mutagenesis of a nucleotide-binding site of an anion-translocating

THE JOURNAL OP BIOLOGICAL. CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 14, Issue of May 15, pp. 7832%Xl%,1990 Printed in U.S.A.

Mutagenesis of a Nucleotide-binding Site of an Anion-translocating ATPase*

(Received for publication, November 20, 1989)

Cyrus E. Karkaria, Chih Ming ChenS, and Barry P. RosenO From the Department of Biochemistry, Wayne State University, School of Medicine, Detroit, Michigan 48201

The ars operon of the conjugative R-factor R773 confers resistance to arsenicals by coding for an anion pump for extrusion of arsenicals from cells of Esche- richia coli. The operon encodes three structural genes arsA, arsB, and arsC. The anion pump requires only two polypeptides, the ArsA and ArsB proteins. Puri- fied ArsA protein exhibits oxyanion-stimulated ATP- ase activity and was demonstrated to bind ATP by photoaffinity labeling with [w~~P]ATP. Analysis of the amino acid sequence deduced from the nucleotide sequence of the arsA gene suggests that the ArsA protein contains two potential nucleotide binding folds, one in the N-terminal half and one in the C-terminal half of the protein. A combination of site-directed and bisulfite mutagenesis was used to alter the glycine-rich region of the N-terminal putative nucleotide-binding sequence G15KGGVGKTS23. Four mutant proteins (G1,+D, Gls+R, G20+S, and T224) were analyzed. Strains bearing the mutated plasmids were all arsenite sensitive and were unable to extrude arsenite. Each purified mutant protein lacked oxyanion-stimulated ATPase activity and ATP binding. These results sug- gest that the N-terminal sequence is part of a nucleotide-binding domain required for catalysis.

Resistance to antibiotics and toxic agents in bacteria is often mediated by extrachromosomal resistance factors (Fos- ter, 1983). A frequent stratagem for resistance is simple extrusion of the toxic compound out of the cell (Mobley and Summers, 1987). Thus, the intracellular concentration re- mains subtoxic. A variety of plasmid-encoded resistance systems use metabolic energy for extrusion of the toxic chemical. Some extrusion systems are secondary antiporters, for ex- ample tetracycline (Levy and McMurry, 1978) and ethidium (Jones and Midgley, 1985) efflux systems are coupled to the electrochemical proton gradient via exchange with protons. Two plasmid resistance determinants encode primary ion pumps. The cadmium resistance carried by plasmids of Gram- positive bacteria has been shown to encode a protein with significant homology to members of the family of E1E2 cation translocating ATPases (Nucifora et al., 1989). We have shown previously that the resistance determinant to arsenite, arsenate, and antimonite carried by plasmids of Gram-negative bacteria encodes an ATP-driven anion pump which catalyzes extrusion of the toxic oxyanions from the cell, resulting in

* This work was supported by United States Public Health Service Grant AI19793. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement ” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address: Dept. of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115.

§ To whom requests for reprints should be addressed.

resistance (Mobley and Rosen, 1982; Silver and Keach, 1982; Rosen and Borbolla, 1984). This system is unique in being the first identified member of a new family of anion-translo- eating ATPases.

The arsenical resistance operon of resistance factor R773 encodes four genes, a regulatory gene arsR and three structural genes arsA, am& and arsC (Mobley et al., 1983; Chen et al., 1986; Hsu and Rosen, 1989a). Only two of the gene products, the ArsA and ArsB proteins, are required for arsenite (antimonite) resistance and transport; the ArsC protein is required to modify the oxyanion specificity to allow for arsenate resistance and transport (Rosen and Borbolla, 1984, Chen et al., 1986). The 45.5-kDa ArsB protein is a hydropho- bic protein located in the inner membrane of Escherichia coli (San Francisco et al., 1989). It is the membrane anchor for the catalytic subunit of the pump, the 63-kDa ArsA protein, and is postulated to be the subunit responsible for anion conduction (Chen et al., 1986).

