YybT is a signaling protein that contains a cyclic dinucleotide phosphodiesterase domain and a GGDEF domain with ATPase activity

Article abstract of DOI:10.1074/jbc.M109.040238

The cyclic dinucleotide c-di-GMP synthesized by the diadenylate cyclase domain was recently discovered as a messenger molecule for signaling DNA breaks in Bacillus subtilis. By searching bacterial genomes, we identified a family of DHH/DHHA1 domain proteins (COG3387) that co-occur with a subset of the diadenylate cyclase domain proteins. Here we report that the B. subtilis protein YybT, a member of the COG3387 family proteins, exhibits phosphodiesterase activity toward cyclic dinucleotides. The DHH/DHHA1 domain hydrolyzes c-di-AMP and c-di-GMP to generate the linear dinucleotides 5′-pApA and 5′-pGpG. The data suggest that c-di-AMP could be the physiological substrate for YybT given the physiologically relevant Michaelis-Menten constant (K_m) and the presence of YybT family proteins in the bacteria lacking c-di-GMP signaling network. The bacterial regulator ppGpp was found to be a strong competitive inhibitor of the DHH/DHHA1 domain, suggesting that YybT is under tight control during stringent response. In addition, the atypical GGDEF domain of YybT exhibits unexpected ATPase activity, distinct from the common diguanylate cyclase activity for GGDEF domains. We further demonstrate the participation of YybT in DNA damage and acid resistance by characterizing the phenotypes of the ΔyybT mutant. The novel enzymatic activity and stress resistance together point toward a role for YybT in stress signaling and response.

Full text of DOI:10.1074/jbc.M109.040238

Enzyme Catalysis and Regulation:
YybT Is a Signaling Protein That Contains
a Cyclic Dinucleotide Phosphodiesterase
Domain and a GGDEF Domain with
ATPase Activity
Feng Rao, Rui Yin See, Dongwei Zhang,
Delon Chengxu Toh, Qiang Ji and Zhao-Xun
Liang
J. Biol. Chem. 2010, 285:473-482.
doi: 10.1074/jbc.M109.040238 originally published online November 9, 2009
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 1, pp. 473–482, January 1, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
YybT Is a Signaling Protein That Contains a Cyclic
Dinucleotide Phosphodiesterase Domain and a GGDEF
□
S
Domain with ATPase Activity*
Received for publication, July 1, 2009, and in revised form, October 6, 2009 Published, JBC Papers in Press, November 9, 2009, DOI 10.1074/jbc.M109.040238
Feng Rao, Rui Yin See, Dongwei Zhang, Delon Chengxu Toh, Qiang Ji, and Zhao-Xun Liang1
From the School of Biological Sciences, Nanyang Technological University, Singapore 637551
The cyclic dinucleotide c-di-GMP synthesized by the diade-
nylate cyclase domain was recently discovered as a messenger
molecule for signaling DNA breaks in Bacillus subtilis. By
searching bacterial genomes, we identified a family of DHH/
DHHA1 domain proteins (COG3387) that co-occur with a sub-
set of the diadenylate cyclase domain proteins. Here we report
that the B. subtilis protein YybT, a member of the COG3387
family proteins, exhibits phosphodiesterase activity toward
cyclic dinucleotides. The DHH/DHHA1 domain hydrolyzes
c-di-AMP and c-di-GMP to generate the linear dinucleotides
5ꢀ-pApAand 5ꢀ-pGpG. Thedatasuggestthatc-di-AMP could be
the physiological substrate for YybT given the physiologically
relevant Michaelis-Menten constant (Km) and the presence of
YybT family proteins in the bacteria lacking c-di-GMP signaling
network. The bacterial regulator ppGpp was found to be a strong
competitive inhibitor of the DHH/DHHA1 domain, suggesting
that YybT is under tight control during stringent response. In
addition, the atypical GGDEF domain of YybT exhibits unex-
pected ATPase activity, distinct from the common diguanylate
cyclase activity for GGDEF domains. We further demonstrate
the participation of YybT in DNA damage and acid resistance by
characterizing the phenotypes of the ⌬yybT mutant. The novel
enzymatic activity and stress resistance together point toward a
role for YybT in stress signaling and response.
protein from Bacillus subtilis (4, 5). It was found that c-di-AMP
was synthesized by the diadenylate cyclase (DAC) domain of
DisA via condensation of two ATP molecules. Witte et al. (5)
suggested that c-di-AMP is involved in signaling DNA damage
considering that the DNA integrity scanning protein DisA
scouts the chromosome for DNA double-stranded breaks. Sub-
sequent genomic mining revealed that the DAC domain pro-
teins are widespread in bacteria and archaea, with many of
them associated with putative sensor domains (6). The wide
occurrence and domain architecture of the DAC domain
proteins hinted that c-di-AMP may be another hidden
nucleotide messenger mediating various cellular functions
and phenotypes.
Currently, there is no report of c-di-AMP degrading or
exporting proteins for controlling cellular c-di-AMP level. To
identify potential c-di-AMP degrading proteins, we searched
bacterial genomes for phosphodiesterase or phosphoesterase
proteins that co-occur with the DAC domain-containing pro-
teins (6). We found that a group of multidomain proteins seems
to co-occur with a subset of the DAC domain proteins, which
include the homologs of YojJ and YbbP from B. subtilis. This
group of proteins (COG3387), as represented by the B. subtilis
protein YybT, contains two N-terminal transmembrane heli-
ces, a region that shares minimum sequence homology with
some Per-Arnt-Sim (PAS) domains, a highly modified GGDEF
domain, and a DHH/DHHA1 domain (see Fig. 1). We were
particularly attracted to this family of proteins because the
DHH domain proteins are known as phosphatases or phos-
phoesterases for hydrolyzing a wide variety of substrates rang-
ing from inorganic pyrophosphate to single-stranded (ss) DNA
(7). The DHH family proteins, which were named after the con-
served Asp-His-His motif in the active site, are divided into two
subfamilies based on the C-terminal subdomain. Subfamily 1
contains a DHHA1 domain at the C terminus, as represented by
the single-stranded DNA exonuclease RecJ and 3Ј-phosphoaden-
osine 5Ј-phosphate (pAp) phosphatase (2, 8). Subfamily 2
contains a DHHA2 domain at the C terminus, as exemplified
by the type II inorganic pyrophosphatase (9), yeast cytosol
exopolyphosphatase (10), and the eukaryotic c(A/G)MPase
protein Prune (11). The C terminus of YybT belongs to the
DHHA1 subfamily because it contains the conserved GGGH
motif and shares moderate sequence homology with the C ter-
mini of RecJ and pAp phosphatase (7). YybT homologs can be
identified in pathogens such as Staphylococcus aureus, Staphy-
lococcus epidermidis, and Clostridium difficile. Interestingly,
the DHH/DHHA1 domain of the YybT family protein (GdpP)
The cyclic dinucleotide c-di-GMP2 has been firmly estab-
lished as a major bacterial messenger molecule in recent years,
with the cellular level of c-di-GMP regulated by diguanylate
cyclase and phosphodiesterase domain proteins (1–3). In con-
trast, the existence of the structurally similar 3Ј,5Ј-cyclic digua-
nylate (c-di-AMP) in living cells was unknown until the recent
serendipitous discovery of the dinucleotide bound by the DisA
* This work is supported by Biomedical Research Council of Singapore Grant
06/1/22/19/464.
