Efflux by Small Multidrug Resistance Proteins is Inhibited by Membrane-Interactive Helix-Stapled Peptides [Membrane Biology]

November 25th, 2014 by Bellmann-Sickert, K., Stone, T. A., Poulsen, B. E., Deber, C. M.

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 ca. 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 90GLALIVAGV98); 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)3-88VVGLXLIZXGVVV100KKKNH2 (X = 2(4pentenyl)alanine at positions 92,96; Z = Val, Gly, or Asn at position 95)). The Asn95 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 vs. an unstapled counterpart. 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.