The nucleotide sequence of the ars operon has been determined (Chen et al., 1986). From analysis of the sequence, it is clear that the ArsA protein is the result of a gene duplication of the common ancestor of both the arsA and nigh genes (Hsu and Rosen, 1989a). Both halves of the ArsA protein contain a sequence with similarity to the consensus region of nucleotide-binding proteins (Walker et al., 1982). The purified ArsA protein exhibits oxyanion-stimulated ATPase activity and can be photocross-linked with [a-32P]ATP (Rosen et al., 1988). Although ATP hydrolysis required the oxyanionic substrate, binding was independent of the presence of oxyanion.

The sequence in the N-terminal half of the protein with similarity to a nucleotide-binding fold is GKGGVGKTS from residues 15 to 23. Using both site-directed and bisulfite mutagenesis, 3 residues within this sequence were changed, one at a time, to produce four mutant proteins. The altered proteins were produced in normal amounts. The proteins were purified and shown to be catalytically inactive and unable to bind [a-32P]ATP. These results support the model in which the ArsA protein is the catalytic subunit of the oxyanion pump.

EXPERIMENTAL PROCEDURES

Preparation of ArsA Proteirz--E. cob strain SG20136 (ion-) (Trisler and Gottesman, 1984) transformed with the appropriate plasmid, or strain WB373 (Barnes et al., 1983), infected with the appropriate Ml3 phage were used as the source of ArsA protein. Cultures were grown at 37 “C overnight with aeration in 200 ml of LB medium (Miller, 1972) containing either 40 rg/ml ampicillin or 35 rg/ml chloramhenicol, depending on the resistances of the plasmids or phage. The cultures were diluted into 2.5 liters of fresh prewarmed medium lacking antibiotics in B-liter flasks and grown at 37 “C for 3- 4 h with aeration. The flasks were then chilled in ice water and the cells harvested by centrifugation at 4 “C. The cells were frozen in a dry ice-ethanol bath and stored at -70 “C. Cells were lysed by a single passage through a French pressure cell at 20,000 pounds/square inch. Wild type and mutant ArsA proteins were purified from the cytosolic

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Mutagenesis of the ArsA Protein 7833

fraction by the method of Rosen et al. (1988), as modified by Hsu and Rosen (1989b).

Bisulfite Mutagenesis-The 4.3-kilobase Hind111 fragment of plasmid pUM3 containing the structural genes of the ars operon (Mobley et al., 1983) was inserted into the unique Hind111 site of plasmid pACYC184 (Chang and Cohen, 1978) to produce pUM11. Point mutations in the arsA gene were generated by the procedure of Shortle and Botstein (1983). A single strand nick was introduced into the ars operon of pUMl1 (10 fig) by restriction enzyme digestion with 125 units of BamHI in the presence of 75 fig/ml ethidium bromide in 0.1 ml of restriction enzyme buffer at 37 “C for 2.5 h in the dark. The reactions were stopped by addition of 25 mM EDTA, pH 8.0, and 0.25 M NaCl. The contents were deproteinized, ethanol precipitated, dried, and suspended in 10 ~1 of 10 mM Tris-HCl, pH 8, containing 1 mM EDTA. The nicked DNA was mixed with 12.5 ~1 of a buffer consisting of 0.5 M Tris-HCl, pH 8, 5 mM MgCL, 10 mM 2-mercaptoethanol, and 1 mg/ml gelatin), with water added to total volume of 0.125 ml. The mixture was incubated at 45 “C for 5 min, then 5 units of exonuclease III were added. Incubation was continued at 45 “C for 4 min. The reaction was terminated by adding 25 ~1 of 0.5 M NaCl, 10 mM EDTA, pH 8, and the contents deproteinized and precipitated. The dried DNA was suspended in 0.1 ml of 15 mM NaCl, 1.5 mM sodium citrate, pH 7. A sodium bisulfite solution (4 M, pH 6) was made freshly by adding 0.234 g of sodium bisulfite and 96 mg of sodium sulfite to 0.645 ml of distilled water. The DNA solution was mixed with 0.3 ml of 4 M sodium bisulfite and 4 ~1 of 50 mM hydroquinone and incubated at 37 “C for 2 h. The contents were dialyzed, and the DNA recovered by ethanol precipitation. The mu- tagenized, gapped DNA was repaired with the Klenow fragment of DNA polymerase I and ligated with T4 DNA ligase. The DNA was transformed into E. coli strain HBlOl, and chloramphenicol-resistant transformants were further screened for loss of arsenite resistance.