□S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Table S1 and Figs. S1–S8.
1 To whom correspondence should be addressed: School of Biological Sci-
ences, Nanyang Technological University, 60 Nanyang Dr., Singapore
637551. Tel.: 65-63167866; Fax: 65-67913856; E-mail: zxliang@ntu.edu.sg.
2 The abbreviations used are: c-di-GMP, cyclic di-GMP; c-di-AMP, cyclic di-
AMP; DAC, diadenylate cyclase; DGC, diguanylate cyclase; pApA, 5Ј-phos-
phoadenylyl-(3Ј35Ј)-adenosine; pGpG, 5Ј-phosphoguanylyl-(3Ј35Ј)-
guanosine; PAS, Per-Arnt-Sim domain; ppGpp, tetraphosphate guanosine;
ss, single-stranded; pAp, 3Ј-phosphoadenosine 5Ј-phosphate; HPLC, high
pressure liquid chromatography; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-
(hydroxymethyl)propane-1,3-diol; CHES, 2-(cyclohexylamino)ethanesul-
fonic acid; AMPPNP, adenosine 5Ј-(␤,␥-iminotriphosphate).
JANUARY 1, 2010•VOLUME 285•NUMBER 1
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Enzymatic Activities of YybT
in S. aureus has been speculated to be a c-di-GMP hydrolyzing 25,000 rpm for 30 min, the supernatant was filtered and then
phosphodiesterase domain (12).
incubated with 2 ml of nickel-nitrilotriacetic acid resin (Qia-
In this report, we present results from the study of the cyto- gen) for 1 h at 4 °C. The resin was washed with 50 ml of W1
plasmic portion of YybT from B. subtilis. We demonstrate that buffer (lysis buffer with 20 mM imidazole) and 20 ml of W2
the DHH/DHHA1 domain exhibited in vitro phosphodiester- buffer (lysis buffer with 50 mM imidazole). The proteins were
ase activity toward the cyclic dinucleotides c-di-AMP and c-di- eluted using a step gradient method with elution buffers con-
GMP among potential physiological substrates. We present taining 50 mM Tris, pH 8.0, 150 mM NaCl, 5% glycerol, and 200,
biochemical and bioinformatic evidence to argue that the phys- 300, or 500 mM imidazole. After the analysis by SDS-PAGE,
iological substrate could be c-di-AMP. We demonstrate that fractions with purity higher than 95% were pooled together.
the stringent alarmone ppGpp is a strong competitive inhibitor Size exclusion chromatograph was carried out at 4 °C using the
of the DHH/DHHA1 domain. In addition, we show that the AKTA fast protein liquid chromatography system equipped
GGDEF domain of YybT possesses unprecedented ATPase with a Superdex 200 HR 16/60 column (Amersham Bio-
activity rather than diguanylate cyclase (DGC) activity. The sciences). The buffer used for gel filtration was comprised of 50
biological function of YybT and the evolutionary relation- mM Tris-HCl, pH 8.0, 0.15 M NaCl, 5% glycerol. The proteins
ships of the DHH/DHHA1 and GGDEF domains are dis- were stored in a Ϫ80 °C freezer after flash freezing, and concen-
cussed in light of the enzymatic activities and the effect of tration measurement was by the Bradford assay method.
⌬yybT mutation on DNA damage and acid resistance.
All of the protein mutants were generated using a site-di-
rected mutagenesis II kit (Stratagene) according to the manu-
facturer’s instruction manual. The mutations were verified
EXPERIMENTAL PROCEDURES
Chemicals were purchased from Sigma with the exception of using a BigDye Terminator v3.1 cycle sequencing kit on ABI
sodium tripolyphosphate (Alfa Aesar), ssRNA and DNA (Euro- Prism 3100 Genetic Analyzer (Applied Biosystems). The
gentec), c-di-AMP (BioLog), ppGpp (TriLink), and c-di-GMP, mutant proteins were expressed, purified, and stored under the
which is synthesized using the thermophilic DGC enzyme in same conditions as wild type.
the lab (13). The proteins were cloned, expressed, and purified
Bioinformatics and Structure Modeling—Sequence and
following standard producer as described in the supplemental genomics analysis was performed using STRING and ClustalW
material. (14, 15). In total, 184 YybT homologs (defined as proteins that
Genomic DNA Isolation—B. subtilis strain 168 was grown in share the same domain architectures) are found in bacterial
LB medium. The cells from 5 ml of overnight culture were pel- genomes. The hidden Markov models (HMM) logo was gener-
leted and resuspended in Tris-EDTA buffer. The suspension ated from the alignment file. The structural model of the
was incubated for 1 h with 6% volume of 10% SDS and 0.6% DHHYybT was built using Swiss-Model sever with the structure
volume of 20 mg/ml proteinase K at 37 °C. After incubation, an of a DHH family protein (Protein Data Bank entry 3DMA) as
equal volume of phenol/chloroform was added to the mixture. template (16).
The DNA phenol mixture was spanned at 14,000 rpm for 2 min.
Substrate Screening by Enzymatic Activity Assays—p-Nitro-
The upper aqueous phase was mixed with an equal volume of phenol-related substrates pNPP, bis-p-nitrophenol phosphate,
1
phenol/chloroform and centrifuged again. A ⁄10 volume of thymidine monophosphate p-nitrophenol ester, and phosphe-
sodium acetate (3 M, pH 5.2) was then added to the upper aque- nolpyruvate were assayed by monitoring the formation of p-ni-
ous phase, followed by 0.6 ml of isopropanol to precipitate the trophenol at 410 nm using a UV-visible spectrophotometer. A
genomic DNA. DNA was pelleted by centrifugation and washed control with the protein storage buffer but not the enzyme solu-
by using 70% ethanol. Washed DNA was redissolved in TE tion was used for base-line correction. The enzymatic activity
buffer for PCR cloning of the YybT gene.
