The recently resolved structures of Sav1866 (3
) and crosslinking studies (4
) provide insights into the coupling interface of ABC proteins. The coupling of ATP hydrolysis (at the NBDs) to transport (at the TMDs) is one of the most critical steps of the transport cycle of ABC proteins. Characterizing the coupling interface in a functional protein, however, is challenging. We previously developed a genetic screen coupled with biochemical assays to identify residues associated with the coupling interface in the yeast ABC transporter Pdr5 (6
). In that study, we exploited a mutation at the extracellular end of TMH2 (S558Y). ATP hydrolysis and molecular movements in the TMHs (which bring about transport) are uncoupled in the mutant Pdr5, which shows both ATP hydrolysis and binding of the transport substrate, IAAP, but no drug resistance. Our strategy was to screen for second-site mutations that restore drug resistance, because these suppressors should identify interacting residues. We reported that the double-mutant S558Y, N242K exhibited almost a complete reversal of the null phenotype of S558Y. The location of N242K was consistent with the NBD face of the coupling interface surmised from the X-ray crystallographic structure of the bacterial ABC multidrug transporter Sav1866 (3
) as well as with crosslinking studies on P-glycoprotein (4
). In the current study we extended our work to further elucidate the coupling interface of Pdr5, identifying key residues in both the NBD face of the protein and ICL1, which connects TMHs 2 and 3.
We used the mutant S558Y to screen for clones that reverse the drug-sensitive phenotype of this mutant as described in detail previously (6
). Upon sequencing the Pdr5 gene in the revertants, we identified nine second-site mutations (including N242K from the previous study), representing 5 unique point mutations and one triplet-codon deletion. Four of these occurred in the NBD and were located almost contiguous to each other at positions 242, 244, and 246 (one independent mutant each of N242K and E244G and two independent mutants with D246del). The residue Glu-244 is equivalent to the conserved glutamine that is used to identify the Q-loop within the NBDs of ABC transporters. Thus, four of the nine second-site mutations appear to define the NBD face of the coupling interface and are proximal to or at the Q-loop. An additional 3 are in Ser-597, which is in the ICL1. The structure of Sav1866 suggested that conserved Q might interact with the ICL1 to couple ATP hydrolysis to drug transport. The residues we identified lie in the same region of Pdr5. Moreover, the residue Glu-244 in Pdr5 lies at the same position as the conserved Q in other ABC transporters, and the glutamic acid at this position is completely conserved in the Pdr5 family (see sequence alignment, Figure S1 in the supporting information
). Our results are consistent with the suggestion put forward by several authors that the Q-loop is implicated in intradomain coupling (1
). Beside the second-site mutations in the NBD, we identified three additional point mutations in ICL1, and all were in the same residue, Ser-597. Thus, both faces of the interface—the NBD as well as the ICL—were recovered in our collection. Although ICL1 is predicted to be approximately 21 amino acids long, the identification of Ser-597 as a second-site mutant in three independently derived colonies suggests that relatively few residues in this loop actually participate in critical contact with the ATP-binding sites. Alternatively, the codons specifying Ser-597 may have a higher likelihood of undergoing mutation than those specifying the other ICL 1 residues.
The genetic screen therefore provides a functional confirmation of the suggestion prompted by the structure of Sav1866 that the NBDs interact with the TMDs via ICLs. This is found in all ABC transporters analyzed to date. However, the functional, genetic evidence from the current study suggests that the Pdr5 signaling interface is in the cis configuration—at least when transporting clo. The inferred pathway is N-terminal NBD Q-loop region (Asn-242, Glu-244, Asp-246) to ICL-1 (Ser-597) to TMD1 (Ser-558, Met-679). An atomic model of Pdr5 shows that E244 lies very close to ICL1, but it is also directly under ICL4, which connects TMH 10 and 11 in TMD2 (R. Rutledge, unpublished observations). Thus, a trans configuration is also theoretically feasible. However, we did not recover any NBD2 (trans) suppressors of S558Y, but we found 4 independent mutations in NBD1. Similar results were obtained by selecting cyhr
suppressors of N242K in NBD1 (see Figure S2 in the supporting materials
). It remains to be seen whether the trans configuration is ever used physiologically. An interesting future experiment would be to determine whether selection of S558Y suppressors on other Pdr5 transport substrates leads to second-site mutations in NBD2.