The 4.3-kilobase Hind111 fragment of the mutant plasmids was cloned into the Hind111 site of mWB2348 (Barnes et al., 1983), and the recombinants in which the insert was in the right orientation were selected for DNA sequence analysis using the method of Sanger et al. (1977). A set of synthetic oligonucleotides covering the entire arsA gene was used for primers, allowing identification of the single point mutation in the arsA gene for each bisulfite mutant.

Oligonucleotide~directed Mutagenesis-Phage mCEK18 (Gia+R) was derived by mutagenesis of mCMC49 using the procedure of Taylor et al. (1985). The Hind111 fragment in the Ml3 phage derivative mCMC49 (Chen et al., 1986) was used as template. The mutagenic oligonucleotide for G18-+Rls change was 5’.CCCACGCGTCCTTTA- 3 (mismatch underlined). The ratio of the mutage& primer to template was 2O:l. Potential mutants were identified by dot blotting with “P-labeled mutagenic oligonucleotide (Zoller and Smith, 1983), and the location of the single base change was confirmed by DNA sequencing (Sanger et nl., 1977).

Electrophoresis and Western Blot Procedures-Cultures (1 ml) of cells with the appropriate plasmids were grown overnight in LB medium and centrifuged to pellet the cells. The pelleted cells were suspended in 0.1 ml of sodium dodecyl sulfate (SDS)’ sample buffer and boiled for 5 min. SDS-polyacrylamide gel electrophoresis was performed according to the procedure of Laemmli (1970) using 12% acrylamide gels. Proteins were transferred electrophoretically to 0.45. pm pore size nitrocellulose paper for 12-16 h at 50 mA. Unoccupied sites on the nitrocellulose paper were blocked with 5% nonfat dried milk in phosphate-buffered saline (21 mM KH2POI, 0.138 M NaCl, 2.5 mM KCl, pH 7.4) (PBS) for 1 h. The blot was then incubated with rabbit anti-ArsA serum diluted 1:2000 for 1 h at 37 “C with gentle shaking and then washed three times for 10 min each time with 5% milk-PBS at 3 “C. The antigen-antibody complex was de- tected by incubation with horseradish peroxidase-conjugated goat antirabbit IgG diluted 1:5000 at 37 “C for 1 h, followed by three washes with PBS, and visualization by addition of hydrogen peroxide (0.03%) and 4-chloro-1-naphthol (3 mg/ml) in PBS.

Transport of 73As02--‘“As0, transport in cells bearing wild type and mutant plasmids was performed as described previously (Rosen and Borbolla, 1984). 73As0i- was purchased from Oak Ridge National Laboratory, Oak Ridge, TN and reduced to 73As0; prior to use (Rosen and Borbolla, 1984).

Photolabeling of the ArsA Protein with [01-32P]ATP-Purified wild type and mutant ArsA proteins were photocross-linked as described

’ The abbreviations used are: SDS, sodium dodecyl sulfate; PBS, phosphate-buffered saline; MOPS, 4-(2-hydroxyethyl)piperazine- ethanesulfonic acid.

previously (Rosen et al., 1988) using the method of Yue and Schimmel (1977).

ATPase Actiuity-Oxyanion-stimulated ATPase activity was measured from a decrease in NADH concentration at 340 nm using a coupled assay (Vogel and Steinhart, 1976). The reaction mixture (1 ml) contained 50 mM MOPS-KOH buffer, pH 7.5, 0.25 mM Na2EDTA, 5 mM ATP, 1.25 mM phosphoenolpymvate, 0.25 mM NADH, 10 units of pyruvate kinase (Sigma), and lactate dehydrogen- ase (Sigma) with or without 0.1 mM potassium antimony1 tartrate (antimonite), all prewarmed to 37 “C. ArsA protein was added to the cuvette in a final concentration of lo-25 fig/ml and preincubated in the assay mixture for 10 min at 37 “C. The reaction was initiated by addition of 2.5 mM MgCl,. The reaction was linear for 5-15 min, and the linear steady-state rates were used to calculate the specific activity.

