From the Department of Biochemistry and Molecular Biology, Wayne
State University, School of Medicine, Detroit, Michigan 48201
ArsA, the catalytic subunit of an
anion-translocating ATPase, has two consensus nucleotide binding sites,
one N-terminal and one C-terminal. A mutation producing a G15C
substitution in the N-terminal domain resulted in substantial
reductions in arsenite resistance, transport, and ATPase activity. A
second site revertant (A344V) adjacent to the C-terminal nucleotide
binding site was previously shown to restore arsenite resistance,
suggesting the interaction of the nucleotide binding sites in ArsA (Li,
J., Liu, S., and Rosen, B. P. (1996) J. Biol.
Chem. 271, 25247-25252). In this study, it is shown that
alteration of Ala-344 to bulkier residues, including Cys, Thr, Pro,
Asp, Leu, Phe, Tyr, or Arg, also suppressed the G15C substitution.
However, A344G or A344S substitutions only marginally suppressed the
primary mutation. Alteration of Gly-15 to Ala, Cys, Asp, Tyr, or Arg
each resulted in decreased arsenite resistance. The larger the residue
volume of the substitution, the lower the resistance, with a G15R
substitution producing the least resistance. Resistance in a strain
expressing an arsA gene encoding the G15R substitution
could be rescued by A344S, A344T, A344D, A344R, or A344V second site
suppressors. The larger the residue is then the greater the suppression
is. The in vitro ArsA ATPase activities from purified wild
type, G15A, G15C, and G15R exhibits an inverse relationship between
activity and residue volume. Purified G15A and G15C exhibited both an
increase in the Km for ATP and a decrease in
Vmax. The results are consistent with a
physical interaction of the two nucleotide binding domains and indicate
that the geometry at the interface between the N- and C-terminal
nucleotide binding sites places spatial constraints on allowable
residues in that interface.
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INTRODUCTION |
Escherichia coli plasmids R773 (1) and R46 (2) carry
arsenical resistance (ars) operons encoding a pump that
produces arsenite and antimonite resistance by ATP-coupled extrusion
(3). This ATP-coupled oxyanion transporter consists of two types of subunits. The 63-kDa ArsA subunit is the catalytic component, with
As(III)/Sb(III)-stimulated ATPase activity, and is allosterically activated by binding of As(III) or Sb(III) to a triad of cysteine thiols (4, 5). The 45.5-kDa intrinsic membrane ArsB component has 12 transmembrane
-helices (6) and functions as both the membrane anchor for ArsA and the anion-translocating sector of the pump
(7).
ArsA has two homologous halves, A1 and A2, most likely the result of an
ancestral gene duplication and fusion (1). Each half has a consensus
glycine-rich sequence similar to a Walker A motif or P-loop (8-10). In
ArsA, the P-loop sequences are G15KGGVGKT and
G334KGGVGKT, respectively. P-loops are usually flanked by a
hydrophobic
-sheet and an
-helix (11). The results of genetic complementation (12) and biochemical reconstitution (13) suggested that
an A1 and A2 domain interacted to form a catalytic unit. The isolation
of intragenic suppressors supported the concept that a single catalytic
site was formed at the interface of the A1 and A2 ATP binding sites
(14).
In this study, amino acid residues 15 and 344 of ArsA were substituted
with a variety of other residues. In single mutants, the smaller the
residue at position 15, the greater the phenotypic resistance, with a
G15R substitution producing the least resistance. In contrast, the
larger the residue at position 344, the greater the phenotypic
suppression of arsAG15C and
arsAG15R mutations. Wild type, G15A, and G15C ArsAs
were purified and characterized. There was an inverse relationship
between ATPase activity and residue volume at position 15, consistent
with the resistance phenotype of the mutants. Enzymes with larger
residues at position 15 had an increased Km for ATP
and a decrease in Vmax. These results suggest
that there are spatial constraints in allowable residues at the
interface of the A1 and A2 nucleotide binding sites.
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EXPERIMENTAL PROCEDURES |
Media and Growth Conditions--
E. coli strains and
plasmids used in this study are described in
Table I. Cells were grown in
Luria-Bertani medium (15) at 37° C. Ampicillin (125 µg/ml) and
tetracycline (10 µg/ml) were added as required. Sodium arsenite,
potassium antimonyl tartrate, and isopropyl
-D-thiogalactopyranoside were added at the indicated concentrations.
