Directed evolution of P-glycoprotein cysteines reveals site-specific, non-conservative substitutions that preserve multidrug resistance
| Autor: | Guillermo A. Altenberg, Anukriti Singh, Douglas J. Swartz, Sri K. Botta, Ina L. Urbatsch, Leo Mok |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2014 |
| Předmět: |
non-conservative cysteine substitutions
Mutant Amino Acid Motifs Biophysics lcsh:Life lcsh:QR1-502 TMD transmembrane domain DDM n-dodecyl-β-D-maltopyranoside protein evolution site-saturation mutagenesis ICL intracellular loop Biology Biochemistry S2 ABC ATP-binding-cassette lcsh:Microbiology Serine Mice Pgp P-glycoprotein Animals CFTR cystic fibrosis transmembrane conductance regulator ATP Binding Cassette Transporter Subfamily B Member 1 Cysteine NBD nucleotide-binding domain Molecular Biology Alanine chemistry.chemical_classification Original Paper CL Cys-less Walker motifs multidrug transporter Cell Biology Directed evolution Molecular biology WT wild-type Drug Resistance Multiple 3. Good health Amino acid lcsh:QH501-531 chemistry Amino Acid Substitution Glycine SEC size exclusion chromatography ABC transporter Directed Molecular Evolution CP-MTS 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin yeast drug resistance |
| Zdroj: | Bioscience Reports, Vol 34, Iss 3, p e00116 (2014) Bioscience Reports |
| ISSN: | 1573-4935 |
| Popis: | Pgp (P-glycoprotein) is a prototype ABC (ATP-binding-cassette) transporter involved in multidrug resistance of cancer. We used directed evolution to replace six cytoplasmic Cys (cysteine) residues in Pgp with all 20 standard amino acids and selected for active mutants. From a pool of 75000 transformants for each block of three Cys, we identified multiple mutants that preserved drug resistance and yeast mating activity. The most frequent substitutions were glycine and serine for Cys427 (24 and 20%, respectively) and Cys1070 (37 and 25%) of the Walker A motifs in the NBDs (nucleotide-binding domains), Cys1223 in NBD2 (25 and 8%) and Cys638 in the linker region (24 and 16%), whereas close-by Cys669 tolerated glycine (16%) and alanine (14%), but not serine (absent). Cys1121 in NBD2 showed a clear preference for positively charged arginine (38%) suggesting a salt bridge with Glu269 in the ICL2 (intracellular loop 2) may stabilize domain interactions. In contrast, three Cys residues in transmembrane α-helices could be successfully replaced by alanine. The resulting CL (Cys-less) Pgp was fully active in yeast cells, and purified proteins displayed drug-stimulated ATPase activities indistinguishable from WT (wild-type) Pgp. Overall, directed evolution identified site-specific, non-conservative Cys substitutions that allowed building of a robust CL Pgp, an invaluable new tool for future functional and structural studies, and that may guide the construction of other CL proteins where alanine and serine have proven unsuccessful. Directed evolution of Cys (cysteines) in the multidrug transporter Pgp identified novel non-conservative substitutions, revealed important domain interactions and produced a CL protein that is more robust and active than previous CL Pgp built solely on conservative alanine substitutions. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|