The Role of Pro-596 and Val- 656 in Pdr5 Domain Communication.

摘要

ABC transporters contribute to drug resistance in a wide variety of cells from human cancer tissue to parasites, bacteria and fungi. Though a signal interface has been defined in Pdr5, the mechanisms that lead to increased resistance in Pdr5 gain-of-function mutants are not understood. The biochemical study of Pdr5 mutations will lead to a better understanding of domain coupling in ABC transporters. An understanding of the mechanics of ABC transporters such as Pdr5 could lead to therapies targeted against these efflux pumps allowing drugs to be utilized in successfully treating patients suffering from cancer and parasitic, bacterial or fungal infections. Sequence alignments of several members of the Pdr5 subfamily of ABC transporters show specific conserved amino acid residues are present in the intracellular loops and a portion of NBD1 known as the Q-loop. It has also been shown that interdomain communication takes place through the coordination of some of these conserved residues.;The first goal of this project was to determine whether Pro-596 (ICL1) and Val-656 (ICL2) and Glu-244 (Q-loop of NBD1) in the Pdr5 signaling interface interact to promote communication between ATP binding sites and substrate transport sites. A second goal was to investigate the mechanism for the increase in resistance of yeast strains containing amino acid substitutions at residues 596 and 656. This resistance may be explained by an increase in protein concentration in the plasma membrane, an increase in ATPase or GTPase activity of the Pdr5 transporter or alteration of the efficiency of signaling between domains.;While Val-656 had previously been shown as an essential residue for substrate transport, Pro- 596 does not seem to be essential due to the fact that alanine replacement at this position does not result in a transport deficient protein. Also, replacement with threonine, an amino acid that is more flexible and polar, results in increased resistance to most substrates tested when compared to the wild type and P596L mutant transporters.;Pro- 596 takes part in intradomain communication between ICL1 and ICL2. The P596L mutation suppresses the hypersensitivity of the profoundly signal-deficient V656A residue thus establishing a genetic interaction between ICL1 and ICL2 in Pdr5. The double mutant P596L, E244G however, showed significant decreases in resistance to substrates tested, indicating a negative, non-additive interdomain interaction.;Western blotting indicates that changes in substrate resistance in mutant Pdr5 transporter strains were not due to changes in protein expression. Biochemical assays measuring NTPase activity however, did indicate differences when compare to the wild type. P596L, E244G transporter has a significantly decreased ATPase activity similar to that of the single mutant E244G (Ananthaswamy, et al., 2012). P596L, V5656A, unlike the single mutant V656A transporter, has an ATPase activity similar to that of the wild type showing the adding the P596L mutation restores the activity and in turn partially suppresses the null phenotype of the V656A mutation. P596L transporters have a significantly decreased ATPase activity while retaining GTPase activity similar to the wild type transporter. Therefore, the NTPase activity of this mutant transporter is uncoupled.;Inhibition of ATP hydrolysis in mutant transporters tested is also unaffected indicating that while the ATPase activity of some mutant transporters are significantly decreased when compared to the wild type, the effect of clotrimazole on the ATPase activity remains unchanged. This is another example of the uncoupling of hydrolysis and allosteric inhibition in the transporter when the mutation P596L is present. Rather than having an increase in resistance to allosteric inhibition seen with other transport deficient mutations that have been previously described (Downes, et al., 2013), the transporter remains affected at a concentration of clotrimazole comparable to that of the wild type.;Rhodamine G assays indicate that Pdr5 mutant yeast strains that have similar or greater resistance than the wild type to some substrates such as cycloheximide are also more resistant to R6G. Mutant Pdr5 containing yeast strains that have decreased resistance to substrates also show a decrease in resistance to R6G. Direct measured of R6G transport in whole cells indicates that while the P596L mutation causes a decrease in ATPase activity of the transporter, this level of activity is high enough for transport of R6G and that the P596L, V656A mutant shows markedly improved efflux capability relative to V656A returning the R6G fluorescent reading to a level similar to that of the wild type strain, confirming the phenotype seen in IC50 assays.