DNA Manipulations--
The conditions for plasmid isolation, DNA
restriction endonuclease analysis, ligation, and transformation have
all been described (16). Restriction enzymes and nucleic acid modifying
enzymes were obtained from Life Technologies, Inc. WizardTM
Plus minipreps DNA purification system and
WizardTM DNA clean-up system (Promega) were used to prepare
plasmid DNA for restriction enzyme digestion and recovering DNA
fragments from low melting agarose gels, respectively.
Oligonucleotide-directed Mutagenesis--
Mutations in the
arsA gene were introduced by site-directed mutagenesis using
the Altered SitesTM in vitro mutagenesis system
(Promega). Plasmid pALTER-AB (4) containing the arsA and
arsB genes was used as the template to obtain G15X mutants,
and pG15C.1 (Table I) to obtain pG15C/A344X double mutants. The
mutagenic oligonucleotides used were as follows: G15A, G15R, and G15Y,
CGCCTCCTTT(A/C)(A/C/G/T)(A/C/G/T)CGTAAAAAACAG; G15D, CGCCTCCTTTA(A/T)CCGTAAAAAACAG; A344P,
A344S, A344T, A344Y, ACAGCAATGGCAGC(A/C)(A/C/G/T)(A/C/G/T) CATCGTGGTTTTCC;
A344R and A344L,
ACAGCAATGGCAGCA(A/ C)(A/G)CATCGTGGTTTTCC;
A344F,
ACAGCAATGGCAGCA(A/C/ T)ACATCGTGGTTTTCC;
A344G and A344D,
ACAGCAATGGCAGCA(C/T)CCATCGTGGTTTTCC; and A344C,
GCAATGGCAGCACACATCGTGGTTTTC.
Oligonucleotides synthesized with a degeneracy have the bases present
in the mixture indicated in parentheses. Substitutions that resulted in
mutation are underlined. The identity of each mutation was
confirmed by DNA sequencing of the entire mutated gene. Double-stranded
plasmid DNA was prepared using the QIAGEN DNA Purification System.
Sequencing was performed using the Pharmacia Cy5 labeled autosequence
kit (Pharmacia Biotech Inc.) and ALFexpress apparatus by the
method of Sanger et al. (17). Other single or double mutants
were constructed by molecular subcloning using a combination of two
restriction endonucleases, either HindIII and
SphI or SphI and KpnI (Table I).
Inhibition by arsenite was tested on solid Luria-Bertani media. The
minimal inhibitory concentration is the concentration at which no
growth was detected. Cells were grown overnight and streaked to single
colonies on agar plates containing varying concentrations of sodium
arsenite. Growth was monitored after 24 h at 37 °C.
Purification and Assay of the ArsA ATPase--
Proteins were
purified from the cytosol of 2.5 liters of induced cultures as
described previously (4, 14). Each ArsA was judged to be greater than
95% homogeneous by Coomassie Blue staining of samples separated by
sodium dodecyl sulfate electrophoresis on 8% polyacrylamide gels (18).
The concentrations of purified ArsAs were determined using a
bicinchoninic acid protein assay (Pierce) or from the absorption at 280 nm using a molar extinction coefficient of 33,480 (19). ATPase activity
was measured using a coupled assay (20), as described previously
(21).
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RESULTS |
Arsenite Resistance Phenotype of G15X Mutants--
To examine the
effect of residue volume at position 15 on arsenite resistance, Gly-15
was altered to Ala, Asp, Cys, Tyr, and Arg. An inverse relationship was
observed (Fig. 1). The larger the residue
then the resistance was less, with the phenotype of cells expressing an
arsAG15R mutation similar to cells with vector
alone.

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Fig. 1.
Effect of
arsAG15X mutations on arsenite
resistance. Overnight cultures of E. coli strain
JM109 bearing plasmids with wild-type and mutant arsA genes
and a wild-type arsB gene were diluted 100-fold into fresh
Luria-Bertani medium containing varying concentrations of sodium
arsenite. A600 nm was measured after 8 h
of growth at 37°C and the values normalized to growth in the absence
of arsenite. Cells had the following plasmids: , pALTER-AB
(arsAB); , pG15A
(arsAG15AB); , pG15C
(arsAG15CB); , pG15D
(arsAG15DB); , pG15Y
(arsAG15YB); , pG15R
(arsAG15RB); and , vector plasmid
pALTERTM-1.
