Functional Characterization of N-Terminal Nucleotide Binding Domain (NBD-1) of a Major ABC Drug Transporter Cdr1p of Candida albicans:  Uncommon but Conserved Trp326 of Walker B Is Important for ATP Binding

Abstract

Using purified N-terminal NBD (NBD-512) domain of Cdr1p, a major multidrug extrusion pump of human pathogenic yeast Candida albicans, we show the relevance of the unique positioning of an atypical Trp326 residue. Similar to Cys193 in Walker A, Trp326 in the Walker B motif of Cdr1p is also a conserved feature of other fungal ATP Binding Cassette (ABC) transporters. By employing fluorescence spectroscopy, chemical modification, and site-directed mutagenesis, we demonstrate that of the five Trp residues in the NBD-512 domain, Trp326 alone is important for nucleotide binding and subsequent conformational changes within the domain. Furthermore, mutation of Trp326 to Ala results in an increased K(M) without appreciably affecting V(max) of ATPase activity. Thus, Trp326 in NBD-512 appears to be important for nucleotide binding and not for its hydrolysis. Additionally, the role of Trp326 in ATP binding is independent of the presence of the adjacent well-conserved Asp327 residue which, like Cys193, has a catalytic role in ATP hydrolysis. Considering that Trp326 of Cdr1p is a typical feature of fungal transporters alone, our study suggests that these ABC transporters may reflect mechanistic differences with regard to nucleotide binding and hydrolysis as compared to their counterparts of non-fungal origin.

Conserved Asp327 of Walker B Motif in the N-Terminal Nucleotide Binding Domain (NBD-1) of Cdr1p ofCandida albicansHas Acquired a New Role in ATP Hydrolysis

“…Fungal ABC transporters pose additional challenges since they have typical amino acid substitutions in conserved motifs of NBDs which suggest possibility of mechanistic differences in ATP catalysis cycle (20). In an attempt to understand the molecular basis of ATP hydrolysis mediated by major ABC multidrug transporter of C. albicans, Cdr1p, we have demonstrated that the uncommon and atypical conserved Trp326 in the Walker B motif of N-terminal NBD is crucial for ATP binding (23). We have also shown that another uniquely replaced residue Cys193 in the Walker A motif of N-terminal NBD is critical for ATP hydrolysis (22).…”

“…To begin defining the functional significance of the conserved substitutions in N-terminal NBD of Cdr1p, we have recently demonstrated that the replacement of Cys193 with Ala gravely impaired the ATP hydrolysis without affecting its ability to bind the nucleotide (21,22). On the other hand, substitution of Trp326 with Ala resulted in the loss of ATP binding to purified N-terminal NBD (NBD-512) (23). A mutagenesis screen of another conserved residue underscored the importance of the highly conserved, putative catalytic carboxylate residue Asp327 of the Walker B motif in the purified N-terminal NBD of Cdr1p.…”

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).…”

Relative Contributions of the Candida albicans ABC Transporters Cdr1p and Cdr2p to Clinical Azole Resistance

“…It was shown that the two transporters localize at the plasma membrane (35,37), bind rhodamine 6G (R6G) (12), export their substrates in an energy-dependent manner, and possess ATPase and phospholipid translocase activities (15,39). Expression systems in S. cerevisiae have also proved useful for structure-function studies of the transmembrane and ATPbinding domains of Cdr1p (14,26,31).…”