ATP Binding to Hemoglobin Response Gene 1 Protein Is Necessary for Regulation of the Mating Type Locus in Candida albicans

Abstract

HBR1 (hemoglobin response gene 1) is an essential gene inCandida albicans that positively regulates mating type locus MTL␣ 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 ϳ2 M. 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 ␥-phosphate. ATP bound somewhat more avidly than ATP␥S 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 MTl␣2 expression in an HBR1/hbr1 mutant, indicating that ATP binding to the P-loop is necessary for this function of Hbr1p.

Hbr1 Activates and Represses Hyphal Growth in Candida albicans and Regulates Fungal Morphogenesis under Embedded Conditions

“…HBR1 heterozygosis leads to an increased rate of opaque cell formation accompanying a turnoff of the MTL α2 gene and the ability to mate as an ‘a’ cell [ 18 , 19 ]. Hbr1 possesses a nucleotide-binding P-loop that is ATP-specific and is necessary for trypsin-resistance of the purified protein [ 20 ]. The human ortholog AD-004 (CINAP, ADK6) was originally identified as a binding partner of the Cajal body component coilin.…”

“…Adk6 possesses both adenylate kinase and ATPase activities [ 21 , 22 ]. However, Hbr1 possesses neither of these activities [ 20 ]. The S .…”

“…The Hbr1 protein has a negatively charged carboxyl terminus that is the main contributor to the net negative charge of Hbr1 (calculated −47, pI 3.9) but is unique among Hbr1 orthologs. This region is predicted to be an intrinsically disordered domain that only assumes structure in the appropriate cellular context [ 20 ]. Members of this protein group include Histone H1, the Gcn4 nucleotide-binding domain, eIF4E binding proteins 1 and 2 and heat shock transcription factor [ 25 ].…”

Site-Directed and Random Insertional Mutagenesis in Medically Important Fungi

“…This study identified amino acid residues important for mating locus regulation, but not for vegetative growth. Thus, amino acids identified to be important in model fungal species do not always translate to related pathogenic fungi (Peterson et al, 2011).…”

“…It was known that HBR1 induced mating type genes, but mating is not an essential process in C. albicans (Peterson et al, 2011). Sites required for optimal growth and the oxidative stress response in the homologous gene in Saccharomyces were targeted in the C. albicans gene.…”