two inactive monomers by intermolecular association of their four transmembrane helices. Here, we have used peptides that correspond to each of the four wild type transmembrane helices of the Halobacterium salinarum protein Hsmr and a corresponding library of mutant peptides to determine the interactive surfaces that likely contribute to protein oligomerization. Hsmr peptides were examined for strong (sodium dodecyl sulfate-resistant) and weaker (perfluorooctanoate-resistant) helix-helix interactions, in conjunction with circular dichroism, fluorescence energy transfer measurements, and molecular modeling. The results are compatible with a scheme in which two faces of helix four permit self-assembly via a higher affinity asymmetric pairing and a lower affinity symmetric interaction, resulting in a discrete tetramer. Our finding that two surfaces of helix four can contribute to the stability of small multidrug resistance protein assembly provides a molecular basis for the design of therapeutics that target this antibiotic resistance mechanism.
临床上，细菌对药物和细胞毒性化合物的显著耐药性可由嵌入细菌细胞膜中的蛋白质催化的毒物能量依赖性外排赋予。其中一类蛋白质，即小多药耐药家族，是药物/质子反向转运体，必须寡聚化才能发挥作用，这一过程需要通过其四个跨膜螺旋的分子间缔合组装至少两个无活性单体。在这里，我们使用了与嗜盐菌蛋白Hsmr的四个野生型跨膜螺旋相对应的肽段以及相应的突变肽库，以确定可能有助于蛋白质寡聚化的相互作用表面。结合圆二色性、荧光能量转移测量和分子建模，检测了Hsmr肽段之间强的（耐十二烷基硫酸钠）和较弱的（耐全氟辛酸）螺旋-螺旋相互作用。结果与一种模式相符，即螺旋4的两个面通过更高亲和力的不对称配对和更低亲和力的对称相互作用实现自组装，形成一个离散的四聚体。我们发现螺旋4的两个表面有助于小多药耐药蛋白组装的稳定性，这为针对这种抗生素耐药机制的治疗药物设计提供了分子基础。