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Suppression of G15C or G15R by A344X Second Site
Alterations--
By random mutagenesis, an A344V alteration was found
to suppress the arsenite-sensitive phenotype of an
arsAG15C mutant (14). The effect of single mutations
at residue 344 on arsenite resistance was examined. Cells expressing
six single arsAA344X genes (X = G,
S, T, D, R and V) each exhibited an arsenite-resistant phenotype, with
only slight differences in resistances at higher concentrations of
arsenite (Fig. 2).

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Fig. 2.
Effect of arsAA344X
mutations on arsenite resistance. Phenotypes of cells expressing
single arsAA344X mutations were determined as
described in the legend to Fig. 1. Cells had the following
plasmids: , pALTER-AB (arsAB); , pA344T
(arsAA344TB); , pA344G
(arsAA344GB); , pA344V
(arsAA344VB); , pA344S
(arsAA344SB); , pA344D
(arsAA344DB); , pA344R
(arsAA344RB); and , vector plasmid
pALTERTM-1.
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To examine the relationship of residues 15 and 344 in more detail, the
codon for Ala-344 in the arsAG15C gene was changed
to 11 different codons by site-directed mutagenesis with mixtures of
oligonucleotides, producing ArsA derivatives G15C/A344X, where
X = G, S, C, T, V, P, D, L, F, Y, or R. Cells harboring the mutated arsA genes and wild-type arsB gene
were phenotypically characterized for arsenite resistance (Fig.
3). Cells expressing the wild-type
arsAB genes could tolerate up to 10 mM
NaAsO2; cells containing only vector were unable to grow
when the concentration of arsenite was more than 1 mM.
Alteration of Ala-344 to bulkier residues, including Cys, Thr, Pro,
Asp, Leu, Phe, Tyr, or Arg each suppressed the G15C substitution.
Moreover, the larger the residue at position 344, the higher the
concentration of arsenite required to inhibit growth. However, the
smaller A344G did not suppress, and a substitution of serine, which has
a volume near that of alanine, only marginally suppressed the primary
mutation. Similarly, when the G15R substitution was combined with
substitutions at residue 344, the larger the residue at position 344, then the higher was the resistance of the double mutant (Fig.
4). While the A344R substitution
suppressed both G15C or G15R, the level of suppression was somewhat
less than expected from a simple steric effect, suggesting a slight
negative effect of a basic residue at position 344. Overall, however,
these results suggest that the primary effect of the side chain at
residue 344 is a steric one, rather than that of charge or
polarity.

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Fig. 3.
Effect of residue volume at position 344 on
suppression of the arsAG15C
mutation. Phenotypes of cells expressing
arsAG15C/A344X double mutations were determined as
described under "Experimental Procedures." The minimal inhibitory
concentration is the concentration of sodium arsenite at which no
growth was observed after 8 h at 37°C. Amino acid residue
volumes are from the partial volume in solution (34).
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Fig. 4.
Suppression of an
arsAG15R mutation by
arsAA344X mutations.
Phenotypes of cells expressing arsAG15R/A344X
double mutations were determined as described in the legend to Fig.
1. Cells had the following plasmids: , pALTER-AB (arsAB);
, pG15R/ A344V (arsAG15R/A344VB); , pG15R/ A344R (arsAG15R/A344RB);
, pG15R/ A344D (arsAG15R/A344DB); , pG15R/ A344T (arsAG15R/A344TB);
, pG15R/ A344S (arsAG15R/A344SB); , pG15R (arsAG15RB); and ,
vector plasmid pALTERTM-1.
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Effect of G15X and A344X Substitutions on ArsA ATPase
Activity--
It was of interest to examine the effect of single and
double mutations on the catalytic activity of the ArsA ATPase. None of
the mutations reported here resulted in reduced synthesis of ArsA (data
not shown). However, just as was reported by the A344V substitution
(14), most A344X derivatives formed inclusion bodies, with very little
enzyme found in the cytosol. Attempts to purify the residual soluble
portions were unsuccessful; the altered proteins appeared to degrade to
smaller polypeptides during chromatography. The formation of inclusion
bodies and increased degradation may result from defective folding of
the abnormal proteins. However, the G15A, G15C, and G15R singly
substituted derivatives could be purified. Consistent with the
phenotypic effect of arsAG15R, the G15R enzyme was
catalytically inactive. When the catalytic parameters of G15C and G15R
were compared with wild-type enzyme, their affinity for ATP was
decreased from 0.05 to 0.7 mM, and the
Vmax for G15A and G15C was decreased 60 and
90%, respectively.