Proteolysis with Trypsin-Trypsin digestion was performed at room temperature in 50 mM MOPS-KOH buffer, pH 7.5, at an ArsA concentration of 0.2 mg/ml. The ratio of protein/trypsin was 1OO:l. Trypsin was added after a lo-min preincubation of protein with the indicated substrate. Proteolysis was terminated at various times by addition of a 2-fold excess of soybean trypsin inhibitor. Samples were analyzed by SDS-polyacrylamide gel electrophoresis using a 10% polyacrylamide gel and Coomassie Blue staining.

RESULTS

Isolation of Mutants in the AFSA Protein-sodium bisulfite mutagenesis was used to produce semi-site-directed mutations within specific regions of the arsA gene. The location of three arsenite sensitive mutations were found to be within the N- terminal consensus nucleotide-binding sequence (Table I). The mutated ArsA protein encoded by pCMC1079 had a G18-, D alteration. Plasmid pCMC1065 encoded an ArsA protein with a G,,+S change. Plasmid pCMClOl0 had a T22-+I mutation in the ArsA protein. The site-directed mutant mCEK18 encoded an ArsA protein with a G1s-+R alteration. The maximum concentration of arsenite on which cells bearing these plasmids could grow was determined (Table I). Strains bearing plasmids or Ml3 phage derivatives grew in the presence of 15 mM arsenite. Without a resistance determinant the maximum arsenite concentration was 1 mM. Cells

bearing mutated plasmids or phage were resistant to 2.5 mM

arsenite but sensitive to 5 mM. The slight increase in resistance with the mutated plasmids may indicate sufficient oxyanion binding by the highly expressed mutant ArsA proteins to produce low level arsenite resistance. Similarly, a low level of arsenate resistance was observed in cells overproducing the ArsC protein in the absence of the ArsA and ArsB proteins, even though high level arsenate resistance requires all three proteins (Chen et al., 1985).

73AsO;1 lhWZSpOrt-we have shown previously that the

TABLE I

Prowerties of E. coli strains bearing wild &De and mutant ars Penes

Phage/plasmid” Nucleotide change+

pACYC184 (vector) pUMl1 (wild type) pCMC1079 G,s - A pCMC1065 C&n--A pCMClOl0 C,, - T

mWB2348 (vector) mCMC49 (wild type) mCEK18 G,,-+C

Amino acid Arsenite alteration resistance’

?7lM

1 15

G,,-+D 2.5 G,,-+S 2.5 Tz+1 2.5

1 12.5

GM-R 2.5

’ The host for plasmids was E. coli strain HBlOl. For Ml3 phage the host strain was E. coli WB373.

b Numbering is from the first nucleotide of the coding sequence of the arsA gene.

‘Maximum concentration of arsenite in solid medium in which cells bearing the indicated plasmids or phage will grow.


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7834 Mutagenesis of the ArsA Protein

ability to pump arsenite out of cells corresponds with arsenite resistance and with low net accumulation in uptake assays. Sensitive cells, which are unable to extrude arsenite, exhibit higher net accumulation (Rosen and Borbolla, 1984). Uptake assays are experimentally simpler than extrusion assays be- cause starvation to reduce endogenous energy reserves and loading with isotope are not required for the former. To correlate loss of arsenical resistance with loss of activity of the oxyanion pump, uptake of 7”As0;’ was assayed in the cells bearing the wild type and mutant ars genes (Fig. 1). Strains expressing wild type ars genes exhibited substantially reduced net uptake compared with strains without the ars operon. Strains with mutant ars genes accumulated 73As0;’ in amounts similar to those without the operon, indicating an inability to extrude arsenite.

Production of Mutant ArsA Proteins-The amounts of wild type and mutant ArsA proteins in cells in the steady state was estimated by Western blot analysis using antiserum to wild type ArsA protein (Fig. 2). Each mutant protein was produced in approximately the same amounts and migrated with the same mobility as the wild type. No abnormal degra- dative products were observed. These results demonstrate that the mutant proteins are translated normally and have turn- over rates not greatly different from the wild type protein. This suggests that the overall conformation of the mutant proteins is not greatly different from that of the wild type protein.