assays with nucleotides and oligonucleotides were carried out
Protein Cloning, Expression, and Purification—The gene by using a HPLC system (Agilent LC1200) with a RP C-18 col-
(yybT) that encodes the full-length YybT was first amplified by umn as previously described for c-di-GMP activity assay (28,
PCR from the genomic DNA using the Expand high fidelity kit 29). The nucleotides and oligonucleotides tested are listed in
(Roche Applied Science). Subsequently, the DNA fragments supplemental Table S1. The activity toward RNA and ssDNA
that encode the cytoplasmic portion of YybT (YybT84–659), the was monitored by detecting the possible products 1-mer and
GGDEF-DHH/DHHA1 di-domain (YybT150–659), and the 2-mer nucleotide along with standards. For inorganic pyro-
PAS-GGDEF di-domain (YybT84–303) were cloned into the ex- phosphate and polyphosphate, the assay was conducted using
pression vector pET-28(aϩ) (Novagen) between the NdeI and the EnzCheck phosphate assay kit (Invitrogen) by measuring
XhoI restriction sites. The plasmids harboring the DNA con- the release of Pi in solution. The assay conditions for substrate
structs and the His6 tag encoding sequence were transformed into screening were: 100 mM Tris, pH 8.3, 20 mM KCl, 0.5 mM
Escherichia coli strain BL21(DE3).
MnCl2, 1–2 ␮M enzyme, and 100 ␮M substrates (10 ␮M for
For protein expression, 1 liter of bacterial culture (LB 24-mer RNA and T7 promoter ssDNA and 1 mM for pyrophos-
medium) was grown up to OD ϭ 0.8 before inducing with 0.8 phate and tripolyphosphate). The reactions were incubated for
mM isopropyl ␤-D-thiogalactopyranoside. The culture was kept up to 2 h for detecting enzymatic activity.
shaking at 16 °C for ϳ12 h before it was pelleted by centrifuga-
Metal and pH Dependence—The assay conditions used for
tion. The cells were lysed in 20 ml of lysis buffer (50 mM Tris, pH metal screening were: 100 mM Tris, pH 8.3, 20 mM KCl, 10 mM
8.0, 150 mM NaCl, 5% glycerol, 0.1% ␤-mercaptoethanol, and 1 [Metal2ϩ], 1 ␮M enzyme, 20 ␮M c-di-AMP. For pH screening,
mM phenylmethylsulfonyl fluoride). After the centrifugation at assay buffer contains 100 mM bis-Tris (for pH 6.7–7.3), Tris (for
474 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 1•JANUARY 1, 2010

Enzymatic Activities of YybT
pH 7.3–8.8), or CHES (pH 8.8–10) with 0.5 mM MnCl2 and 20
mM KCl.
Kinetic Measurement of the DHH Domain Activity—The
measurement of steady-state kinetic parameters were carried
out by monitoring the formation of 5Ј-pApA/5Ј-pGpG (for
DHH/DHHA1 domain) using HPLC. The assay buffer condi-
tions are the same as described above. Initial velocity at a cer-
tain substrate concentration was obtained from a series of reac-
tions that were stopped at various time points within linear
range. Initial velocity was measured at seven or eight substrate
concentrations. The kinetic parameters kcat and Km were
obtained by fitting the initial velocity at various substrate con-
centrations to a Michaels-Menten equation using the software
Prism (GraphPad). For ppGpp inhibition assay, the initial
velocity for c-di-AMP hydrolysis (at 20 ␮M) was first measured
in the presence of 0–2 mM ppGpp under the same reaction
conditions used for steady-state kinetic measurement. The IC50
was obtained by fitting the data to the dose-response model
using the software Prism (GraphPad). The steady-state kinetics
in the presence of two ppGpp concentrations was measured
with KI obtained from data fitting.
FIGURE 1. Domain architecture of YybT family proteins (COG3387) with
the three protein constructs studied in this work indicated.
agar plates (number of colonies Ͻ 500/plate). The whole exper-
iment was repeated three times to ensure reproducibility. The
spore formation efficiency was estimated by calculating the
percentage of spores, which equals (number of colonies with
heating)/(number of colonies without heating) ϫ 100.
Measurement of the Cellular c-di-AMP Concentration—The
procedure for measuring the cellular concentration of c-di-
AMP in B. subtilis was essentially the same as the one reported
by Simm et al. (20). Briefly, the vegetative and sporulating cells
harvested from the casein hydrolysate and sporulation medium
were pelleted and resuspended in double distilled H2O (100 mg
of cells/300 ␮l of water). The suspension was heated at 95 °C for
15 min, followed by sonication for 5 min. 99% ethanol was then
added to the sample to a final concentration of 70%. After cen-
trifugation, the supernatant was pooled, frozen, and subse-
quently lyophilized overnight. The precipitate was dissolved in
500 ␮l of double distilled H2O, filtered, and loaded to the HPLC
system. Because no clear c-di-AMP peak was observed on
HPLC trace, a 4-min fraction corresponding to the retention
time of c-di-AMP (Ϯ2 min) was collected from HPLC, lyophi-
lized, and redissolved into 5–10 ␮l of double distilled H2O. For
mass spectrometry, the sample was mixed with an equal volume
of matrix (␣-cyano-4-hydroxycinnamic acid, 20 mg/ml) before
being spotted onto 384-well plate to crystallize. Mass spectra
were recorded with a 4800 matrix-assisted laser desorption ion-
ization time-of-flight analyzer (Applied Biosystems) in both
positive and negative modes.
Kinetic Measurement of the ATPase Activity—The ATPase
activity of the GGDEF domain was assayed by two methods.
The hydrolysis of ATP was monitored by measuring the release
of Pi in solution using the EnzCheck phosphate assay kit
(Invitrogen) and a UV-visible spectrophotometer or directly
monitoring ADP formation using HPLC with a solvent system
described by Ryjenkov et al. (17). The measurement of steady-
state kinetic parameters was carried out by monitoring the for-
mation ADP using HPLC. All of the kinetic measurements were
carried out at 23 °C with the assay conditions: 50 mM Tris, pH
7.5, 1 mM MgCl2, 0.05–8 mM ATP, 2.9 ␮M enzyme. Competitive
inhibition was carried out in the presence of 250 ␮M AMPPNP.
The dissociation constants (KD) were calculated using the
equation: KD ϭ [Inhibitor]/(Km(I)/Km Ϫ 1).
