(Received for publication, January 4, 1996; and in revised form, February 6, 1996)
From the
The ars operon of the Escherichia coli plasmid
R773 that confers arsenical and antimonial resistance is negatively
regulated by the ArsR repressor. ArsR residues Cys-32 and Cys-34 were
previously identified as involved in induction by arsenite and
antimonite, suggesting coordination between As(III) and the two
cysteine thiolates. However, in small molecule thiolate-As(III)
complexes, arsenic is frequently three-coordinate. A site-directed
mutagenic approach was employed in a search for a third arsenic ligand.
ArsR proteins with C32G, C34G, and C32G/C34G substitutions were active
repressors, but were not inducible in vivo. In vitro,
the altered repressor-ars DNA complexes could not be
dissociated by inducers. Alteration of Cys-37 and Ser-43, residues
located in or near the putative helix-turn-helix DNA-binding region of
the protein, had no effect on the inducibility of the operon. While
these results indicated that neither the thiolate of Cys-37 nor the
hydroxyl oxygen of Ser-43 is required for induction, they did not
eliminate either atom as a potential arsenic ligand. Another approach
involved reaction with an alternative inducer, phenylarsine oxide,
which can form only two coordinations. Phenylarsine oxide was shown to
be as effective as or more effective than arsenite or antimonite in
induction in vivo. In vitro, the organic arsenical
was more effective than either arsenite or antimonite in dissociating
the repressor-promoter complex. Thus, two ArsR-arsenic bonds are
sufficient for induction. The interaction of ArsR proteins with As(III)
was examined using a phenylarsine oxide affinity resin. ArsR proteins
containing any two of the three cysteine residues Cys-32, Cys-34, and
Cys-37 bound to the resin. Alteration of any two of the three resulted
in loss of binding. Arsenic x-ray absorption spectroscopy of ArsR
treated stoichiometrically with arsenite confirmed the average arsenic
coordination as AsS. These results suggest that all three
cysteine thiolates are arsenic ligands, but binding to only two, the
Cys-32 and Cys-34 thiolates, is required to produce the conformational
change that results in release of the repressor from the DNA and
induction.
Plasmid R773-mediated resistance to arsenite, arsenate, and antimonite in Escherichia coli is catalyzed by an arsenite extrusion system whose components are encoded by the ars operon (for recent reviews, see Rosen et al.(1995) and Dey and Rosen(1995)). Transcription of the ars operon is regulated by a substrate-responsive repressor, the ArsR protein. As metalloids, As(III) and Sb(III) can potentially interact with proteins as nonmetallic oxyanions do (Quiocho et al., 1987), or they can react as soft metals, forming covalent bonds with cysteine thiolates (Freeman, 1975). Among the proteins of the R773 ars operon, the ArsA ATPase, the catalytic component of the Ars pump, is allosterically activated by As(III)/Sb(III) interaction with three cysteine thiolates (Bhattacharjee et al., 1995). In contrast, there are no essential cysteine residues in ArsB, the membrane sector of the pump, indicating that the substrates of the pump are the oxyanions arsenite and antimonite (Chen et al., 1996).
Using a novel selection for arsR mutants that encoded active repressors that were unable to be induced by arsenicals or antimonials, we previously isolated three hydroxylamine mutants in the codons for Cys-32 and Cys-34, resulting in ArsR proteins C32Y, C32F, and C34Y (Shi et al., 1994). All three altered ArsR proteins still bound specifically to the ars operator, but inducers were considerably less effective in dissociating in vitro ArsR-DNA complexes. In vivo, each mutant arsR gene repressed expression of a reporter gene controlled by the ars promoter, and addition of inducer did not relieve repression. Thus, these altered proteins retained repressor function, but had reduced response to inducer, suggesting that As(III)/Sb(III) can react as soft metals with the thiolates of Cys-32 and Cys-34. Among members of the ArsR family of metalloregulatory proteins, this cysteine pair and adjacent residues are highly conserved; we have proposed that this region forms a portion of the inducer recognition site and that the cysteine pair is required for metal binding (Shi et al., 1994).
How does binding of arsenite result in induction? It is
reasonable to consider that coordination of As(III) with the cysteine
thiolates results in a conformational change in the repressor that
disrupts its interaction with the operator DNA. Both sulfur and oxygen
can be arsenic ligands. For example, in the arsenite-glutathione
complex, sulfur thiolates from three glutathiones serve as arsenic
ligands forming As(GS) (Delnomdedieu et al.,
1994), and in the arsenite-dithiothreitol complex, As(III) forms soft
metal bonds with the two sulfur thiolates and one hydroxyl oxygen
(Cruse and James, 1972). In addition to Cys-32 and Cys-34, ArsR
contains three other cysteine residues, Cys-37, Cys-108, and Cys-116.
