Insertion of an arginine residue into the transmembrane segments corrects protein misfolding

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• Cystic fibrosis transmembrane conductance regulator (CFTR) misfolding due to Phe-508 deletion causes disease.
• P-glycoprotein (P-gp) misfolding, similar to CFTR, can be rescued by drug substrates, but the mechanism remains unclear.
• The role of drug-substrate binding to transmembrane (TM) segments versus indirect effects on protein folding is debated.

Purpose of the Study:

• To investigate if mutations in P-gp's TM segments can mimic drug rescue effects.
• To identify specific residues and their properties that suppress P-gp misfolding.
• To determine if TM segment mutations can correct misprocessing mutations in other P-gp domains.

Main Methods:

• Computational prediction of residues prone to hydrogen bonding within P-gp's drug-binding pocket.
• Site-directed mutagenesis of Ile-306 (TM5) and Phe-343 (TM6) in P-gp.
• Assessment of mature DeltaY490 P-gp levels using Western blotting or similar techniques.
• Analysis of suppressor mutation effects on various P-gp mutants with defects in different domains.

Main Results:

• Arginine substitutions at positions 306 (TM5) or 343 (TM6) significantly increased mature DeltaY490 P-gp levels (6-fold).
• Other amino acid substitutions at these positions did not enhance DeltaY490 P-gp maturation.
• The I306R mutation also rescued misprocessing in mutants with defects in nucleotide-binding domains, cytoplasmic loops, linker regions, and TM segments.

Conclusions:

• Arginine residues in P-gp's transmembrane domains can effectively suppress protein misfolding.
• These arginine mutations mimic the effects of drug substrates, suggesting a role for TM segment interactions in rescue.
• TM domain mutations offer a potential strategy for correcting P-gp processing defects beyond the drug-binding site.