title: Modulation of substrate efflux in bacterial small multidrug resistance proteins by mutations at the dimer interface.
abstract: Bacteria evade the effects of cytotoxic compounds through the efflux activity of membrane-bound transporters such as the small multidrug resistance (SMR) proteins. Consisting typically of ca. 110 residues with four transmembrane (TM) α-helices, crystallographic studies have shown that TM helix 1 (TM1) through TM helix 3 (TM3) of each monomer create a substrate binding "pocket" within the membrane bilayer, while a TM4-TM4 interaction accounts for the primary dimer formation. Previous work from our lab has characterized a highly conserved small-residue heptad motif in the Halobacterium salinarum transporter Hsmr as (90)GLXLIXXGV(98) that lies along the TM4-TM4 dimer interface of SMR proteins as required for function. Focusing on conserved positions 91, 93, 94, and 98, we substituted the naturally occurring Hsmr residue for Ala, Phe, Ile, Leu, Met, and Val at each position in the Hsmr TM4-TM4 interface. Large-residue replacements were studied for their ability to dimerize on SDS-polyacrylamide gels, to bind the cytotoxic compound ethidium bromide, and to confer resistance by efflux. Although the relative activity of mutants did not correlate with dimer strength for all mutants, all functional mutants lay within 10% of dimerization relative to the wild type (WT), suggesting that the optimal dimer strength at TM4 is required for proper efflux. Furthermore, nonfunctional substitutions at the center of the dimerization interface that do not alter dimer strength suggest a dynamic TM4-TM4 "pivot point" that responds to the efflux requirements of different substrates. This functionally critical region represents a potential target for inhibiting the ability of bacteria to evade the effects of cytotoxic compounds.
authors: Division of Molecular Structure & Function, Research Institute, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8.
authors: Bradley E
authors: BE
authors: Poulsen
authors: Fiona
authors: F
authors: Cunningham
authors: Kate K Y
authors: KK
authors: Lee
authors: Charles M
authors: CM
authors: Deber
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000235:genetics* / Q000378:metabolism*; D020816:Amino Acid Motifs; D019943:Amino Acid Substitution / Q000235:genetics; D000900:Anti-Bacterial Agents / Q000378:metabolism; D024881:Drug Resistance, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004996:Ethidium / Q000378:metabolism; D019617:Halobacterium salinarum / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization
chemical_list: D018435:ATP Binding Cassette Transporter, Subfamily B; D000900:Anti-Bacterial Agents; D004996:Ethidium