title: The assembly motif of a bacterial small multidrug resistance protein.
abstract: Multidrug transporters such as the small multidrug resistance (SMR) family of bacterial integral membrane proteins are capable of conferring clinically significant resistance to a variety of common therapeutics. As antiporter proteins of approximately 100 amino acids, SMRs must self-assemble into homo-oligomeric structures for efflux of drug molecules. Oligomerization centered at transmembrane helix four (TM4) has been implicated in SMR assembly, but the full complement of residues required to mediate its self-interaction remains to be characterized. Here, we use Hsmr, the 110-residue SMR family member of the archaebacterium Halobacterium salinarum, to determine the TM4 residue motif required to mediate drug resistance and SMR self-association. Twelve single point mutants that scan the central portion of the TM4 helix (residues 85-104) were constructed and were tested for their ability to confer resistance to the cytotoxic compound ethidium bromide. Six residues were found to be individually essential for drug resistance activity (Gly(90), Leu(91), Leu(93), Ile(94), Gly(97), and Val(98)), defining a minimum activity motif of (90)GLXLIXXGV(98) within TM4. When the propensity of these mutants to dimerize on SDS-PAGE was examined, replacements of all but Ile resulted in approximately 2-fold reduction of dimerization versus the wild-type antiporter. Our work defines a minimum activity motif of (90)GLXLIXXGV(98) within TM4 and suggests that this sequence mediates TM4-based SMR dimerization along a single helix surface, stabilized by a small residue heptad repeat sequence. These TM4-TM4 interactions likely constitute the highest affinity locus for disruption of SMR function by directly targeting its self-assembly mechanism.
authors.affiliation: Division of Molecular Structure and Function, Research Institute, Hospital for Sick Children, Toronto, Ontario M5G 1X8.
authors.forename: Bradley E
authors.initials: BE
authors.lastname: Poulsen
authors.forename: Arianna
authors.initials: A
authors.lastname: Rath
authors.forename: Charles M
authors.initials: CM
authors.lastname: Deber
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D019281:Dimerization; D024881:Drug Resistance, Bacterial; D018432:Drug Resistance, Multiple; D004996:Ethidium / Q000494:pharmacology; D019617:Halobacterium salinarum / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry*; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009154:Mutation; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
chemical_list: D008565:Membrane Proteins; D004996:Ethidium