Acid Stress Resistance—The procedure was adopted from a
previous report (18). The wild type and ⌬yybT mutant B. sub-
tilis cells (obtained from Prof. K. Kobayashi, Nara, Japan) grown
from an overnight culture at pH 7.0 were inoculated into LB
RESULTS
Three protein constructs with different domain combination
medium (pH 4.0, 3.5, or 3.0) at final A600 nm ϭ 0.1. To determine were cloned, expressed, and purified as His6-tagged proteins
viability by colony formation, the cells were plated on LB agar (Fig. 1). YybT84–659 contains the whole cytoplasmic portion,
plate (pH 7.0) with proper dilution both prior to (T0) and after whereas YybT150–659 and YybT84–303 contain the GGDEF-
acid challenge for 2 h (T2). Relative survival was calculated as DHH/DHHA1 and PAS-GGDEF segments, respectively. The
the ratio of colony-forming units/ml at time T2 to that at T0.
recombinant proteins were purified by nickel-nitrilotriacetic
Sporulation of Wild Type and ⌬yybT Mutant in the Presence acid affinity and size exclusion chromatography to homogene-
of Nalidixic Acid—The spore formation efficiency of the wild ity (supplemental Fig. S1). The purified proteins were not
type B. subtilis strain 168 and YybT mutant strain (⌬yybT) were bound by RNA, DNA, or small nucleotide ligands as detected by
examined by using an established procedure (4). ⌬YybT was HPLC and spectroscopic analysis of the denatured protein
obtained from a complete mutant library of B. subtilis 168 with solution.
an insertion of pMUTIN3MCS after the codon for amino acid
The DHH/DHHA1 Domain of YybT Exhibits Specific Phos-
88 (19). Growth and sporulation were carried out with shaking phodiesterase Activity—We first tested the activity of YybT
at 32 °C in casein hydrolysate medium and sporulation toward four p-nitrophenol substrates that are known to be
medium, respectively (4, 37). Sporulation was induced by trans- easy substrates for phosphodiesterases and phosphatases. In
ferring the early log-phase cells from the casein hydrolysate the presence of Mg2ϩ or Mn2ϩ, YybT84–659 hydrolyzed bis-p-
medium to the sporulation medium with the DNA double nitrophenol phosphate and thymidine monophosphate p-ni-
break inducing nalidixic acid (sigma) at the indicated concen- trophenol ester but not p-nitrophenol phosphate and phos-
tration. After 22 h, the cells were heated at 80 °C for 10 min for phenolpyruvate (Fig. 2B), indicating that the enzyme is a
spore selection and then plated with proper dilutions on LB phosphodiesterase rather than a phosphatase. This conclusion
JANUARY 1, 2010•VOLUME 285•NUMBER 1
JOURNAL OF BIOLOGICAL CHEMISTRY 475

Enzymatic Activities of YybT
Several naturally occurring cyclic
nucleotide substrates were tested
for phosphodiesterase activity. No
activity was detected with 3Ј,5Ј-
cAMP and cGMP even after pro-
longed incubation with Mg2ϩ or
Mn2ϩ. In contrast, the incubation of
YybT with c-di-AMP and c-di-GMP
resulted in the disappearance of the
cyclic dinucleotides and the forma-
tion of new products. Based on
HPLC analysis, YybT hydrolyzes
c-di-AMP and c-di-GMP to pro-
duce exclusively the linear dinucle-
otides 5Ј-pApA and 5Ј-pGpG, with-
out generating the 3Ј-dinucleotide
(3Ј-pApA or 3Ј-pGpG) or mono-
phosphate (AMP or GMP) (Fig. 3A
and supplemental Figs. S2 and S4).
Steady-state kinetic measurement
yielded a turnover number (kcat) of
0.55 Ϯ 0.02 sϪ1 and Michaelis-Men-
ten constant (Km) of 1.3 Ϯ 0.3 ␮M
for c-di-AMP (Fig. 3B). In compari-
son, YybT exhibited a comparable
kcat (0.23 Ϯ 0.02 sϪ1) but a much
greater Km (349 Ϯ 55 ␮M) for c-di-
FIGURE 2. Specific phosphodiesterase activity of YybT84–659. A, partial alignment of DHH-DHHA1 domain
sequences with the conserved residues for metal ion coordination highlighted. B, enzymatic activity of YybT
against nonphysiological p-nitrophenol substrates. C, structural model of DHHYybT active site with the metal
ions shown as balls and the coordinating residues as sticks.
is further supported by the observation that YybT did not GMP. The difference in Km indicates that the substrate-binding
hydrolyze monophosphate ribonucleotides and deoxyribo- pocket prefers c-di-AMP over c-di-GMP. The cyclic dinucle-
nucleotides (supplemental Table S1). Moreover, YybT did not otide specific activity of YybT could also be detected with the
act on 3Ј-phosphoadenosine 5Ј-phosphate (PAP), suggesting construct (YybT150–659) that contains the GGDEF-DHH/
that YybT differs from the DHH family phosphatase YtqI (21). DHHA1 segment, although YybT150–659 exhibited a ϳ10-fold
YybT also did not exhibit activity toward sodium tripolyphos- decrease in kcat (supplemental Table S2). According to
phate and pyrophosphate, excluding it as a pyro- or polyphos- sequence comparison and the structural model of the DHHYybT
phatase (10, 22, 23).
subdomain, the residues Asp420 and Asp499 are predicted to be
After ruling out YybT as a phosphatase, we examined the the metal ion-coordinating residues (Fig. 2, A and C). The
activity of YybT toward a wide range of possible physiological D420A mutant only exhibited residual activity against c-di-
substrates for phosphohydrolyase or phosphodiesterase as AMP, c-di-GMP, bis-p-nitrophenol phosphate, and thymidine
listed in supplemental Table S1. Di- and tri-phosphate nucleo- monophosphate p-nitrophenol ester (Fig. 2B), whereas the
ϩ
tides, deoxyribonucleotides, and NADH, NADPH, NAD , double mutation D420A/D499A totally abolished the catalytic
ϩ
NADP , and FAD were first tested. No phosphohydrolyase activity. These observations confirm that the DHH domain is
activity was observed for all of the compounds with the excep- responsible for the specific phosphodiesterase activity.
tion of ATP. We found that ATP was slowly converted to ADP.
