A novel catalytic mechanism for ATP hydrolysis employed by the N-terminal nucleotide-binding domain of Cdr1p, a multidrug ABC transporter of Candida albicans

Abstract

Although essentially conserved, the N-terminal nucleotide-binding domain (NBD) of Cdr1p and other fungal transporters has some unique substitutions of amino acids which appear to have functional significance for the drug transporters. We have previously shown that the typical Cys193 in Walker A as well as Trp326 and Asp327 in the Walker B of N-terminal NBD (NBD-512) of Cdr1p has acquired unique roles in ATP binding and hydrolysis. In the present study, we show that due to spatial proximity, fluorescence resonance energy transfer (FRET) takes place between Trp326 of Walker B and MIANS [2-(4-maleimidoanilino) naphthalene-6-sulfonic acid] on Cys193 of Walker A motif. By exploiting FRET, we demonstrate how these critical amino acids are positioned within the nucleotide-binding pocket of NBD-512 to bind and hydrolyze ATP. Our results show that both Mg2+ coordination and nucleotide binding contribute to the formation of the active site. The entry of Mg2+ into the active site causes the first large conformational change that brings Trp326 and Cys193 in close proximity to each other. We also show that besides Trp326, typical Glu238 in the Q-loop also participates in coordination of Mg2+ by NBD-512. A second conformational change is induced when ATP, but not ADP, docks into the pocket. Asn328 does sensing of the gamma-phosphate of the substrate in the extended Walker B motif, which is essential for the second conformational change that must necessarily precede ATP hydrolysis. Taken together our results imply that the uniquely placed residues in NBD-512 have acquired critical roles in ATP catalysis, which drives drug extrusion.

The Signaling Interface of the Yeast Multidrug Transporter Pdr5 Adopts a Cis Conformation, and There Are Functional Overlap and Equivalence of the Deviant and Canonical Q-Loop Residues

“…Analogous results were found with P-gp (17). In reporting on a study that used FRET (Forster resonance energy transfer) Rai et al suggested a role for the equivalent Cdr1 residue (Glu-237) in coordinating the Mg ion during ATP hydrolysis, but this study was carried out with a single, purified NBD and did not look directly at ATPase activity or determine a drug phenotype (18). …”

Efflux pump proteins in antifungal resistance

“…Conformational changes induced by ATP binding and/or hydrolysis are transmitted from the NBDs to the TMDs, which can result in drug translocation, drug efflux, or resetting of the reaction cycle (Gupta et al, 2011). In addition to powerhouse of the protein, several residues in NBDs have also been found to alter drug specificity toward many drugs as shown in Figure 3 (Jha et al, 2003, 2004; Shukla et al, 2003; Rai et al, 2005, 2006, 2008; Kumar et al, 2010; Prasad et al, 2012). The typical feature of all yeast ABC transporters of PDR family is that they exhibit high basal ATPase activity and that the most commonly observed feature of drug stimulated ATPase activity component is completely absent from yeast proteins.…”

The ABCs of Candida albicans Multidrug Transporter Cdr1

“…The experiments that followed to investigate the functional relevance of sequence degeneracy in isolated domains revealed that atypical C193 of Walker A and W326 of Walker B of N-NBD are in close proximity in the ATP binding pocket, where the former residue participates in hydrolysis while the latter impacts the binding of the nucleotide (35,36). Interestingly, well-conserved D327, which is otherwise the catalytic carboxylate in other ABC transporters, appears to have a new role and behaves as a catalytic base involved in ATP hydrolysis in this transporter (37).…”