Introduction of the Most Common Cystic Fibrosis Mutation (ΔF508) into Human P-glycoprotein Disrupts Packing of the Transmembrane Segments

Abstract

The most common mutation in cystic fibrosis (deletion of phenylalanine 508 (⌬F508) in the cystic fibrosis conductance transmembrane regulator (CFTR) gene) causes defective synthesis of CFTR protein. To understand how this deletion interferes with protein folding, we made the equivalent deletion (⌬Y490) in P-glycoprotein (P-gp). A Cys-less P-gp with cysteines in transmembrane (TM) 4 or TM5 can be cross-linked with a cysteine in TM12. Deleting Tyr 490 in P-gp resulted in an inactive and defectively processed mutant in which no crosslinking between TM4 or TM5 and TM12 was detected. Expression of the ⌬Y490 mutant in the presence of a chemical chaperone corrected the processing defect and yielded active P-gp mutants that could be cross-linked between TM4 or TM5 and TM12. Cross-linking between TM4 or TM5 and TM12 was also detected when residues 483 TIAENIRYG 491 in P-gp were replaced with residues 501 TIKENIIFG 509 from CFTR (P-gp/CFTR). Deleting Phe 508 in the P-gp/CFTR chimera, however, caused defective processing of the mutant protein and no detectable cross-linking between TM4 or TM5 and TM12. The processing defect was corrected with a chemical chaperone and yielded active P-gp/CFTR mutant proteins that could be cross-linked. These results show that deletion at residue 490 disrupts packing of the TM segments possibly by affecting interaction between the first nucleotide-binding domain (Tyr 490 ) and the first cytoplasmic loop (Glu 184 ).

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

“…9B). Processing mutations such as G251V appear to act as thermodynamic hurdles to inhibit packing of the TM segments between TMD1 and TMD2 (6). Introduction of an arginine into position Met-68 may help to restore packing of the TM segments between TMD1 and TMD2 by forming hydrogen bonds with Tyr-950 and/or Tyr-953 in TM11.…”

“…One explanation is that introduction of arginine at positions 64, 65, 68, and 71 promotes folding of P-gp by affecting the orientation of TM1in the lipid bilayer. We previously showed that packing of the TM segments between the two TMDs is a rate-limiting state in maturation of ABC transporters such as P-gp and CFTR (6,46). Processing mutations such as G251V may alter the orientation of one or more TM segments in the membrane to interfere with subsequent packing of the TM segments at the interface between TMD1 and TMD2.…”

“…The second P-gp cDNA was modified to contain a 10-histidine tag at the COOH-terminal end to facilitate purification of the expressed protein by nickel-chelate chromatography (14). Mutations were introduced into wildtype or processing mutant G251V (13) P-gp cDNAs as described previously (6,15). To perform arginine-scanning mutagenesis of TM1, the cDNA of mutant G251V P-gp was modified to create a set of mutants that contained one arginine at positions 52-72.…”

“…The mutant protein is delivered to the cell surface in a functional form. Processing mutations appear to inhibit folding by disrupting packing of TM segments at the interface between the TMDs (6), a defect that can be repaired by expression in the presence of drug substrates (6,7). Drug substrates appear to rescue P-gp processing mutants through direct interactions with the TM segments because their effects can be mimicked by introducing arginine mutations into positions predicted to line the drug binding domain such as position 65 in TM segment 1 (8,9).…”

The Dileucine Motif at the COOH Terminus of Human Multidrug Resistance P-glycoprotein Is Important for Folding but Not Activity

“…Expression of misprocessed P-gp mutants in the presence of a drug substrate, however, promotes maturation of the protein so that the protein is trafficked to the cell surface in an active form (21). Drug substrates appear to rescue misprocessed mutants by promoting domain-domain interactions (22,23).…”