Metal and pH Dependence of the Phosphodiesterase Activity—
However, this activity was attributed to the GGDEF domain The phosphodiesterase activity of YybT is strictly dependent on
rather than the DHH domain as we will discuss later. We next divalent metal ions such as Mn2ϩ, Mg2ϩ, Ni2ϩ, and Co2ϩ ions
tested whether YybT functions as an oligoribonuclease or ribo- (Fig. 4A). Meanwhile, Zn2ϩ ion inhibits catalytic activity in the
nuclease for degrading RNA or ssDNA (21, 24). Neither the presence of Mn2ϩor Mg2ϩ, whereas Ca2ϩ only inhibits catalysis
ssDNA and RNA substrate nor the 16 oligoribonucleotides in the presence of Mg2ϩ and not Mn2ϩ (Fig. 4B). Mn2ϩ and
and oligodeoxyribonucleotides of various lengths could be Mg2ϩ are most likely the physiological metal ion for YybT, con-
degraded by YybT in the presence of Mn2ϩ or Mg2ϩ. Moreover, sidering that DHH family proteins tend to use Mn2ϩ or Mg2ϩ
Yybt did not degrade diadenosine tetraphosphate (AppppA), for catalysis (24). We examined the Mg2ϩ and Mn2ϩ ion
triphosphate (ApppA), or pentaphosphate (ApppppA), sug- dependence of the catalytic activity for identifying the physio-
gesting that it is not a bis(5Ј-adenosyl) tri-, tetra-, or penta- logical metal ion for YybT (Fig. 4C). Mn2ϩ ion exhibits a bell-
phosphatase (25, 26). Lastly, the small nucleotide (p)ppGpp is shaped profile with the optimal contraction in the range of 10
known as a bacterial and plant messenger for signaling starva- ␮M to 10 mM, with the activity strongly inhibited at high [Mn2ϩ
]
tion and other stresses (27). The enzymatic assay showed that concentration (Ն10 mM). By contrast, the catalytic activity
YybT did not degrade ppGpp and thus is not a ppGpp-specific remained low for Mg2ϩ at 1 mM [Mg2ϩ] and did not increase
phosphohydrolyase or phosphatase.
significantly until [Mg2ϩ] reached 10 mM. Because the cellular
476 JOURNAL OF BIOLOGICAL CHEMISTRY
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Enzymatic Activities of YybT
FIGURE 3. Degradation of c-di-AMP by YybT. A, HPLC analysis of the hydro-
lysis of c-di-AMP by YybT84–659. The assay conditions are described under
“ExperimentalProcedures”. B, steady-statekineticanalysisofthehydrolysisof
c-di-AMP catalyzed by YybT84–659
.
Mn2ϩ and Mg2ϩ concentrations are approximately 10 ␮M and 1
mM, respectively, in B. subtilis (28), the results support Mn2ϩ
ion as the physiological metal ion for the DHH/DHHA1
domain of YybT. In addition, the pH dependence study showed
a strong preference for alkaline conditions with the optimal pH
between 8.5 and 9.0 (supplemental Fig. S3), which is in line with
a two-metal ion-assisted catalytic mechanism (29).
FIGURE 4. Metal and pH dependence of c-di-AMP degradation by
YybT84–659. A, metal dependence of YybT-catalyzed c-di-AMP hydrolysis.
The enzymatic assay conditions are described under “Experimental Proce-
dures.” B, Mn2ϩ and Mg2ϩ concentration dependence of c-di-AMP hydrolysis.
C, pH dependence of DHHYybT domain activity.
The GGDEF Domain of YybT Binds and Hydrolyzes ATP—
Initial enzymatic assays suggested that the GGDEF domain of
YybT is neither a functional DGC domain for converting GTP
into c-di-GMP nor a GTPase for hydrolyzing GTP. During sub-
strate screening for the DHH/DHHA1 domain of YybT, we
noticed that the concentration of ATP decreases with time in ture GGDEF motif and the guanosine-binding residues critical
the presence of YybT and Mg2ϩ (or Mn2ϩ). HPLC analysis for the DGC activity (30). To test whether the degenerate
revealed that the depletion of ATP was accompanied by the GGDEF domain is responsible for the ATPase activity, we
formation of ADP, suggesting that YybT catalyzes the hydroly- cloned and expressed another protein construct (YybT84–303
)
sis of ATP. Surprisingly, the D420A/D499A double mutation in that only contains the PAS-GGDEF didomain. Although the
the DHH domain did not affect ATP hydrolysis, hinting that purified YybT84–303 protein did not show any activity against
DHH/DHHA domain is not responsible for the ATPase activ- GTP, it could still hydrolyze ATP to yield ADP exclusively (Fig.
ity. Meanwhile, sequence alignment showed that YybT con- 5A). An enzymatic assay with the EnzCheck phosphate assay kit
tains a highly modified GGDEF domain, which lacks the signa- confirmed that the inorganic phosphate was released into the
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Enzymatic Activities of YybT
FIGURE 5. ATPase activity of the GGDEFYybT domain. A, HPLC analysis of ATP hydrolysis catalyzed by YybT84–303. B, steady-state kinetic measurement of
ATPase domain activity in the presence and absence of nonhydrolyzable ATP analog. C, relative ATPase activity of YybT84–303 and its mutants in the presence
of Mg2ϩ and Mn2ϩ. D, sequence comparison of the GGDEFYybT domain with some characterized GGDEF domains. The three residues mutated in this study are
indicated by the vertical arrows, whereas the residues for guanosine binding in orthodox GGDEF domains are indicated by asterisks. GdpP (S. aureus) is a
homolog of YybT. The GGDEF domain of GdpS (S. aureus) exhibits residual GTPase activity. The GGDEF domains of PleD (C. crescentus) (30), WspR (P. aeruginosa)
(54), and AxDGC2 (Acetobacter xylinum) function as diguanylate cycles (55). The GGDEF domain of Cc3396 binds GTP as a regulatory domain.
solution, suggesting that the GGDEF is not a kinase that trans- ing of the adenosine group remain to be defined, the mutagen-
fers the phosphate onto a substrate (supplemental Fig. S7). esis results suggested that the metal ion and phosphate-binding
Steady-state kinetic measurement yielded a kcat of 0.59 Ϯ 0.03 sites in the GGDEF domain may be preserved despite the diver-
minϪ1 and Km of 0.90 Ϯ 0.12 mM for the ATPase in the presence gence of function.
of Mg2ϩ ion (Fig. 5B). The metal ion Mg2ϩ or Mn2ϩ is required
Inhibition of the Phosphodiesterase Activity by ppGpp—
for the ATPase activity with Mg2ϩ as the preferred metal ion, in Screening of Lactococcus lactis mutants revealed that the YybT
contrast to the metal preference for the DHH/DHHA1 domain family protein llmg1816 is involved in acid and starvation stress
(Fig. 5C). To further provide evidence for the preferred binding resistance, with the ⌬llmg1816 mutant strain exhibiting a higher
of ATP by the divergent GGDEF domain, we measured the survival rate under low pH and starvation conditions (18). It
dissociation constant (KD) for the nonhydrolyzing analog of was postulated that the deletion of llmg1816 may cause change
ATP (adenylyl imidodiphosphate AMPPNP) by competitive in the (p)ppGpp level (18). (p)ppGpp is a global messenger that
inhibition experiments (Fig. 5B). The measurement revealed a KD plays a major role coordinating cellular responses under star-
of 44.0 Ϯ 14.6 ␮M for AMPPNP. The presence of the AMPPNP vation stresses. The level of (p)ppGpp is significantly elevated
did not exert any significant effect on the DHH/DHHA1 during stringent responses, resulting in the binding of
domain activity, suggesting that the GGDEF domain is not an (p)ppGpp to its protein targets such as RNA polymerase and
ATP-binding regulatory domain. Finally, although the signa- inosine monophosphate dehydrogenase (18, 33, 34).
