W1038 near D-loop of NBD2 is a focal point for inter-domain communication in multidrug transporter Cdr1 of Candida albicans

Abstract

Candida drug resistance 1 (Cdr1), a PDR subfamily ABC transporter mediates efflux of xenobiotics in Candida albicans. It is one of the prime factors contributing to multidrug resistance in the fungal pathogen. One hallmark of this transporter is its asymmetric nature, characterized by peculiar alterations in its nucleotide binding domains. As a consequence, there exists only one canonical ATP-binding site while the other is atypical. Here, we report suppressor analysis on the drug-susceptible transmembrane domain mutant V532D that identified the suppressor mutation W1038S, close to the D-loop of the non-catalytic ATP-binding site. Introduction of the W1038S mutation in the background of V532D mutant conferred resistance for most of the substrates to the latter. Such restoration is accompanied by a severe reduction of ATPase activity, of about 85%, while that of the V532D mutant is half-reduced. Conversely, alanine substitution of the highly conserved aspartate D1033A in that D-loop rendered cells selectively hyper-susceptible to miconazole without an impact on steady-state ATPase activity, suggesting altogether that ATP hydrolysis may not hold the key to restoration mechanism. Analysis of the ABCG5/ABCG8-based 3D-model of Cdr1p suggested that the W1038S substitution leads to the loss of hydrophobic interactions and H-bond with residues of the neighbor NBD1, in the non-catalytic ATP-binding site area. The compensatory effect within TMDs accounting for transport restoration in the V532D-W1038S variant may, therefore, be mainly due to an increase in NBDs mobility at the non-catalytic interface.

ABC Transporter Genes Show Upregulated Expression in Drug-Resistant Clinical Isolates of Candida auris: A Genome-Wide Characterization of ATP-Binding Cassette (ABC) Transporter Genes

“…This separation of clusters within a subfamily corroborates previous studies, which suggested that a common ancestor gives rise to each half of the proteins within each subfamily, and also may allocate different functions (Daumke and Knittler, 2001). More recent data also point to this functional asymmetry between N- and C-terminal NBDs (Banerjee et al, 2018). Notably, the presence and the positional order of TMDs and NBDs in each subfamily are specific, and this domain arrangement creates a topological difference among different members of subfamilies.…”

All about CDR transporters: Past, present, and future

“…A recent study by Banerjee et al (Banerjee, Haseeb, Kumari, Moreno, & Falson, ; unpublished results) further reinforced the point, where through suppressor screens against TMH mutants, the authors dissected two important residues that act as focal point for interdomain communication. The two primary mutants, V532D and L529A, characterized earlier are part of TMH1 and close to the extracellular side of the membrane.…”

“…When S. cerevisiae strains overexpressing the respective mutants were grown in the presence of toxic concentrations of xenobiotics, spontaneous resistant colonies harbouring intragenic mutations could be isolated. Whereas the suppressor for V532D mapped to a site very close to the D‐loop of C‐NBD and part of the noncanonical NBS (Banerjee et al, ), the suppressor for L529A mapped within the signature sequence of noncanonical NBS (Banerjee et al, ; unpublished results). Interestingly, in both cases, it was observed that the second‐site mutations did not correct the primary defects of the mutants but instead modulated the interdomain crosstalk.…”

ABCG: a new fold of ABC exporters and a whole new bag of riddles!

“…This is the case, for example of the human heterodimer ABCG5/G8 displayed in Figure 1, or that of the full transporters Pleiotropic drug resistance protein 5 (Pdr5p) of S. cerevisiae or Candida drug resistance protein 1 (Cdr1p) of C. albicans. Initially thought to be degenerated to save energy, mutagenesis studies of Pdr5p and Cdr1p now point to a specific role of this non-hydrolytic site that modulates both the ATPase and drug-transport activities (see section 3) (Banerjee et al, 2018(Banerjee et al, , 2020Ernst et al, 2008;Prasad et al, 2019;Saini et al, 2006). (Alam et al, 2019;Kim & Chen, 2018) and of ABCG2 (bottom, PDB ID: 6VXF and 6HBU, respectively) Orlando & Liao, 2020) illustrate the differences in interdomains connections between the two types of exporters and the major conformational switch involved in substrate transport.…”

“…S558Y in Pdr5p is rescued by the secondary mutations N242K, E244G, D246, S597I/T, M679L, P596L or G1233D; N242K is rescued by K1016I, V656L, P596L, L1367F, A670S, or I1063M; E244G is rescued by V656L (Sauna et al, 2008;Ananthaswamy et al, 2010;Downes et al, 2013). E597A, V532D and L529A in Cdr1p are rescued by C1041W, R1008I and L1032F for the first one and W1038S and Q1005H, respectively, for the last two (Banerjee et al, 2018;Banerjee et al, 2020) Shah et al identified three suppressors for one of the ICL1 mutant, E597A, that mapped to the non-catalytic NBS (Shah, Banerjee, et al, 2015;. Out of the three, while two were located close to the D-loop, one mapped close to the Signature sequence.…”