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Functional analysis of H-loop residues of Cdr1p, an ABC
transporter of human fungal pathogen Candida albicans
Antresh Kumar 1 and Rajendra Prasad 1
1 School of Life Sciences, Jawaharlal Nehru University, New Delhi, INDIA
Nucleotide Binding Domains (NBDs) of multidrug transporter of Candida albicans,
Cdr1p possess unique divergent amino acids in their conserved motifs. For
example, NBD1 possesses divergent H-loop (IYQ) while the same motif is
conserved (IHQ) in NBD2.
We report here the contribution of these conserved and divergent H-loop motifs of
Cdr1p in ATP catalysis and drug transport. For this, we mutagenized two residues
of divergent (IYQ) and conserved H-loop region (IHQ) either by replacing them with
alanines or replacing residues with equiposition residues of another H-loop. These
mutations were introduced into GFP-tagged Cdr1p which was stably overexpressed
at the PDR5 locus in a heterologous host S. cerevisiae mutant strain, AD1-8u - which
lacks seven major ABC transporters.
All the Cdr1p mutant variants of H-loop region were properly expressed and
localized to the cell surface similar to wild-type protein. Alanine mutants of H-loop
region, Y361A in NBD1 has no effect on Cdr1p function and showed normal R6G
transport and ATPase activity whereas corresponding mutation (H1059A) in NBD2
abolished R6G transport without any significant loss of ATPase activity. Moreover,
cells expressing mutation like Q362A severely abrogated Cdr1p function while
Q1060A mutation in NBD2 showed no effect on Cdr1p function and measured
normal transport and ATPase activity similar to wild type. Though, Arginine
substitution of glutamine residue present in H-loop region of Cdr1p (Q362R and
Q1060R) displayed deferential effect on Cdr1 transport. These results suggest that
not only the conserved residue like H1059 but divergent Q362 is also crucial for
Cdr1p function.
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