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DISCUSSION |
The glycine-rich P-loop is conserved in purine binding enzymes
from prokaryotes to eukaryotes (8). Similar three-dimensional structures from RecA (22), myosin (23), the ras oncogene
product p21 (24) and F1-ATPase (25) imply that the function
of the P-loop is conserved in each enzyme, even though the primary
sequence of the P-loop for each specific purine NTPase is variant, and each enzyme has different biological activity (26). Using cassette mutagenesis, Shen et al. (26) substituted each residue of
the primary sequence of the yeast mitochondrial F1-ATPase
-subunit P-loop and found that there were constraints imposed by
only four residues in the P-loop
(G190XXXXGKT197). Furthermore, stereochemical constraints were observed in the P-loops of the E. coli ATP
synthase
-subunit (27) and dinucleotide binding proteins (28). An inverse relationship was observed between the volume of
-subunit residue 174 of the E. coli ATP synthase and ATPase activity,
suggesting that residue 174 was spatially near the ATP binding site
(29), a prediction that was subsequently verified in the crystal
structure (25).
ArsA, the catalytic subunit of the Ars pump, has two P-loops (1)
and an allosteric effector binding site (4, 5). Previous studies
demonstrated the necessity for Gly-15 (14), Gly-18, Gly-20, Thr-22
(30), and Lys-21 (31) in the A1 P-loop (G15KGGVGKT22). Substitutions of residues
Gly-334 (14) and Lys-340 (31) in the A2 nucleotide binding domain
(G334KGGVGKT341) were better tolerated than the
equivalent substitutions in the A1 P-loop. From a combination of
results from genetic complementation (12), biochemical reconstitution
(13), and intragenic suppression (14), a model has been proposed in
which the A1 and A2 nucleotide binding domains of ArsA must physically
interact for catalysis to occur, requiring the two domains to be
located near each other in the quaternary structure of the enzyme.
In the absence of structural information on ArsA, this hypothesis was
tested genetically. An intragenic suppressor of a G15C substitution in
the A1 P-loop was found to be in the codon for Ala-344 (14). In
nucleotide binding sites, the P-loop is usually preceded by a
-sheet
and followed by an
-helix (11). Ala-344 is at the beginning of a
putative
-helix which follows the A2 P-loop. In this study, the
effect of mutation of the codon for Ala-344 to the codons for eleven
different residues was examined. The substitutions include residues
representing each type of side group: nonpolar (Gly, Ala, Val, Pro, and
Leu), polar (Ser, Thr, and Cys), aromatic (Phe and Tyr), negatively
charged (Asp), and positively charged (Arg). The ability to suppress
the phenotype of the primary mutation correlated only with size and not
charge or polarity, with bulkier residues at position 344 providing
better suppression of the change at position 15 (Figs. 3 and 4).
The effect of side group substitution at position 15 was examined. A
series of single mutants were created by changing the codon for Gly15
in the A1 P-loop to codons for nonpolar (Ala), polar (Cys), aromatic
(Tyr), negatively charged (Asp), and positively charged (Arg). Again,
the effects correlated primarily with side chain volume and not charge
or polarity (Fig. 1). In contrast to the effect of residue size at
position 344, there was an inverse effect of residue 15 side group size
on arsenite resistance. However, considering that the volumes of
arginine (197 Å3) and tyrosine (191 Å3) are
similar, an arginine substitution produced less resistance than
anticipated. Nevertheless, the G15R phenotype could be effectively rescued by introduction of a larger residue at position 344 (Fig. 4).
Thus the primary effect is one of size. These results demonstrate that the interaction of the two nucleotide binding domains can be
spatially constrained by the steric effects of residue side chain
volume, presumably at an interface between the two domains.
Many transport ATPases have multiple nucleotide binding domains,
including ArsA (1), the F1-ATPase (25) and ABC transporters such as the P-glycoprotein (32). The existence of multiple sites may
provide control of catalysis. For example, multisite catalysis of
F1 results from positive cooperativity between nucleotide
binding sites (25). P-glycoprotein has two drug binding sites and an allosteric site for modulators of drug transport (32). Moreover, conformational communication between a drug binding site and the nucleotide binding domains of the P-glycoprotein has also been reported
(33). Information flow between metal or drug binding sites and
catalytic sites in transport ATPases is both expected and necessary for
function, both in P-glycoprotein and ArsA. We postulate that this
transduction of information is most likely communicated by
conformational changes in the enzymes that bring the nucleotide binding
sites together, with catalysis accelerated at the interface of the two
domains.