The latter two have been shown to be unnecessary for repressor function
(Wu and Rosen, 1991). In addition, in the putative helix-turn-helix
DNA-binding domain, there are two serine residues, Ser-43 and Ser-48.
In this study, the requirement for the sulfur thiolate of Cys-37 and
the two serine hydroxyls was explored.
It was also not clear from
the previous study whether the loss of inducer recognition stemmed from
the loss of the sulfur thiolates of Cys-32 and Cys-34 or from the
introduction of the bulky aromatic rings. In this study, a series of
single and double mutants in the codons for these residues were
isolated by site-directed mutagenesis, where the residues were altered
to residues with smaller side groups. In confirmation with the previous
study (Shi et al., 1994), only alterations of Cys-32 and
Cys-34 altered inducer recognition. In addition, using both in vivo and in vitro assays, the arsenite analog phenylarsine
oxide (PAO) ()was found to be the most effective inducer of
the ars operon. Since PAO can form only two
arsenic-thiol bonds with ArsR, dicoordinate binding must be
sufficient for induction.
Using PAO affinity chromatography as an
assay for arsenic-ArsR interaction, Cys-32, Cys-34, and Cys-37 were
each found to be required for ArsR binding. These results suggest that
the thiolates of Cys-32, Cys-34, and Cys-37 form a tricoordinate
AsS site. Arsenic x-ray absorption fine structure (EXAFS)
was used to probe directly the coordination of As(III) in ArsR treated
stoichiometrically with arsenite. AsS
coordination is
confirmed in these experiments. Thus, while the Cys-37 thiolate is able
to form a bond with As(III), binding to the sulfurs of only Cys-32 and
Cys-34 is required to produce the conformational change in ArsR that
results in dissociation from the operator DNA and transcriptional
derepression.
Plasmid pAltR was constructed by inserting a 0.73-kilobase
pair EcoRI-HindIII fragment containing the ars operator/promoter, the arsR gene, and a portion of the arsD gene from plasmid pWSU1 (San Francisco et al.,
1990) into the multiple cloning site of vector plasmid
pALTER-1 (Promega). The pAltR series of plasmids was
derived from pAltR by site-directed mutagenesis of the arsR gene. Each of the plasmids in the pT
R series was
derived from the corresponding plasmid of the pAltR series by cloning
an EcoRI-HindIII fragment containing arsR into expression vector pT7-5 (Tabor and Richardson, 1985). To
create the plasmid pBGD23 series, the EcoRI-BclI
fragment from the corresponding plasmid of the pAltR series was
inserted into the EcoRI-BamHI site of vector pMLB1034
(Silhavy et al., 1984). Plasmid pBGD
R1 was constructed by
deleting a DraI-StuI fragment from arsR in
plasmid pBGD23.
All oligonucleotides were synthesized in the Macromolecular Core Facility of the Wayne State University School of Medicine. Each mutant gene was sequenced to confirm the desired mutation and to exclude introduction of additional mutations.
Wild-type and altered
ArsR proteins were purified from E. coli strain HMS174(DE3)
bearing pTR series plasmids. Cytosol from 2 liters of a
culture induced with 0.25 mM isopropyl-
-D-thiogalactopyranoside was applied to a
120-ml S-Sepharose column (Pharmacia Biotech Inc.) pre-equilibrated
with buffer consisting of 10 mM sodium phosphate, pH 7.0,
containing 1 mM EDTA and 14.3 mM
-mercaptoethanol. Proteins were eluted with a linear gradient
of 0.15-0.60 M NaCl in the same buffer. ArsR-containing
fractions were pooled, concentrated, and loaded onto a Bio-Gel P-30
column (1.5
100 cm; Bio-Rad). The column was eluted with the
same buffer containing 0.1 M NaCl. Each ArsR protein was
judged to be >90% homogeneous by SDS-PAGE and stored at -70
°C until use. Protein concentration was determined using the
bicinchoninic acid assay (Pierce).
Extraction of structural data used curve fitting of raw EXAFS data based on scattering parameters extracted from the EXAFS data for the model compounds arsenite (three oxygens at 1.85 Å) and arsenite + glutathione (three sulfurs at 2.25 Å in solution) (Delnomdedieu et al., 1994). Such analysis typically yields bond distances with an accuracy of ±0.02 Å.
