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The tyrosine89 residue of yeast FKBP12 is required for rapamycin binding
P L Koser1, W K Eng, M J Bossard
1Department of Biomolecular Discovery, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406.
Abstract:
Rapamycin (Rm) is a macrolide antifungal agent related to FK506 that exhibits potent immunosuppressive properties which are mediated through interaction with specific cytoplasmic receptors (FKBPs or RBPs, for FK506- and Rm-binding proteins, respectively). These proteins possess peptidyl-prolyl cis-trans isomerase (PPIase) activity in vitro which is inhibited by the binding of Rm and FK506. In Saccharomyces cerevisiae, Rm sensitivity (Rms) is mediated by binding of the drug to RBP1, a homolog of the 12-kDa human FK506-binding protein (FKBP12); null mutations in the yeast RBP1 gene result in a recessive drug resistance phenotype. To identify missense mutations that define amino acid (aa) residues in RBP1 involved in drug sensitivity, we selected and genetically characterized over 250 independent RmR rbp1 mutants and screened them for both RBP1-specific mRNA and protein expression. Whereas all rbp1 mutants expressed abundant levels of RBP1 mRNA, stable RBP1 protein production was detected in only one mutant strain. The RBP1 gene was PCR-generated (in triplicate) from several rbp1 mutants and independent clones were sequenced. Most of the immunoblot-negative alleles were found to contain various types of null mutations; however, some alleles contained specific missense mutations that apparently affect protein stability in vivo. The single immunoblot-positive allele was found to contain a mutation altering a specific residue (Tyr89) which is conserved among the known FKBPs, and which, based on the solution and x-ray structures of human FKBP12, has been proposed to be part of a hydrophobic drug-binding pocket for FK506 and Rm.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Rapamycin resistance in yeast involves mutations in the RBP1 gene. A specific mutation at Tyr89 in RBP1 affects protein stability and drug binding, impacting rapamycin sensitivity.
Area of Science:
- Molecular Biology
- Immunology
- Mycology
Background:
- Rapamycin (Rm) is an antifungal macrolide with immunosuppressive properties.
- Its action involves binding to FKBP/RBP proteins, inhibiting peptidyl-prolyl cis-trans isomerase (PPIase) activity.
- In yeast, rapamycin sensitivity (Rms) is mediated by RBP1, a homolog of human FKBP12.
Purpose of the Study:
- To identify specific amino acid residues in yeast RBP1 critical for rapamycin sensitivity.
- To characterize missense mutations affecting RBP1 protein stability and drug interaction.
Main Methods:
- Genetic characterization of over 250 independent rapamycin-resistant (RmR) rbp1 mutants in Saccharomyces cerevisiae.
- Screening for RBP1 mRNA and protein expression using immunoblotting.
- PCR amplification and sequencing of the RBP1 gene from selected mutants.
Main Results:
- Most rbp1 mutants showed abundant RBP1 mRNA but lacked stable protein.
- Several missense mutations were identified that appear to destabilize the RBP1 protein in vivo.
- A single drug-sensitive allele contained a mutation at Tyr89, a conserved residue in the proposed FKBP drug-binding pocket.
Conclusions:
- Specific amino acid residues, like Tyr89 in RBP1, are crucial for maintaining protein stability and drug sensitivity.
- Mutations affecting these residues can lead to altered protein stability and rapamycin resistance.
- This study provides insights into the structural basis of rapamycin-binding protein interactions.