Abstract
Bacterial cell membranes contain several protein pumps that resist the toxic effects of drugs by efficiently extruding them. One family of these pumps, the small multidrug resistance proteins (SMRs), consists of proteins of about 110 residues that need to oligomerize to form a structural pathway for substrate extrusion. As such, SMR oligomerization sites should constitute viable targets for efflux inhibition, by disrupting protein-protein interactions between helical segments. To explore this proposition, we are using Hsmr, an SMR from Halobacter salinarum that dimerizes to extrude toxicants. Our previous work established that (i) Hsmr dimerization is mediated by a helix-helix interface in Hsmr transmembrane (TM) helix 4 (residues (90)GLALIVAGV(98)); and (ii) a peptide comprised of the full TM4(85-105) sequence inhibits Hsmr-mediated ethidium bromide efflux from bacterial cells. Here we define the minimal linear sequence for inhibitor activity (determined as TM4(88-100), and then "staple" this sequence via Grubbs metathesis to produce peptides typified by acetyl-A-(Sar)3-(88)VVGLXLIZXGVVV(100)-KKK-NH2 (X = 2-(4'-pentenyl)alanine at positions 92 and 96; Z = Val, Gly, or Asn at position 95)). The Asn(95) peptide displayed specific efflux inhibition and resensitization of Hsmr-expressing cells to ethidium bromide; and was non-hemolytic to human red blood cells. Stapling essentially prevented peptide degradation in blood plasma and liver homogenates versus an unstapled counterpart. The overall results confirm that the stapled analog of TM4(88-100) retains the structural complementarity required to disrupt the Hsmr TM4-TM4 locus in Hsmr, and portend the general validity of stapled peptides as therapeutics for the disruption of functional protein-protein interactions in membranes.
Highlights
Helix-helix interactions commonly mediate a host of biological functions in membrane proteins
Synthesis of Hsmr transmembrane helix 4 (TM4)-based Peptides—The sequences for the peptides synthesized in this study were derived from TM4 of the efflux pump Hsmr found in the archaea H. salinarum [30]
Given that Poulsen and Deber [25] had created a linear 28-residue peptide based on the full-length of the Hsmr TM4 that inhibited EtBr efflux by more than 60%, we first sought to find shorter linear sequences derived from TM4 that displayed similar inhibiting activity
Summary
Helix-helix interactions commonly mediate a host of biological functions in membrane proteins. Results: A stapled peptide sequentially complementary to a helix-helix interaction motif disrupted a bacterial multidrug resistance efflux pump. Bacterial cell membranes contain several protein pumps that resist the toxic effects of drugs by efficiently extruding them. One family of these pumps, the small multidrug resistance proteins (SMRs), consists of proteins of about 110 residues that need to oligomerize to form a structural pathway for substrate extrusion. SMR oligomerization sites should constitute viable targets for efflux inhibition, by disrupting protein-protein interactions between helical segments. To explore this proposition, we are using Hsmr, an SMR from Halobacter salinarum that dimerizes to extrude toxicants. The overall results confirm that the stapled analog of TM4(88 –100) retains the structural complementarity required to disrupt the Hsmr TM4TM4 locus in Hsmr, and portend the general validity of stapled peptides as therapeutics for the disruption of functional protein-protein interactions in membranes
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