Figure 1: Autoregulation of ArsR proteins. Exponentially growing cultures of E. coli strain JM109 bearing pAltR series plasmids with the indicated arsR mutations were grown for 2 h with (+) or without(-) 10 µM sodium arsenite. Expression of ArsR proteins was monitored by SDS-PAGE followed by immunoblotting with anti-ArsR serum.
Since As(III) can form tricoordinate
complexes with sulfur or oxygen atoms, the involvement of a third
residue was investigated. Possible residues include cysteine thiolates
and oxygens of serine or threonine hydroxyls. In R773 ArsR, there are 5
cysteine, 10 serine, and 2 threonine residues (Fig. 2).
Comparison of the R773 repressor with the four other known ArsR
homologs demonstrates that Cys-32, Cys-34, Cys-108, Ser-43, and Ser-48
are conserved (Fig. 2). Cys-108 has been shown previously not be
required for ArsR function (Wu and Rosen, 1991). Ser-43 and Ser-48 are
within the predicted helix-turn-helix DNA-binding domain of the
repressor (Shi et al., 1994); coordination with As(III) could
cause a conformational change in that domain, producing dissociation
from the DNA. For this reason, the codons for Ser-43 and Ser-48 were
altered by site-directed mutagenesis to produce S43A, S43P, S48A, and
S48T. The nonconserved Cys-37 was also changed to an alanine residue,
and an arsR double mutant was constructed.
The single Cys-37 and Ser-43 substitutions inducibly regulated their
own synthesis from the ars promoter (Fig. 1). The
Ser-48 substitutions resulted in a constitutive phenotype, and the arsR
double mutant exhibited uninducible
phenotypes similar to that of the arsR
single
mutant.
Figure 2:
The
ArsR repressors. Shown are the ArsR proteins of plasmids R773 (San
Francisco et al., 1990), R46 (D. F. Bruhn, J. Li, S. Silver,
F. Roberto, and B. P. Rosen, unpublished data;
GenBank/EMBL accession number U38947), pI258 (Ji and
Silver, 1992), and pSX267 (Rosenstein et al., 1992) and
chromosomal E. coli ArsR (Xu et al., 1996). The
location of the putative helix-turn-helix DNA-binding motif is
indicated. Residues altered in this study are numbered and shaded.
Figure 3:
Effects of altered proteins on induction
of the ars operon. Top, the relevant portion of the
pBGD23 plasmid series is shown. A lacZ gene was fused in frame
to arsD to form a arsD::lacZ fusion under control of
the ars promoter (Pars). Bottom, cells
bearing plasmids of the pBGD23 series were induced with the indicated
concentrations of sodium arsenite. -Galactosidase assays were
performed as described under ``Materials and
Methods.''
Figure 4:
Affinity of ArsR proteins for promoter
DNA. Gel mobility shift assays were carried out as described under
``Materials and Methods'' using 0.25 µM purified
DNA probe. The gels were dried, and the amount of free probe and
probe-protein complex was quantified with an AMBIS radioactive analysis
system. A, wild type (), C32G (
), C34G (
),
and C37A (
); B: wild type (
), S48A (
),
C32G/C34G (
), and C32G/C37G
(
).
To demonstrate that the altered proteins bound to the same site on the DNA, the DNase I footprints of the wild-type and C32G/C34G repressors were compared (Fig. 5). The doubly substituted C32G/C34G protein protected the same regions as the wild type, corresponding to nucleotides -61 to -37 on the coding strand and nucleotides -64 to -40 on the noncoding strand. While addition of the inducer arsenite or PAO (see below) dissociated the wild-type repressor from the DNA, C32G/C34G was not dissociated by inducers.
Figure 5: DNase I footprint analysis. DNase I footprint assays were performed with both the coding (lanes 1-7) and noncoding (lanes 8-14) strands as described under ``Materials and Methods.'' Proteins were as follows: none (lanes 1 and 8), wild-type ArsR (lanes 2-4 and 9-11), and C32G/C34G (lanes 5-7 and 12-14). Inducers were as follows: none (lanes 1, 2, 5, 8, 9, and 12), 0.5 mM sodium arsenite (lanes 3, 6, 10, and 13), and 0.5 µM PAO (lanes 4, 7, 11, and 14). Protected sequences are indicated by boxes.
