ATP Binding to Hemoglobin Response Gene 1 Protein Is
Necessary for Regulation of the Mating Type Locus in
Alexander W. Peterson1, Michael L. Pendrak, and David D. Roberts2
Bethesda, Maryland 20892-1500
HBR1 (hemoglobin response gene 1) is an essential gene in
Candida albicans that positively regulates mating type locus
MTL? gene expression and thereby regulates cell type-specific
(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 Kd?2 ?M.
ATP but not ADP, AMP, GTP, or dATP specifically protected
the highly conserved P-loop lysine (K22Q) and the less con-
served 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 char-
activities have been reported for the apparent human ortholog
of Hbr1p, assays for adenylate kinase activity, autophosphory-
lation, 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 bind-
ing to the P-loop is necessary for this function of Hbr1p.
Candida albicans typically resides as a commensal in the
human gastrointestinal tract but becomes an opportunistic
tion (1). Adaptation to each host organ may require changes in
phenotype that are triggered by specific host environmental
observed at some sites of infection (2, 3). Opaque phase cells,
mating in C. albicans (4). However, suppression of this switch-
ing during vascular dissemination may help C. albicans elude
host defenses. HBR1 (hemoglobin response gene 1) is a gene
that represses white opaque switching and may play a key role
to understanding C. albicans survival in a host.
HBR1 was identified based on its differential induction in
cells cultured with hemoglobin and discovered to positively
regulate MTL? genes (5). MTL? genes, in turn, control white
opaque switching (4). HBR1 shows haplo-insufficiency for
MTL? gene expression, indicating sensitivity of the MTL sig-
naling pathway to levels of Hbr1p (5).
mating is not sufficient to explain its requirement during vege-
tative growth. In fact, the mating genes in C. albicans are not
essential for viability (6, 7), suggesting that Hbr1p plays addi-
sion is maximal during early exponential growth, attenuated
during the diauxic transition, and weak in the stationary phase
Hbr1p may serve a regulatory role at the level of protein-
protein interactions. The yeast ortholog Fap7 is also an essen-
tial gene (8) and was initially shown to interact with Krr1p, the
product of a gene required for ribosomal RNA processing (9).
FAP7 heterozygosis also leads to an 18S rRNA processing
its predicted phosphate-binding loop (P-loop)3(11). In addi-
tion, a G19S mutation prevented oxidative stress-induced acti-
vation of a Gal4-Pos9 hybrid transcription factor, indicating an
important functional role for this motif (8).
Hbr1p from C. albicans and Candida dubliniensis shares
68% amino acid sequence conservation with Fap7p and more
predicted P-loop motif (12) that is characteristic of ATP- and
GTP-binding proteins (13, 14). The P-loop pattern in Hbr1p
(GTPGCGKS) most closely resembles that of the archaeal
derived AK sequence (GXPGXGK(T/S)) (15). The absence of a
terminal glycine that follows the invariable lysine, which is a
unique feature of the archaeal AK family (15), is also a distin-
guishing feature of the AK-6 group from higher eukaryotes
(16–18) (Fig. 1).
The defining member of the nuclear-localized AK group is
human AD-004 (CINAP; AK-6), and its crystal structure has
been determined (16). The positions of the P-loop, the NMP-
binding domain, and the lid domain are indicated in the Hbr1p
primary sequence (Fig. 1). Both ATPase and AK activities have
* This work was supported, in whole or in part, by the National Institutes of
Health through the National Cancer Institute Center for Cancer Research
Intramural Research Program, ZIA SC 009173.
1Present address: National Institute of Standards and Technology, Chemical
burg, MD 20899.
2To whom correspondence should be addressed: National Institutes of
Health, Bldg. 10, Rm. 2A33, 10 Center Dr., Bethesda, MD 20892-1500. Tel.:
301-496-6264; Fax: 301-402-0043; E-mail: firstname.lastname@example.org.
3The abbreviations used are: P-loop, phosphate binding loop; AK, adenylate
small ubiquitin-related modifier; NMP, nucleoside monophosphate.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 16, pp. 13914–13924, April 22, 2011
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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 3? ends (21).
Despite major similarities, 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 car-
boxyl terminus (Fig. 1). We examine here the nucleotide bind-
ing specificity and potential enzymatic activities of Hbr1p. We
also report effects of site-directed mutations in the P-loop on
Construction of HBR1 Expression Vectors—Thrombin-His6-
tagged versions of Hbr1p were constructed in two steps. A
clone containing the entire HBR1 open reading frame (5) was
amplified by PCR using PFX polymerase (Invitrogen) and the
CCAAG to introduce a KpnI cloning site at the 5? end of the
gene and a 3? primer containing coding sequences for a throm-
bin cleavage site followed by a His6tag sequence TAATGGT-
ACCAATTGTGCAATATCTTCTGTATGC (1.65 kDa). The
purified PCR fragment was cloned into pCR4-Blunt TOPO
(Invitrogen) using the manufacturer’s standard protocol to
generate plasmid pR1-THis. Second, the sequence cloned in
pR1-THis was changed to standard codon usage by converting
serine in standard usage, CCT (22). Primers GGAAATCATC-
TCATTCCTCATGTTTAGTTTCTCAACTC and GAGTT-
used with the QuikChange II XL system (Stratagene, La Jolla,
CA) to generate a codon-corrected version of the tagged HBR1
gene in plasmid pR1-THis-ls.
consensus (K22Q) and nonconsensus (G19S) residues in the P-loop motif. A K66R mutation in the NMP-binding domain was designed to disrupt a predicted
a dashed boxed region. The lack of a terminal P-loop glycine that is a defining characteristic of the nuclear-localized group of adenylate kinase enzymes is
indicated by a (?). The acidic carboxyl terminus contributes to the overall net charge of the molecule (?19.4 to ?47).
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Alexander W. Peterson, Michael L. Pendrak and David D. Roberts
of the Mating Type Locus in
ATP Binding to Hemoglobin Response Gene 1 Protein Is Necessary for Regulation
doi: 10.1074/jbc.M110.180190 originally published online March 3, 2011
2011, 286:13914-13924. J. Biol. Chem.
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