ture GGDEF motif and the residues for guanosine group bind-
To test whether the YybT protein is a previously unknown
ing are absent, the residues that coordinate the Mg2ϩ ions in target for (p)ppGpp, the phosphodiesterase and ATPase activ-
orthodox GGDEF domains, with one in the signature GG(D/ ities were examined in the presence of ppGpp. Remarkably,
E)EF motif and another at the end of ␤-strand 1, seem to be although ppGpp did not inhibit or stimulate the ATPase activ-
conserved in GGDEFYybT (Glu225 and Asp183) (31, 32) (Fig. 5D ity, it significantly suppressed the c-di-AMP hydrolyzing activ-
and supplemental Fig. S6, A and B). We found that the mutation ity of the DHH/DHHA1 domain. An apparent IC50 value of 234
of the conserved residues Asp183, Glu225, and the conserved ␮M was derived from the activity versus [ppGpp] plot (Fig. 6A).
␥-phosphate binding Arg291 reduced the catalytic activity to Initial velocity measurement at various ppGpp concentrations
below 5% (Fig. 5C). Although the structural motifs for the bind- indicated that the inhibition is competitive in nature, with an
478 JOURNAL OF BIOLOGICAL CHEMISTRY
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Enzymatic Activities of YybT
FIGURE 6. Inhibition of the phosphodiesterase activity by ppGpp.
A, ppGpp concentration dependence of the c-di-AMP hydrolyzing activity.
The IC50 value is determined from the midpoint of the plot. B, double-recip-
rocal plots of c-di-AMP hydrolyzing activity at various ppGpp concentrations.
Theexperimentalconditionsaredescribedunder“ExperimentalProcedures.”
FIGURE 7. Acid and DNA damage resistance. A, relative survival of wild type
and ⌬yybT strain under acid stress conditions. B, spore formation efficiency
forthewildtype B. subtilis and⌬yybTmutantinthepresenceofDNAdamage-
inducing agent nalidixic acid. The spores (%) were obtained as described
under “Experimental Procedures.”
inhibition constant (KI) of 35.9 Ϯ 7.2 ␮M (Fig. 6B). Considering gesting that YybT does not play a significant role in DNA repair
that bacterial cells can rapidly accumulate ppGpp up to a mil- during vegetative growth. However, when we compared the
limolar level under starvation conditions (27, 35), the inhibition sporulating cells, a significant difference was observed between
of the phosphodiesterase activity by ppGpp is likely to be phys- the wild type and mutant strains. As shown in Fig. 7B, when
iologically relevant. No such inhibition effect was observed with both the wild type and yybT mutant cells were induced to enter
other cyclic or linear nucleotides or on the c-di-GMP hydrolyz- sporulation without exposure to nalidixic acid, the spore for-
ing activity of the EAL domain proteins such as RocR (36, 37), mation efficiency was comparable, suggesting that the gene
indicating that the inhibition of the DHH/DHHA1 domain by knock-out does not significantly affect sporulation. However,
ppGpp is rather specific.
in the presence of nalidixic acid, the mutant strain consistently
Effect of ⌬yybT Mutation on Acid and DNA Damage exhibited more colonies, which were derived from the spores
Resistance—L. lactis contains an yybT-like gene (llmg1816). that survive the heat treatment. The discrepancy in spore for-
The ⌬llmg1816 mutant was found to be more resistant under mation efficiency widens between the wild type and the yybT
acid stress and also to exhibit enhanced long term (2-day) sur- mutant with increasing nalidixic acid concentration. This is in
vival (18). We found that the ⌬yybT mutant strain of B. subtilis sharp contrast to the observation for the ⌬disA mutant, which
is also more resistant than the wild type against acid stress (Fig. formed less spores than the wild type strain (4). These observa-
7A). Because the c-di-AMP synthesizing DisA has been pro- tions indicate that YybT plays a role in DNA damage signaling
posed to signal DNA damage (5), we further tested whether and repair during sporulation.
YybT plays a role in mediating DNA damage resistance. No
Measurement of the Cellular c-di-AMP Concentration—To
significant survival advantage or disadvantage was observed test whether YybT affects global c-di-AMP level, we set out to
when the wild type B. subtilis and ⌬yybT mutant were treated measure the cellular concentration of c-di-AMP for the wild
with the topoisomerase inhibitor nalidixic acid (DNA gyrase type and ⌬yybT strains during vegetative growth and sporula-
inhibitor) or ␥-ionizing irradiation (supplemental Fig. S8), sug- tion. Intriguingly, although we could readily detect the c-di-
JANUARY 1, 2010•VOLUME 285•NUMBER 1
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Enzymatic Activities of YybT
AMP standard in the spiked sample as well as c-di-GMP in also revealed that the Arg residues critical for the binding of
E. coli and Pseudomonas aeruginosa by using the mass spec- polyphosphate, RNA, or ssDNA in other DHHA1 domains (e.g.
trometry method described by Simm et al. (20), we could not RecJ and YtqI) are not conserved in YybT. In contrast, only one
observe any c-di-AMP peak above the background in both the conserved Arg (Arg606) was found in YybT homologs (supple-
wild type and the mutant strains after repeated attempts. The mental Fig. S5). This Arg is likely to interact with the phosphate
results led us to suspect that the steady-state concentration of group of c-di-AMP according to our structural model. Thus,
c-di-AMP in B. subtilis is rather low in bacterial cells.
the uniqueness of the DHHA1 subdomain of YybT seems to be
in agreement with its unprecedented substrate specificity.
