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.
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.
Rai
Versha
V
Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India.
Shukla
Sudhanshu
S
Jha
Sudhakar
S
Komath
Sneha Sudha
SS
Prasad
Rajendra
R
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D000409:Alanine / Q000235:genetics; D020816:Amino Acid Motifs / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D001974:Bromosuccinimide / Q000737:chemistry; D002176:Candida albicans / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence / Q000235:genetics; D025141:Drug Resistance, Fungal / Q000235:genetics; D005456:Fluorescent Dyes / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D013053:Spectrophotometry; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics
D018528:ATP-Binding Cassette Transporters; C094693:CDR1 protein, Candida albicans; D005456:Fluorescent Dyes; D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D010446:Peptide Fragments; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D008274:Magnesium; D001974:Bromosuccinimide; D000409:Alanine