ATPase Actiuity-In accordance with the model of the ArsA protein serving as the catalytic component of the oxyanion pump, the ArsA protein exhibits oxyanion-stimulated ATPase activity (Rosen et al., 1988). The ability of the mutant proteins to hydrolyze ATP was examined (Table II). None exhibited significant levels of ATPase activity, either with or without

123456789 w-- -Mm

Frc. 2. Immunoblot analysis of mutant ArsA proteins. West- ern blotting was performed using 6 ~1 of cell suspensions boiled in SDS sample buffer and bearing the indicated plasmids or phage, as described under “Experimental Procedures.” Lane 1, pUMl1 (wild type ArsA); lane 2, pCMC1079 (G,R-+D); lane 3, pCMC1065 (G20-t S); lane 4, pCMClOl0 (Tp2+J); lane 5, pACYC184 (vector with no insert); lane 6, mWB2348 (Ml3 phage with no insert); lane 7, mCMC49 (wild type ArsA); lane 8, mCEK18 (G,a+R), lane 9, 1 pg purified wild type ArsA protein.

TABLE II

ATPuse activity of wild type and mutant ArsA proteins

ATPase activity ArsA”

-Antimonite +Antimonite Antimonite- stimulated

~mol/min/mg protein Wild type 0.080 1.100 1.020 $_,D +R 0.014 0.008 0.015 0.008 0.000 0.001

Gg,,+S 0.002 0.002 0.000 T,,+I 0.015 0.015 0.000

“Wild type and mutant ArsA proteins were purified as described under “Experimental Procedures.”

A IA 1

/ ,- .

2 Tim: (mien)

s 10 J

FIG. 1. Plasmid-directed exclusion of arsenite from cells. Cells of E. coli strain HBlOl bearing the indicated plasmids and strain WB373 bearing the indicated phage were assayed for arsenite uptake as described under “Experimental Procedures.” A, no plasmid (A); pACYC184 (vector) (*); pUM3 (wild type) (0); B, pUMl1 (wild type) (m); mCEK18 (G,s-+R) (0); pCMC1079 (G,R*D) (+); pCMC1065 (Gal-S) (Cl); pCMClOl0 (T,,+I) (0).

FIG. 3. ATP photolabeling of wild type and mutant ArsA proteins. Photolabeling was performed as described previously (Ro- sen et al., 1988) using 5 pM [(u-“‘PIATP. Each lane was loaded with 18, 31, or 62 Kg of wild type ArsA and mutant proteins, as indicated. Samples were analyzed by autoradiography after electrophoresis on 15% SDS-polyacrylamide gels. Incorporation of 31P was quantified by densitometry and is expressed as % of the highest value. ArsA proteins: wild type (0); D,, (A); R,, (0); SzO (*); I,, (+).

oxyanions. The apparent lack of activity is not due to an altered K,,, for ATP. The K, for ATP was found to be 0.13 mM (Hsu and Rosen, 198913). No activity was observed with the mutant enzymes even with 50 mM ATP (data not shown).

Nucleotide Binding-We have previously shown that wild type ArsA protein can be photocross-linked to [cY~~P]ATP by ultraviolet irradiation (Rosen et al., 1988). Photolabeling was assayed with purified preparations of wild type and each mutant ArsA protein (Fig. 3). Binding was proportional to protein concentration with wild type ArsA. In contrast none of the mutant proteins retained significant nucleotide binding ability, labeling to less than 10% of wild type levels.