Third, the only putative phenotype mediated by c-di-AMP is
DISCUSSION
DHH family proteins function as phosphatase or phosphodi- the signaling of DNA damage. The mutation of DisA signifi-
esterases for hydrolyzing a wide variety of substrates that range cantly retards sporulation in the presence of a DNA damaging
from pyrophosphate to ssDNA. The enzymatic assay results agent. In sharp contrast, the ⌬yybT mutant exhibited higher
suggested that YybT possesses different substrate specificity spore formation efficiency than the wild type (Fig. 7). Third,
from other known DHH family proteins. The biochemical assay DAC domains can be found in 275 bacterial or archaeal species
revealed that YybT is a novel phosphodiesterase that hydro- with many of them belonging to the phylums of firmicutes and
lyzes the cyclic dinucleotides c-di-GMP and c-di-AMP. The ⑀-proteobacteria, whereas YybT family proteins can be found in
substrate specificity is consistent with the low sequence homol- 123 bacterial species that mainly consist of firmicutes (supple-
ogy shared by the substrate-binding DHHA1 subdomains mental Table S3). Importantly, for all the 123 bacterial species
between YybT and other DHH family proteins. c-di-GMP is a that contain the YybT family protein, at least one DAC domain
ubiquitous second messenger in bacteria and is known to be protein can be identified. Based on these considerations, we
degraded by the EAL domain and HD-GYP domain phosphodi- propose that YybT is probably involved in c-di-AMP signaling
esterases (38, 39). Although c-di-GMP could be degraded by by hydrolyzing the cyclic dinucleotide, resembling the role of
YybT in our assay, it is unlikely to be the physiological sub- the EAL domain in c-di-GMP signaling. Meanwhile, the
strates for YybT for two major reasons. First, the cellular c-di- absence of YybT protein in some DAC domain protein-con-
GMP concentration is in the low or submicromolar range taining bacteria seems to hint of the existence of other families
according to the estimated cellular c-di-GMP concentration of c-di-AMP-specific phosphodiesterases.
(40–42). In addition, the Km values for the c-di-GMP degrad-
It has been noticed that YybT family proteins contain a
ing EAL domain proteins are also known to be in the same highly modified GGDEF domain with unknown function (12,
range (36, 37, 43). Hence, the elevated Km (349 Ϯ 54.6 ␮M) for 45). GGDEF domains are homologs of adenylate cyclase
c-di-GMP implies that the DHH/DHHA1 domain is incapable domains and are mainly known as DGC domains for synthesiz-
of hydrolyzing c-di-GMP efficiently under physiological condi- ing c-di-GMP from GTP (17, 46). A large number of catalyti-
tions. Second, considering that the YybT family proteins cally inactive GGDEF domains that lack the signature GG(D/
(COG3387) can be found in the bacteria (e.g. Streptococcus and E)EF motif have been identified, including two divergent
Staphylococcus) that do not seem to harbor c-di-GMP signaling GGDEF domains that can still bind GTP. Jenal and co-workers
network (as judged by the absence of catalytically active EAL/ (43) reported a regulatory GGDEF domain that binds GTP and
HD-GYP and GGDEF domain proteins (12)), the YybT family stimulates the activity of the adjacent EAL domain, whereas
proteins are unlikely to be involved in c-di-GMP signaling. O’Gara and co-workers (12) recently reported a GGDEF
Hence, the physiologically irrelevant Km and the presence of domain with residual GTPase activity. In contrast to these
YybT in bacteria that lack the c-di-GMP network strongly dis- GGDEF domains, the current study revealed that the GGDEF
favor c-di-GMP as the cellular substrate for YybT.
domain of YybT specifically binds and hydrolyzes ATP but not
On the other hand, the biochemical and genetic data seem to GTP. Sequence comparison suggested that the metal ion-bind-
support c-di-AMP as the cellular substrate for YybT. First, the ing residues in GGDEFYybT are probably conserved, but the
small Km of 1.3 Ϯ 0.3 ␮M toward c-di-AMP indicates that YybT residues for the guanosine group binding are absent (Fig. 5D).
can degrade c-di-AMP efficiently even if each B. subtilis cell Based on the mutagenesis results, we speculate that ATP is
only contains 200–600 molecules of c-di-AMP, which corre- probably bound at approximately the same site as GTP in the
sponds to a cellular concentration in the low or submicromolar typical GGDEF domains, although the structural motifs that
range. Second, the fact that YybT hydrolyzes c-di-AMP to gen- confer the binding specificity remain to be identified. Interest-
erate exclusively 5Ј-pApA (not 3Ј-pApA or AMP) suggests that ingly, phylogenetic analysis revealed that the GGDEF domain of
the substrate-binding pocket can differentiate the cyclic dinu- YybT resembles adenylate cyclase domains more than the typ-
cleotide and the linear dinucleotide (i.e. the product pApA). ical GGDEF domains (supplemental Fig. S6C), providing fur-
The crystal structures of several DHH domain proteins ther support for the notion that GGDEF and adenylate cyclase
revealed that the C-terminal subdomain (DHHA1 or DHHA2) domains were evolved from the same ancestor (47). Consider-
is largely responsible for the binding of substrate (10, 24, 44). ing that the YybT family proteins are only found in an early
Sequence alignment of YybT and DHH family proteins revealed branch of the evolutionary tree, the ATPase activity exhibited
that although the DHH subdomain is fairly conserved with all by the GGDEFYybT domain seems to suggest an early diver-
the putative metal ion-binding residues (Fig. 2A), the DHHA1 gence of function for the GGDEF domains during evolution.
subdomain does not share significant sequence homology with Lastly, the intrinsic ATPase activity of the GGDEF domain is
ϩ
any characterized DHH family proteins. Sequence comparison relatively low, reminiscent of the basal activity of the AAA -
480 JOURNAL OF BIOLOGICAL CHEMISTRY
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Enzymatic Activities of YybT
4. Bejerano-Sagie, M., Oppenheimer-Shaanan, Y., Berlatzky, I., Rouvinski,
A., Meyerovich, M., and Ben-Yehuda, S. (2006) Cell 125, 679–690
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167–178
ATPases prior to the stimulation by small molecule or protein
partners (48). It is intriguing to speculate whether the PAS
domain may function as a sensor domain for perceiving and
transducing a signal to further stimulate the activity of the
GGDEF-based ATPase domain.
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The nucleotide ppGpp has been known as the stringent alar-
mone that regulates RNA polymerase and other protein targets
for gene expression and other cellular activities during strin-
gent responses (49). The inhibition of the DHH/DHHA1 do-
main by ppGpp suggests a direct link between the ppGpp and
c-di-AMP signaling networks and implies that the local c-di-
AMP level will increase during stringent responses to starva-
tion and other stresses. The measured IC50 and KI values indi-
cate that the hydrolysis of c-di-AMP will be fully suppressed
during stringent response with the ppGpp level raised above 1
mM. Interestingly, the interference of the ppGpp and c-di-GMP
signaling networks has also been proposed recently (50, 51).
The inhibition of c-di-AMP hydrolysis by ppGpp may repre-
sent another example of cross-talking between two nucleotide
signaling networks.
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In addition to the novel enzymatic activities exhibited by
YybT, the opposite effects of the ⌬disA and ⌬yybT mutation on
sporulation in the presence of a DNA-damaging agent point
toward contrasting roles played by the proteins in DNA damage
resistance. The higher sporulation efficiency for the ⌬yybT
mutant indicates that the mutant strain is more resistant
against DNA damage, whereas the acid resistance experiment
showed that the ⌬yybT mutant strain is more tolerant to acid
stress, consistent with the observation for the L. lactis mutant
(25). Because acid stress can also cause chromosomal DNA
damage in bacteria (52, 53), the observed acid and DNA damage
resistance may be highly related on the molecular level.