Figure 6:
Phenylarsine oxide is an inducer of the
R773 ars operon. A, structure of PAO and its binding
to a vicinal cysteine pair; B, gel mobility shift assays of
the -
P-labeled probe containing the ars operator/promoter and wild-type ArsR (lanes 2-5)
and inducer-independent C32Y (lanes 6-9). Concentrations
of PAO were 0.1 µM (lanes 3 and 7), 0.5
µM (lanes 4 and 8), and 1 µM (lanes 5 and 9).
The affinity of the repressor for the
physiological inducers was determined (Fig. 7). In this assay, 1
µM ArsR was mixed with sufficient DNA to produce 60%
protein-DNA complex and 40% free DNA. To dissociate half of the bound
repressor from the DNA required <1 µM PAO, compared
with 10 µM sodium arsenite or potassium antimonyl
tartrate. These results demonstrate that PAO is a better inducer of the ars operon in vitro than inorganic As(III) or
Sb(III). Since induction in vivo is multifactorial, reflecting
a combination of inducer uptake, transcriptional activation, and
effects of inducer on the activity of the reporter gene product, in
vitro inducibility may be a better measure of the strength of the
inducer-repressor interaction.
Figure 7:
Effects of inducers on the dissociation of
the ArsR-ars operator/promoter complex. Mobility shift assays
were carried out as described in the legend to Fig. 4. ArsR
proteins were added at 1 µM. Inducers were PAO (),
sodium arsenite (
), and potassium antimonyl tartrate
(
).
The effect of PAO on the DNA binding properties of ArsR proteins with substitutions of cysteine residues was determined (Fig. 8). Neither the C32G nor C34G repressor dissociated from the DNA with any concentration of PAO tested. The C37A repressor exhibited the same response to PAO as the wild type. The doubly substituted C32G/C34G and C32G/C37G proteins gave the same response as the C32G protein (data not shown). Similarly, 0.5 µM PAO prevented the wild-type repressor, but not C32G/C34G, from protecting the DNA fragment from DNase I digestion (Fig. 5, lanes 4, 7, 11, and 14). Thus, for the in vitro response of the repressor to PAO, cysteine thiolates appear to be required at residues 32 and 34, but not 37.
Figure 8:
In vitro effect of inducer on DNA
and ArsR altered protein interaction. Mobility shift assays were
performed as described under ``Materials and Methods'' with 1
µM repressor. PAO was added to the reaction mixture at the
indicated concentrations. Repressors were wild type (), C32G
(
), C37A (
), and C34G
(
).
Figure 9:
PAO-ArsR interaction chromatography. PAO
affinity chromatography was performed as described under
``Material and Methods.'' One mg of each repressor protein
was applied to the column, followed by stepwise elution with increasing
concentrations of -mercaptoethanol (
-ME). In each
assay, the height of the bar indicates the concentration of
-mercaptoethanol required for elution. BSA, bovine serum
albumin.
Figure 10:
Determination of arsenic ligands in ArsR.
AsK edge EXAFS (top) and Fourier transform (FT) (bottom; -weighted,
=
3.5-13.0 A-1) data are shown for As(III)-loaded ArsR. Solid
lines, experimental data; dashed lines, the best fit
simulation with three arsenic-sulfur interactions at 2.25 Å; dotted lines, simulation of hypothetical
AsO
S
coordination (arsenic-oxygen
= 1.85 Å; arsenic-sulfur = 2.25
Å).
ArsR is the arsenic/antimony-responsive repressor of the plasmid R773 ars operon (Wu and Rosen, 1991, 1993). The protein is the best characterized member of the ArsR family of metalloregulatory proteins (Shi et al., 1994). In members of this family, there is the highly conserved sequence ELCVCDL located adjacent to the putative helix-turn-helix DNA-binding domain. Using a direct selection of arsR mutants that lost the ability to respond to inducer but retained repression, we previously isolated by chemical mutagenesis three mutants in the cysteine pair of this conserved sequence (C32Y, C32F, and C34Y) (Shi et al., 1994). These altered proteins retained the ability to bind to the ars promoter, but had reduced metal response, suggesting that the cysteine pair Cys-32 and Cys-34 is a component of the metallosensory domain of ArsR, forming soft metal bonds between trivalent arsenic and the sulfur thiolates. However, it was possible that the phenotypes resulted from the introduction of aromatic side chains rather than from loss of the thiolates. In this study, it was shown that glycine substitutions produced the same effects both in vivo and in vitro as the phenylalanine and tyrosine substitutions, supporting the hypothesis that cysteine thiolates are ligands for As(III) coordination (Rosen et al., 1995).