Limited Proteolysis by Trypsin-Trypsin sensitivity has been used to probe the interaction of oxyanion and nucleotide- binding sites (Hsu and Rosen, 198915). The rate at which the wild type ArsA protein loses activity and the rate at which


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Mutagenesis of the ArsA Protein

No Addi tlon +Substratee

Time (min): 0 63J60 0 63080 5-T-- ----- -

Protein: WT

-ZE - - - - - - -

018 --m-s --- _- -w_. _ ---- -

II, cr-b## . ..I- R18 -

--+- .-. --c - *L1

-=, b- r - - -

122 --'r;,-- -~

- --._

FIN. 4. Effect of ATP on the formation of cleavage products by trypsin. Purified wild type and mutant ArsA proteins were proteolyzed for 0,5,30, and 60 min, as described under “Experimental Procedures” and analyzed by SDS-polyacrylamide gel electrophoresis on 10% gels stained with Coomassie Blue. The proteins in the left set of gels were treated with trypsin in the absence of substrates. The samples in the right set of gels were trypsin treated in the presence of 5 mM ATP and 0.5 mM antimonite. The fastest mizratina band in each lane is the trypsin inhibitor used to terminate proteol&is. The location of the ArsA protein (63 kDa) and trypsin inhibitor (21 kDa) are indicated in the right margin.

unique proteolytic fragments are produced was shown to depend on the presence of substrates during proteolysis. ATP partially protected from proteolysis, while antimonite did not. However, the presence of both ATP and antimonite greatly decreased the rate of trypsin inactivation and the disappearance of the native 63-kDa species. These results have been interpreted to demonstrate conformational interaction of oxyanion and nucletoide-binding sites (Hsu and Rosen, 198913). As shown in Fig. 4, in the absence of either antimonite or ATP the wild type ArsA protein is cleaved to one or more polypeptides of 30 kDa. This pattern was unaffected by addition of antimonite (data not shown). Incubation of the wild type protein with both 5 mM ATP and 0.5 mM SbO; substantially decreased the formation of proteolytic fragments. When the mutant ArsA proteins were digested with trypsin under similar conditions, ATP in the presence of SbO; protected the 63-kDa form from degradation. The patterns of digestion were not identical to that of the wild type, especially with mutant DIR. However, the fact that ATP has an effect at all implies that the nucleotide must still bind to each mutant protein, producing a conformation change which alters the accessibility of trypsin sites.

DISCUSSION

A consensus sequence rich in glycine has been found in a variety of different nucleotide-binding proteins including ATPases and oncogenes (Walker et al., 1982). This sequence

is part of a nucleotide-binding fold which has been best studied in adenylate kinase (Fry et al., 1985). Residues 15-22 (GGPGSGKG) of this enzyme form a flexible glycine-rich loop between an Q helix and a p sheet, and KZ1 may be very close to the a- or y-phosphate of ATP.

The ArsA protein, postulated to be the catalytic subunit of the arsenical pump, is an oxyanion-stimulated ATPase (Ro- sen et al., 1988). From the primary sequence of the ArsA protein deduced from its nucleotide sequence two consensus glycine-rich regions are apparent, suggesting two potential nucleotide-binding sites (Chen et al., 1986). From analysis of data bases the ArsA protein shows a distant relatedness to the nigh gene product of the nitrogenase complex (Mevarech et al., 1980). Dinitrogen reductase is a 64-kDa homodimer of two 32-kDa nifH gene products. Two polypeptides of dinitrogen reductase, when aligned as a head-to-tail dimer, show a striking resemblance to the ArsA protein (Hsu and Rosen, 1989a). Both are energy transducing ATPases with similar nucleotide-binding domains. Dinitrogen reductase is also an iron sulfur protein which catalyzes electron transfer. The cysteines which comprise the iron sulfur cage are not conserved in the ArsA protein, suggesting that it is not an iron sulfur protein. Thus, while the two proteins share an evolu- tionary history, their present functions are different. The common ancestor which gave rise to both the arsA and nifH genes probably encoded a 30-kDa polypeptide with ATPase activity. By gene duplication and fusion, this 900-base pair ancestor evolved into the 1749-base pair arsA gene. Support- ing this idea is the internal homology within both the arsA gene and ArsA protein (Chen et al., 1986). Within each half of the protein is a sequence which corresponds to the glycine- rich loop of the nucleotide-binding fold. It is not known whether each is a component of a nucleotide-binding site or whether either site is catalytic. Although the sequences are similar, they are not identical, so that the nucleotide-binding sites need not be equivalent. One method for distinguishing between the two sites and for elucidating their functicn is mutagenesis of conserved residues.