Two possible explanations are strongly implied by these findings. Pdr5 may differ from all prokaryotic and eukaryotic ABC efflux pumps analyzed to date. Structural studies of several importers, however, such as E. coli
ButCD, which influxes vitamin B, and ModBC, which is an archea molybdate pump, appear to have a signal interface in the cis configuration (23
). Because Pdr5 is evolutionarily distant from these, it would be important to determine whether other eukaryotic members of the ABC family have this arrangement. Other studies were conducted in the absence of transport substrate, whereas our mutants were selected on drug plates, so it is also quite possible that both cis and trans exist physiologically. For instance, it is plausible that drug binding results in conformational switching.
As illustrated in and S1, the Walker A and Walker B domains of the N-terminal NBD and the signature region of the second NBD vary significantly in the identity of key conserved residues vis-à-vis other ABC transporters. Thus, together they make up a deviant ATP-binding site. Because Glu-244 replaces the canonical Gln in the Q-loop, we characterized it in an otherwise WT background as well as the equivalent Gln-951 in NBD2.
The effect of site-directed mutagenesis on Q-loop residues was studied in mouse P-gp by Urbatsch et al. (17
). In mouse P-gp, these residues are Gln-471 and Gln-1114 in the NBD1 and 2, respectively. The mutants Gln-471A and Gln-1114A showed a reduction in ATPase activity. However, in no case was the impairment greater than two orders of magnitude, and the Km
(ATP) was not altered, leading the authors to conclude that the mutations had no major effect on the substrate binding or on reaction chemistry (17
). Furthermore, mutations in either of the two P-gp catalytic sites produced the same effects, implying functional symmetry.
Because the most obvious effect of the Gln-471A or Gln-1114A mutation was to reduce stimulation of ATPase activity by transport substrates, it was hypothesized that such residues play a critical role in interdomain communication. Mutations in either the Glu-244 or Gln-951 residue of Pdr5 had similar effects on ATP hydrolysis ( and ); although ATP hydrolysis was diminished, a significant residual activity remained, and the Km was unaffected. We also studied E244G, Q951G, which showed no less ATPase activity than that observed in either of the two single mutants (). GTPase activity, which our previous work suggests has a physiological role to play in Pdr5-mediated transport, decreased even less than ATPase activity. Thus, our data are consistent with these residues being nonessential to the reaction chemistry (such as activation of the attacking water for ATP hydrolysis). However, unlike the Km of ATP hydrolysis, the IC50 for the Pdr5 transport substrates clo and cyh is significantly decreased in both the E244G or Q951G mutant. Moreover, Pdr5-mediated drug resistance is largely abrogated in the double mutant, E244G, Q951G. Its phenotype is almost equivalent to the S558Y strain originally used to isolate the suppressor collection ().
It was evident that the reduced resistance to drug substrates in the E244G, Q951G double mutant was not a consequence of impaired binding of the transport substrate. We demonstrated that the photoaffinity transport substrate analog IAAP crosslinked to WT and mutant Pdr5s with comparable efficiency (). E244G was initially isolated as a suppressor of the faulty-signaling S558Y mutant, and the results from the current study further indicate that the primary role of the conserved residues Glu-244 and Gln-951 is to communicate signals from the ATP sites to the TMDs via the ICLs. Furthermore, the much greater drug sensitivity of the double mutant and the similarity in phenotype of the single mutants strongly implies that these residues are functionally overlapping even though one is from a deviant portion of NBD-1.
Equivalence of function is, of course, a striking feature of symmetric transporters such as P-gp; hence the phenotypic similarity of the Q-loop residue mutants (Q471A and Q1114A). Asymmetric transporters may differ, however. For instance, mutation of the conserved Walker A lysine in Tap1 and the analogous residue in Tap2 (Tap1 and Tap2 make up a peptide-translocating heterodimer) results in different effects on ATP binding and peptide transport (25
). A similar nonequivalence is observed in MRP1. Mutation of the same residue in the Walker A motif of each NBD has disparate effects on nucleotide binding (24
). The observation, therefore, that the Q-loops of Pdr5 are equivalent and overlapping strongly implies that both ATP-binding sites are sending signals to the TMDs.
Pdr5 has a high basal ATPase that is not stimulated further by the addition of exogenous transport substrates (11
). In this regard, it is strikingly different from P-gp. Our data also strongly suggest that Pdr5 is not especially efficient at coupling ATP hydrolysis to transport and they support the contention that much of this activity is uncoupled (19
). For instance, although the S558Y, E244G and S558Y, N242K suppressors restore cyh resistance 8-fold, they show no restoration of ATPase activity. The Pdr5 family of fungal transporters appears distinct from all other eukaryotes and may be a unique, but clinically important evolutionary variant.