Evidence for assembly of small multidrug resistance proteins by a "two-faced" transmembrane helix.
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.
Division of Structural Biology & Biochemistry, Research Institute, Hospital for Sick Children, Toronto, Ontario M5G 1X8.
Arianna
A
Rath
Roman A
RA
Melnyk
Charles M
CM
Deber
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D019281:Dimerization; D018432:Drug Resistance, Multiple / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D019766:Genes, Archaeal; D019617:Halobacterium salinarum / Q000737:chemistry / Q000187:drug effects / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes; D016296:Mutagenesis; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary
D019843:Archaeal Proteins; D046912:Multiprotein Complexes