This approach has been utilized with the glycine-rich loop of the cy and p subunits of the FoF, ATPase of E. coli. When Kis5 of the /3 subunit was mutated to glutamate or glutamine a decrease of 80 and 66% was observed in the steady state rate of ATP hydrolysis, suggesting decreased binding of Mg’+ATP, possibly due to improper interaction with the y- phosphate of ATP (Parsonage et al., 1988). When the analo- gous K171i of the LY subunit was mutated to isoleucine or glutamate, there was a similar 2.5-3-fold decrease in specific activity (Rao et al., 1988). Similarly when either K1c4 in the fi subunit or KIT5 in the LY subunit of the FoFl of the thermophilic bacterium PS3 were changed to isoleucine, the mutated enzyme had lost all its ATPase activity (Yohda et al., 1988). Mutation of A,,,+V in the @ subunit of the E. coli FoFl gave about 6% of wild type steady-state ATPase activity (Hsu et al., 1987). Interestingly, activated (oncogenic) p21 rus proteins have glycine and valine residues at the corresponding Aljl position (Tabin et al., 1982; Reddy et al., 1982) and have 10% of the GTPase activity on the wild type (Sweet et al., 1984).

The effect of substitutions in the first potential nucleotide- binding site of the ArsA protein was examined in four mutants. The effect of mutation could be determined at four levels: resistance, transport, catalysis, and nucleotide binding. Each of the four mutations resulted in loss of arsenite resistance (Table I) and transport (Fig. 1). These effects could be the result of decreased synthesis, increased degradation, or improper interaction of the ArsA protein with ArsB in assembly of the pump. The results from Western analysis (Fig. 2)


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7836 Mutagenesis of the ArsA Protein

imply that the mutant proteins are expressed in the same amount and are not degraded in vivo at an abnormal rate. The suggestion of normal folding of the mutant proteins is further strengthened by the results of experiments with con- trolled proteolysis (Fig. 4). A substrate-related decrease in the rate of disappearance of the 63-kDa form of the mutant proteins produced by trypsin indicates that surface arginyl and/or lysyl residues retain their general location in the inactive ArsA proteins. Finally, the mutant ArsA proteins bind normally to membranes containing the ArsB protein,* suggesting that assembly of the pump takes place in the mutants.

Hsu, C. M., and Rosen, B. P. (1989a) in Highlights of Modern Biochemistrv (Kotvk. A.. Skoda. J.. Paces. V.. and Kostka. V.. eds)

We have previously shown that binding and hydrolysis of ATP are separable steps (Rosen et al., 1988). Photolabeling of the ArsA protein with [cx-~*P]ATP was independent of oxyanion, while ATP hydrolysis required antimonite or arsenite. Mg*+ was required for photolabeling, indicating that di- valent cation is involved in binding of nucleotide. Similarly binding of the nucleotide analogue 2’,3’-O-(2,4,6)trinitro- phenyl adenosine triphosphate was not dependent on the presence of oxyanion but did require Mg’+. The fact that the mutant proteins did not photolabel with [a-32P]ATP suggests that the N-terminal nucleotide-binding fold is involved in or essential for binding nucleotide (Fig. 3).

ATP protection from trypsin digestion still occurred in the mutant proteins (Fig. 4), implying that those proteins are still capable of binding ATP, perhaps at the second potential nucleotide-binding site. It should be noted that the effect of ATP on trypsin inactivation was Mg’+-independent (Hsu and Rosen, 1989b). Moreover, binding of 5’-p-fluorosulfonylben- zoyladenosine to the ArsA protein was also M$+-independent.

In summary, we would propose that the ArsA protein has two nucleotide-binding sites. The first is a Mg”+ATP site and is catalytic, The second does not require Mg*+ and could be either catalytic or regulatory. Isolation of mutations in the second potential nucleotide-binding site is essential for deter- mining the function of each site and is currently in progress.

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C E Karkaria, C M Chen and B P RosenMutagenesis of a nucleotide-binding site of an anion-translocating ATPase.

1990, 265:7832-7836.J. Biol. Chem.

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