Arginines in the First Transmembrane Segment Promote Maturation of a P-glycoprotein Processing Mutant by Hydrogen Bond Interactions with Tyrosines in Transmembrane Segment 11

Abstract

A key goal is to correct defective folding of mutant ATP binding cassette (ABC) transporters, as they cause diseases such as cystic fibrosis. P-glycoprotein (ABCB1) is a useful model system because introduction of an arginine at position 65 of the first transmembrane (TM) segment could repair folding defects. To determine the mechanism of arginine rescue, we first tested the effects of introducing arginines at other positions in TM1 (residues 52-72) of a P-glycoprotein processing mutant (G251V) that is defective in folding and trafficking to the cell surface (20% maturation efficiency). We found that arginines introduced into one face of the TM1 helix (positions 52, 55, 56, 59, 60, 62, 63, 66, and 67) inhibited maturation, whereas arginines on the opposite face of the helix promoted (positions 64, 65, 68, and 71) or had little effect (positions 61, and 69) on maturation. Arginines at positions 61, 64, 65, and 68 appeared to lie close to the drug binding sites as they reduced the apparent affinity for drug substrates such as vinblastine and verapamil. Therefore, arginines that promoted maturation may face an aqueous drug translocation pathway, whereas those that inhibited maturation may face the lipid bilayer. The highest maturation efficiencies (60 -85%) were observed with the Arg-65 and Arg-68 mutants. Mutations that removed hydrogen bond acceptors (Y950F/Y950A or Y953F/Y953A) in TM11 predicted to lie close to Arg-65 or Arg-68 inhibited maturation but did not affect maturation of the G251V parent. Therefore, arginine may rescue defective folding by promoting packing of the TM segments through hydrogen bond interactions.

Human P-glycoprotein Contains a Greasy Ball-and-Socket Joint at the Second Transmission Interface

“…IH2 is in an intracellular loop between TM segments 4 and 5 that connects TMD1 to NBD2. IH2 appears to be particularly important because we showed that a cysteine introduced into IH2 (A266C) could be directly cross-linked to a cysteine in NBD2 (F1086C) but the mutant was inactive (21). By contrast, cysteine mutations at the equivalent sites at the fourth transmission interface (links IH4 in TMD2 to NBD1) (L443C(NBD1)/S909C(IH4) had no effect on activity (21).…”

A Promiscuous Conformational Switch in the Secondary Multidrug Transporter MdfA

“…Better understanding of how Mdr transporters respond conformationally to dissimilar substrates in a transport-competent manner would add mechanistically important clues to how dissimilar compounds are actively exported by such transporters. For example, in a P-glycoprotein mutant that is defective in biogenesis, it has been reported that mutations in the binding site improve protein maturation and that this effect can also be achieved by substrates (27)(28)(29)(30)(31)(32). Thus, binding site modifications also have substrate-mimicking effects in this ATP-binding cassette Mdr transporter.…”

Lipid Regulation of the ABCB1 and ABCG2 Multidrug Transporters

“…Loo and Clarke performed numerous studies in order to define amino acids facing the membrane phase, thus interacting with the lipid environment. In these studies, a construct with the G251V mutation, causing protein instability and improper maturation (processing mutant), was used in combination with Arg mutations in transmembrane helices (Loo, Bartlett, & Clarke, 2008;Loo et al, 2009;Loo & Clarke, 2013). As a result, some "corrector" mutations rescued the processing mutant G251V by promoting its proper folding by Loo et al (2008).…”

“…In these studies, a construct with the G251V mutation, causing protein instability and improper maturation (processing mutant), was used in combination with Arg mutations in transmembrane helices (Loo, Bartlett, & Clarke, 2008;Loo et al, 2009;Loo & Clarke, 2013). As a result, some "corrector" mutations rescued the processing mutant G251V by promoting its proper folding by Loo et al (2008). The authors hypothesized that when the inserted Arg faces the lipid interface, the rescue fails because of the intolerability of the positive charge in the hydrophobic environment (see Fig.…”