Together, the acid and DNA damage resistance exhibited by the
mutant suggest that YybT functions as a signaling protein for
coordinating stress sensing and cellular responses. Finally, con-
sidering that the DHH/DHHA1 domain of YybT is an efficient
c-di-AMP phosphodiesterase under in vitro conditions and
that the c-di-AMP synthesizing DisA is implicated in signaling
DNA damage (5), it is tempting to conclude that the effect of the
⌬yybT mutation is exerted through c-di-AMP. However, it
remains to be fully established in the future whether c-di-AMP
is truly a signaling messenger that regulates the phenotypes
associated with stress responses. The failure to detect c-di-
AMP in both the vegetative and sporulating B. subtilis cells
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This paper is available online at www.jbc.org
ADDITIONS AND CORRECTIONS
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 33, p. 29441, August 19, 2011
© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
VOLUME 282 (2007) PAGES 4288–4300
DOI 10.1074/jbc.A111.608307
VOLUME 284 (2009) PAGES 6079–6092
DOI 10.1074/jbc.A111.806077
Activation of JNK-dependent pathway is required for
HIV Viral Protein R-induced apoptosis in human
monocytic cells. INVOLVEMENT OF ANTIAPOPTOTIC BCL2
AND c-IAP1 GENES.
Preparation and properties of asymmetric vesicles that
mimic cell membranes. EFFECT UPON LIPID RAFT
FORMATION AND TRANSMEMBRANE HELIX ORIENTATION.
Hui-Ting Cheng, Megha, and Erwin London
Sasmita Mishra, Jyoti P. Mishra, and Ashok Kumar
The experiments reported were carried out at 390 mM methyl-␤-
cyclodextrin (M␤CD; 825 mg ϩ 1 ml of water, which gives a volume of
ϳ1.6 ml), not as reported at 625 mM M␤CD (825 mg/ml of solution).
We (MiJin Son and E. L.) have investigated asymmetric vesicle prepara-
tion at the higher M␤CD concentration and found that 625 mM M␤CD
could be used to produce asymmetric small unilamellar vesicles. How-
ever, the results were not as reproducible as with the lower M␤CD
concentration. We recommend the use of 390 mM M␤CD to prepare
asymmetric small unilamellar vesicles.
PAGE 4293:
Fig. 3A(upper right panel) shows an alteration of a lane (10 ␮M PD98059)
that was not detected at the time of manuscript submission and that is
contrary to the Journal of Biological Chemistry guidelines. Herein, we pro-
vide an alternative figure that shows that the ERK inhibitor PD98059 inhib-
ited Vpr-(52–96)-induced ERK activation in a dose-dependent manner.
THP-1 cells were treated with the indicated concentrations of PD98059 for
4 h, followed by stimulation with 1.5 ␮M Vpr-(52–96) peptide for another
2 h. The legend of this figure remains unchanged. The data support the
published observations and therefore do not impact the interpretation of
this figure or the central conclusion of this article.
VOLUME 284 (2009) PAGES 31006–31017
DOI 10.1074/jbc.A109.010736
Ibuprofen impairs allosterically peroxynitrite
isomerization by ferric human serum heme-albumin.
Paolo Ascenzi, Alessandra di Masi, Massimo Coletta, Chiara Ciaccio, Gabriella
Fanali, Francesco P. Nicoletti, Giulietta Smulevich, and Mauro Fasano
Vpr(52-96) + PD (μM)
2
5
10
20
pp-44
pp-42
For graphical reasons, kobs ϭ (kon ϫ 10) ϫ [HSA-heme-Fe(III)] ϩ k0
and kiobs ϭ (koi n ϫ 10) ϫ [HSA-heme-Fe(III)] ϩ ki0. Although the values
of kon and kion are reported correctly in the text and in Tables 1 and 2,
derivation from data reported in Figs. 2, 3, and 5 corresponds to kon ϫ 10
and kion ϫ 10.
p44/42
VOLUME 285 (2010) PAGES 21858–21867
DOI 10.1074/jbc.A110.117291
VOLUME 285 (2010) PAGES 473–482
DOI 10.1074/jbc.A109.040238
Nucleolar targeting of the chaperone Hsc70 is regulated
by stress, cell signaling, and a composite targeting
signal which is controlled by autoinhibition.
Piotr Ban´ski, Hicham Mahboubi, Mohamed Kodiha, Sanhita Shrivastava,
Cynthia Kanagaratham, and Ursula Stochaj
YybT is a signaling protein that contains a cyclic
dinucleotide phosphodiesterase domain and a GGDEF
domain with ATPase activity.
During the continuation of our work on hsc70 trafficking, we have
noticed that there was an error during the preparation of midiprep
plasmid DNA for two constructs that were described in this work. The
error reported by us does not alter the validity of the raw data. The
following two constructs were affected by the error: GFP-hsc70(225–
262) and GFP-hsc70(245–287). These two plasmid DNAs have been
inverted in our article. Following the detection of the error, we have
verified the correctness of all other constructs encoding wild-type or
mutant fragments of domain IIB of hsc70. This error affects the presen-
tation of results shown in Figs. 2B and 3 (A and C) and the interpretation
depicted in Fig. 6A. Thus, the correct interpretation of our data is that
segment 245–287 locates constitutively in the nucleolus under non-
stress and stress conditions. Fragment 225–262 displays weak stress-
induced nucleolar accumulation. This makes residues 225–244 the neg-
ative regulator of hsc70 nucleolar accumulation.
Feng Rao, Rui Yin See, Dongwei Zhang, Delon Chengxu Toh, Qiang Ji,
and Zhao-Xun Liang
PAGE 473:
The first sentence in the Abstract should read as follows: The cyclic
dinucleotide c-di-AMP synthesized by the diadenylate cyclase domain
was discovered recently as a messenger molecule for signaling DNA
breaks in Bacillus subtilis.
In the Introduction, line 21 in the right-hand column should read as
follows: This group of proteins (COG3887), as represented by the
B. subtilis protein YybT, contains two N-terminal transmembrane hel-
ices, a region that shares minimum sequence homology with some Per-
Arnt-Sim (PAS) domains, a highly modified GGDEF domain, and a
DHH/DHHA1 domain (see Fig. 1).
We suggest that subscribers photocopy these corrections and insert the photocopies in the original publication at the location of the origi-
nal article. Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to
carry notice of these corrections as prominently as they carried the original abstracts.
AUGUST 19, 2011•VOLUME 286•NUMBER 33
JOURNAL OF BIOLOGICAL CHEMISTRY 29441