In addition to Cys-32 and Cys-34, there are three other cysteine residues in the ArsR protein: Cys-37, Cys-108, and Cys-116. The last two have been shown not to be required for ArsR function (Wu and Rosen, 1993). In the present study, Cys-37 was altered mutagenically to alanine, and this substitution did not affect the in vivo regulatory or in vitro DNA binding properties. Additionally, the possible participation of the hydroxyl oxygens of Ser-43 and Ser-48, residues conserved in the five known ArsR proteins, was investigated. Alteration of Ser-43 to alanine or proline had no effect on ArsR function in vivo or in vitro, while alteration of Ser-48 resulted in constitutivity in vivo and decreased affinity of DNA in vitro. Although both residues are in the putative helix-turn-helix DNA-binding domain (Shi et al., 1994), Ser-43 is located in the turn region, and its substitution may not be affected by introduction of a helix breaker such as proline. The use of phenylarsine oxide has proven additionally instructive. This organic arsenical can form only two coordinations, the third being an arsenic-carbon bond to the phenyl ring. PAO is not only an effective inducer in vivo; in in vitro DNA binding assays, ArsR exhibited 10-100-fold higher affinity for this organic arsenical than for inorganic arsenite or antimonite. Since a third coordination is not possible with PAO, induction must require only two bonds between inducer and ligands in ArsR. The combination of the results of mutagenesis and PAO induction suggests that only the Cys-32 and Cys-34 pair is involved in the interaction with As(III) that results in induction.
However, it was possible that there are other interactions with As(III) not required for induction. While there appear to be two soft metal bonds to the Cys-32 and Cys-34 sulfur thiolates, the existence or nature of a third coordination in arsenite was unknown. In PAO, it is the phenyl ring; in arsenite, possibilities included other cysteine sulfur thiolates, the oxygens of serine or threonine hydroxyls, or the third hydroxyl group of arsenite itself. PAO-protein interactions can be measured using matrix-bound PAO (Hoffman and Lane, 1992). Wild-type ArsR bound to the PAO resin with extremely high affinity, characteristic of a protein with a vicinal thiol pair. However, ArsR proteins in which either Cys-32 or Cys-34 was altered to glycine still bound strongly to the PAO resin. The C32G/C34G doubly altered protein no longer bound, demonstrating that these residues are involved in binding. But, if binding requires coordination to two groups, what is the additional ligand when only Cys-32 or Cys-34 is present? While C37A bound with affinity equal to that of C32G and C34G, C32G/C37G no longer bound. These results indicate that binding requires two cysteines, but that, in contrast to induction, any combination of Cys-32, Cys-34, and Cys-37 suffices. Note that Cys-108 and Cys-116 are present in all altered proteins and thus do not appear to be able to substitute in proteins lacking any two of the other cysteines. The x-ray absorption spectroscopy studies indicate that there are three sulfur ligands 2.25 Å from the As(III) and that there are no oxygen ligands, confirming that only cysteine residues, and not serine or threonine, are arsenic ligands. Combining these results with the information from the known structures of the arsenite-dithiothreitol (Cruse and James, 1972) and the arsenite-glutathione (Delnomdedieu et al., 1994) complexes, we propose a model for the arsenic-ArsR complex in which As(III) is bound in a cage formed by the three cysteine thiolates at residues 32, 34, and 37 (Fig. 11). Even though coordination to Cys-37 thiolate occurs, the soft metal bonds formed between arsenic and the sulfurs of Cys-32 and Cys-34 are sufficient to produce a conformational change in the DNA-binding domain that results in dissociation of the repressor from the operator DNA.
Figure 11: Model for metalloregulation by the ArsR repressor. The ArsR homodimer binds to the operator/promoter region through a helix-turn-helix domain in each monomer, repressing transcription of the ars operon. Inducers, including arsenite, antimonite, and phenylarsine oxide, bind to Cys-32 and Cys-34 through covalent metal-sulfur bonds (solid lines). This produces a conformational change in the DNA-binding domain that results in dissociation of the repressor from the operator/promoter. Cys-37 is the third ligand for As(III) or Sb(III) (dotted lines), although this is not required for induction. Thus, As(III) is three-coordinate in a pyramidal cage in which the three sulfur thiols form the base and the lone electron pair of arsenic forms the apex.