Amino acid substitutions in the first transmembrane domain (TM1) of P‐glycoprotein that alter substrate specificity

Bartlett

Clarke

2008

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.

Section: Discussionsupporting

confidence: 92%

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

Bartlett

Clarke

2008

“…For example, the L65R mutation only caused a large reduction (57-fold) in the apparent affinity for vinblastine and little change in affinities for cyclosporin A or rhodamine B. The reduction in affinity of mutant L65R(TM1) for vinblastine is consistent with the observation that it had reduced ability to confer resistance to vinblastine (39). The F343R mutation exhibited decreased apparent affinity for rhodamine B (3.6-fold) and cyclosporin A (18-fold) but not for vinblastine (Ͻ2-fold).…”

Section: Discussionsupporting

confidence: 68%

Suppressor Mutations in the Transmembrane Segments of P-glycoprotein Promote Maturation of Processing Mutants and Disrupt a Subset of Drug-binding Sites

Bartlett

Clarke

2007

Defective folding of cystic fibrosis transmembrane conductance regulator protein missing Phe 508 (⌬F508) is the major cause of cystic fibrosis. The folding defect in ⌬F508 cystic fibrosis transmembrane conductance regulator might be correctable because misfolding of a P-glycoprotein (P-gp; ABCB1) mutant lacking the equivalent residue (⌬Y490) could be corrected with drug substrates or by introduction of an arginine residue into transmembrane (TM) segments 5 (I306R) or 6 (F343R). Possible mechanisms of arginine rescue were that they mimicked some of the effects of drug substrate interactions with P-gp or that they affected global folding such that all drug substrate/modulator interactions with P-gp were altered. To distinguish between these mechanisms, we tested whether arginines introduced into other TMs predicted to line the drug-binding pocket (TM1 or TM3) would affect folding. It was found that mutation of L65R(TM1) or T199R(TM3) promoted maturation of processing mutants. We then tested whether arginine suppressor mutations had local or global effects on P-gp interactions with drug substrates and modulators. The L65R(TM1), T199R(TM3), I306R(TM5), or F343R(TM6) mutations were introduced into the P-gp mutant L339C(TM6)/F728C(TM7), and thiol crosslinking was carried out in the presence of various concentrations of vinblastine, cyclosporin A, or rhodamine B. The presence of arginine residues reduced the apparent affinity of P-gp for vinblastine (L65R, T199R, and I306R), cyclosporin (I306R and F343R), or rhodamine B (F343R) by 4 -60-fold. These results show that the arginine mutations affect a subset of drug-binding sites and suggest that they rescue processing mutants by mimicking drug substrate interactions with P-gp.Defective folding of a mutant membrane protein in the endoplasmic reticulum and/or trafficking to its normal cellular destination is responsible for many inherited diseases. The protein usually has a deletion or substitution of an amino acid (1) leading to their retention in the endoplasmic reticulum and subsequent degradation by the endoplasmic reticulum-associated degradation system (2).A classic example of a genetic disease caused by an amino acid change in a membrane protein is cystic fibrosis. The most common cause of cystic fibrosis is deletion of Phe 508 (⌬F508) in the cystic fibrosis transmembrane conductance regulator (CFTR; ABCC7) 2 protein. The ⌬F508 mutation causes misfolding of CFTR such that the amount of mature CFTR at the cell surface is reduced from about 25% (wild type) to less than 1% (3, 4). An important goal in developing a treatment for proteinfolding diseases is to correct folding/trafficking defects in the mutant proteins.The human multidrug resistance P-glycoprotein (P-gp, ABCB1) is a useful model system for studying protein misfolding because processing mutations can be rescued with drug substrates (reviewed in Ref. 5). P-gp is an ABC (ATP-binding cassette) protein that uses ATP to transport a variety of cytotoxic compounds out of the cell (6, 7). It has two homologous halve...

“…Recent crystal structures of these two transporters showed that the amino acids involved in ligand binding are in agreement with those identified through mutational analyses (23,24). The residues in P-gp that are involved in drug binding, however, have been difficult to characterize because a large number of mutations throughout the molecule can alter the substrate specificity (25)(26)(27)(28)(29)(30)(31). It has been difficult to determine whether mutations that affected activity were actually close to the drug-binding site or whether they affected the global structure of the protein (32).…”

mentioning

confidence: 67%

Identification of Residues within the Drug-binding Domain of the Human Multidrug Resistance P-glycoprotein by Cysteine-scanning Mutagenesis and Reaction with Dibromobimane

Clarke²

2000

157

144

P-glycoprotein (P-gp) can transport a wide variety of cytotoxic compounds that have diverse structures. Therefore, the drug-binding domain of the human multidrug resistance P-gp likely consists of residues from multiple transmembrane (TM) segments. In this study, we completed cysteine-scanning mutagenesis of all the predicted TM segments of P-gp (TMs 1-5 and 7-10) and tested for inhibition by a thiol-reactive substrate (dibromobimane) to identify residues within the drug-binding domain. The activities of 189 mutants were analyzed. Verapamil-stimulated ATPase activities of seven mutants (Y118C and V125C (TM2), S222C (TM4), I306C (TM5), S766C (TM9), and I868C and G872C (TM10)) were inhibited by more than 50% by dibromobimane. The activities of mutants S222C (TM4), I306C (TM5), I868C (TM10), and G872C (TM10), but not that of mutants Y118C (TM2), V125C (TM2), and S776C (TM9), were protected from inhibition by dibromobimane by pretreatment with verapamil, vinblastine, or colchicine. These results and those from previous studies (Loo,