Newly identified motifs in Candida albicans Cdr1 protein nucleotide binding domains are pleiotropic drug resistance subfamily-specific and functionally asymmetric.

Manpreet Kaur Rawal,Mohammad Firoz Khan,Brian C Monk,Rajendra Prasad,Ajay Kumar Saxena,Richard D Cannon,Rakesh Bhatnagar,Atanu Banerjee,Sobhan Sen,Abdul Haseeb Shah,Alok Kumar Mondal

doi:10.1038/srep27132

Manpreet Kaur Rawal, Mohammad Firoz Khan + Show 9 more

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https://doi.org/10.1038/srep27132

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An analysis of Candida albicans ABC transporters identified conserved related α-helical sequence motifs immediately C-terminal of each Walker A sequence. Despite the occurrence of these motifs in ABC subfamilies of other yeasts and higher eukaryotes, their roles in protein function remained unexplored. In this study we have examined the functional significance of these motifs in the C. albicans PDR transporter Cdr1p. The motifs present in NBD1 and NBD2 were subjected to alanine scanning mutagenesis, deletion, or replacement of an entire motif. Systematic replacement of individual motif residues with alanine did not affect the function of Cdr1p but deletion of the M1-motif in NBD1 (M1-Del) resulted in Cdr1p being trapped within the endoplasmic reticulum. In contrast, deletion of the M2-motif in NBD2 (M2-Del) yielded a non-functional protein with normal plasma membrane localization. Replacement of the motif in M1-Del with six alanines (M1-Ala) significantly improved localization of the protein and partially restored function. Conversely, replacement of the motif in M2-Del with six alanines (M2-Ala) did not reverse the phenotype and susceptibility to antifungal substrates of Cdr1p was unchanged. Together, the M1 and M2 motifs contribute to the functional asymmetry of NBDs and are important for maturation of Cdr1p and ATP catalysis, respectively.

Highlights

An analysis of Candida albicans adenosine triphosphate (ATP) binding cassette (ABC) transporters identified conserved related α-helical sequence motifs immediately C-terminal of each Walker A sequence
We have shown that these new motifs are functionally asymmetric in both NBDs i.e. the motif in NBD1 is critical for proper localisation or folding of Cdr1p and the motif in NBD2 is required for ATP hydrolysis
We previously used comparative primary sequence alignment to identify among ATP binding cassette (ABC) proteins in C. albicans two NBD-specific variants of a motif characteristic of the pleiotropic drug resistance (PDR) subfamily of ABCG proteins[17]

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An analysis of Candida albicans ABC transporters identified conserved related α-helical sequence motifs immediately C-terminal of each Walker A sequence. They bind and hydrolyze cellular ATP, energizing translocation of a variety of structurally unrelated compounds across the PM8,9 Due to their common substrate (ATP), NBDs from different ABC proteins share extensive amino acid sequence identity and motifs that are considered to be critical for the domain function[10]. The swapping of C193 of NBD1 with the conserved K901 of NBD2, in the context of the full protein, was not tolerated and cells expressing the swapped variants displayed very low ATPase activity and high susceptibility to drugs[14] It has been shown for the closely related ABC transporter Pdr5p that mutations in the divergent ATP binding site did not significantly affect ATPase activity[15].

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