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.
Highlights
Bacteria can evade the toxic effects of antimicrobials via several mechanisms including development of drug efflux proteins [1]
We find that three key residues, Gly90, Gly97, and Val98, define an assembly “hot spot” where replacements are highly disruptive to Hsmr-based drug resistance
Modeling of Hsmr TM4 Dimers—Potential dimerization sites with larger side chains (Val, Leu, Ile, Asn) were replaced with Ala; for the TM4 sequence were identified using the CNS searching small side chains (Gly and Ala) were mutated to Val or of helix interactions (CHI) software suite of the crystallography Met. These replacements were anticipated to disrupt any closeand NMR system (CNS) as described elsewhere (29 –31). packed or knobs-into-holes type interactions in which TM4 may Briefly, two identical ␣-helices were generated from the pri- participate
Summary
Our work defines a minimum activity motif of 90GLXLIXXGV98 within TM4 and suggests that this sequence mediates TM4based 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. The TM4 helix pair has been proposed to represent a strong intermonomer association that does not contribute to conformational change during ligand binding but instead stabilizes the dimer interface [24] This potential dependence of SMR function on TM4 self-assembly suggests that inhibition of the self-interaction of this segment may provide a straightforward means of controlling drug efflux. We find that three key residues, Gly, Gly, and Val, define an assembly “hot spot” where replacements are highly disruptive to Hsmr-based drug resistance
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