A series of site-directed mutagenesis experiments were carried out on specific amino acid residues in the proximal and distal binding pockets to investigate and confirm their roles in vivo. The mutagenesis experiments were carried out on E. coli cells and the effects of these mutations were observed and recorded by quantifying the level of resistance of the cells against erythromycin and doxorubicin. The drug resistance levels of the E. coli mutants were quantified by measuring cell growth at subinhibitory doses of each drug.
Mutagenesis in Proximal Pocket
Key residues, structures and interactions
- Ser134 and Ser135
- Form hydrogen bonds with erythromycin
- Ser79, Thr91 and Asp681
- Do not interact directly with erythromycin but are involved in the physical access of the drug to the pocket
- Ser134Ala, Ser135Ala
- Eimination of hydrogen bonds
- Ser79Arg, Thr91Arg
- Introduction of long side chains
- Asp681Trp
- Introduction of long side chain
- Significantly decreased cell growth at subinhibitory concentrations of erythromycin due to decreased efflux of and resistance to the drug
- Efflux of and resistance to doxorubicin unaffected due to (a) weak interaction between the drug and the proximal pocket and (b) ability of the low MW drug to travel through the pocket
- Inhibition of doxorubicin efflux by erythromycin completely disappeared*
- Affirms the hypothesis that high and low MW drugs that bind the proximal and distal pockets respectively travel through a common translocation pathway
Mutagenesis in the Distal Pocket
Key residues/ structures and interactions
- Phenylalanine cluster region
- Involved in the binding of doxorubicin
- Phe-617 loop
- Involved in the binding of erythromycin as well as doxorubicin
- Phe610Ala, Phe628Ala
- Decreased efflux of and resistance to erythromycin
- Abolished efflux of and resistance to doxorubicin
- Conclusive from both observations that high and low MW drugs travel through the distal pocket of the binding monomer
- Mutation affected doxorubicin efflux to a higher degree due to the correspondingly higher level of interaction of the drug with the distal pocket (compared to erythromycin)
2. Single Mutation (Loop)
- Phe617Ala
- No effect on efflux of and resistance to both erythromycin and doxorubicin
- Alteration of Phe617 at the tip of the loop has no effect on the ability of drugs to bind to either pocket
- Implies that interactions between this residue with bound drugs are not significant
- Gly614Pro, Gly621Pro
- Gly616Pro, Gly619Pro
- Introduction of proline to rigidify the loop and decrease its capacity for movement
- Resistance to erythromycin and doxorubicin close to complete elimination, almost to the level of arcrB defecient strains
- Efflux of doxorubicin completely abolished
- Both observations imply that movement of the loop- linked with conformational change from the access to binding form of AcrB monomers- is involved and important for the efflux of both high and low MW drugs
Further Insight
- Loop is constituted of the segment Gly614- Gly621
- Phe617 located at the tip
- Loop separates distal from proximal binding pocket in AcrB monomer and its orientation affects these pockets
- Orientation influenced by its flexibility which is in turn determined by amino acids at positions 614, 616, 619 and 621
- Glycine: Contains the smallest side chain among all amino acids
- Inclusion in the loop confers upon the structure a wide range of flexibility
- Proline: Cyclic side chain has intrinsic rigidity
- Decreases flexibility when included in loop
- Observed as reduced capacity for motion when double mutations introduced
- Prevented conformational change from access to binding form, hence preventing the drugs from being dispelled
- The nature of residue 617 at tip of loop is inconsequential but its position during conformational change is key
- Position of residue influenced by flexibility of loop
No comments:
Post a Comment