Abstract
HBR1 (hemoglobin response gene 1) is an essential gene in Candida albicans that positively regulates mating type locus MTLα gene expression and thereby regulates cell type-specific developmental genes. Hbr1p contains a phosphate-binding loop (P-loop), a highly conserved motif characteristic of ATP- and GTP-binding proteins. Recombinant Hbr1p was isolated in an oligomeric state that specifically bound ATP with K(d) ∼2 μM. ATP but not ADP, AMP, GTP, or dATP specifically protected Hbr1p from proteolysis by trypsin. Site-directed mutagenesis of the highly conserved P-loop lysine (K22Q) and the less conserved glycine (G19S) decreased the binding affinity for soluble ATP and ATP immobilized through its γ-phosphate. ATP bound somewhat more avidly than ATPγS to wild type and mutant Hbr1p. Although Hbr1p exhibits sequence motifs characteristic of adenylate kinases, and adenylate kinase and ATPase activities have been reported for the apparent human ortholog of Hbr1p, assays for adenylate kinase activity, autophosphorylation, and ATPase activity proved negative. Overexpression of wild type but not the mutant forms of Hbr1p restored MTlα2 expression in an HBR1/hbr1 mutant, indicating that ATP binding to the P-loop is necessary for this function of Hbr1p.
Highlights
Candida albicans typically resides as a commensal in the human gastrointestinal tract but becomes an opportunistic pathogen in immunocompromised hosts
HBR1 is an essential gene in C. albicans [5], but its function in mating is not sufficient to explain its requirement during vegetative growth
Hbr1 Protein Expression—C. albicans Hbr1-His6 was expressed in S. cerevisiae and purified by two sequential nickel affinity chromatographic steps
Summary
P-loop, phosphate binding loop; AK, adenylate kinase; LDS, lithium dodecyl sulfate; MTL␣, mating type-like locus ␣; SUMO, small ubiquitin-related modifier; NMP, nucleoside monophosphate. Nuclear localization of AD-004 may depend upon its interaction with coilin, a major protein component of Cajal bodies [19, 20]. These bodies are extranucleosomal maturation sites of snRNAs, small nucleolar RNAs, and histone mRNA 3Ј ends [21]. The primary amino acid sequence of Hbr1p differs in two aspects from these potential orthologs: (i) a 9-amino acid insertion between the P-loop and predicted NMP-binding domain and (ii) a highly negatively charged carboxyl terminus (Fig. 1). We report effects of site-directed mutations in the P-loop on ligand binding to Hbr1p and functional effects of Hbr on MTL gene expression
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
