introduced to optimize alignment, semicolons indicate identities, and dots indicate conservative aminoacid replacements. The marked proline (*) and aspartate ...
Proc. Natl. Acad. Sci. USA Vol. 86, pp. 3544-3548, May 1989 Biochemistry
Cadmium resistance from Staphylococcus aureus plasmid pI258 cadA gene results from a cadmium-efflux ATPase (EIE2-type ATPase/heavy-metal plasmid resistance)
GIUSEPPINA NUCIFORA, LIEN CHU, TAPAN K. MISRA, AND SIMON SILVER Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago, IL 60680
Communicated by Emanuel Margoliash, January 30, 1989
ABSTRACT Cadmium resistance specified by the cadA determinant of Staphylococcus aureus plasmid p1258 results from the functioning of a cadmium-efflux system. In the nucleotide sequence of the DNA fragment containing the cadA determinant, two open reading frames were identified. The larger one, corresponding to a predicted polypeptide of 727 amino acid residues, is necessary and sufficient for expression of cadmium resistance. Comparison of the CadA amino acid sequence with known protein sequences suggested that CadA is a member of the EjE2 cation-translocating ATPases, similar to the K+-uptake ATPases of Gram-positive and Gram-negative bacteria. The sequence homology is lower but significant with other EjE2-type ATPases, including the H+-efflux ATPases of eukaryotic microbes and the Ca2+- and Na+/K+-ATPases of animals. A frame-shift mutation in the middle of the gene destroys the Cd2+-resistance phenotype. A detailed model for the putative CadA ATPase based on homologies to other EjE2 ATPases is presented and discussed.
Uptake Assays. The cells were grown to a density of 100 Klett turbidity units (no. 54 Kodak Wratten filter) at 37TC with shaking, harvested by centrifugation at 6800 X g for 7 min, and suspended in medium at a density of 25 mg (dry weight)/ml. The final cell density in transport assays was 0.5 mg (dry weight)/ml. Transport assays were at 37°C in broth with aeration by shaking. Samples were filtered through 0.45-,um pore-diameter filters (Bio-Rad) and rinsed twice with 5 ml of NaCl solution (9 g/liter). Radioactive samples were assayed in a liquid scintillation spectrometer (Packard Instrument).' DNA Cloning and Sequencing. For construction of plasmids and phage M13 derivatives containing the cadA determinant, a derivative of pRALl (1, 9) was used. The 3.5-kilobase (kb) Bgl II-Xba I DNA fragment, previously identified by Novick et al. (2) as containing the cadA Cd2+-resistance determinant, was cloned into phage mTMO10 (10) in both orientations. Nested deletions for sequencing were made by using BAL-31 exonuclease (10). The 3.5-kb fragment was also cloned into vector pSK265 (7) (for expression in B. subtilis). Two recombinant plasmids, pGN114 and pGN115, containing the entire 3.5-kb DNA fragment in opposite orientations, were generated. Plasmids pGN116-1 and pGN116-2 [containing the intact larger ORF (ORF2, cadA) cloned in both orientations] were obtained by ligating into the vector plasmid pSK265 a DNA fragment generated by BAL-31 deletion and lacking the first 920 base pairs (bp) of the Bgl II-Xba I fragment shown in Fig. 1. This fragment was introduced also in pGEM-3Zf(+) (Promega Biotec) under control of the T7 phage transcriptional promoter to generate pGN118 [for the expression of the CadA protein in Escherichia coli strain BL21 (11)], containing the T7 RNA polymerase under the control of the inducible
Resistance to Cd2+ is widespread in Staphylococcus aureus (1). There are two separate Cd2+-resistance determinants, cadA and cadB, located on plasmids (2). The cadB gene product may protect the cell by binding Cd2+ (3). The resistance function coded by the cadA determinant results from decreased intracellular accumulation of Cd2+ (4), mediated by an energy-dependent efflux mechanism (5). In S. aureus, Cd2' enters the cell by the Mn2' active transport system (4, 6), but cells that have the cadA gene have lower net accumulation. The CadA-efflux system is sensitive to metabolic inhibitors such as uncouplers but is not affected by agents that eliminate the membrane potential (5). Therefore, it was proposed (5) that the cadA product is an electroneutral antiporter that ejects one Cd2+ while accumulating two protons. We have cloned the cadA determinant from S. aureus plasmid pI258 and expressed it in Bacillus subtilis. The DNA sequence was determined.* It consists of a single open reading frame (ORF) very likely coding for an E1E2 cationtranslocating ATPase. METHODS Bacterial Strains. For cloning of the cadA determinant of plasmid p1258 into the vector pSK265 (7), B. subtilis strain BD224 (8) (trpC2 recE4 thr-5) (BGSC 1 A46, Bacillus Genetic Stock Center, Columbus, OH) was used. The cells were grown in tryptone broth (8 g of Difco Bacto tryptone and 5 g of NaCl per liter) supplemented with threonine and tryptophan (50 ,ug/ml each). When necessary for selection, chloramphenicol (5 ,g/ml) was added to liquid medium or to the tryptone broth agar plates.
lacUV5 promoter. Mutant plasmid pGN117, containing the entire 3.5-kb DNA fragment with a frame-shift mutation in the cadA gene, was obtained by Klenow DNA polymerase conversion of the protruding ends generated by Xho I (see Fig. 1) digestion of pGN114 to blunt ends. Membrane Preparation, Solubilization and Reconstitution of Proteoliposomes, and Transport Assays. The procedure of Ambudkar et al. (12), slightly modified, was used for the preparation of bacterial membranes. Membranes and proteoliposomes were suspended in 20 mM Mops, pH 7/200 mM KCI and used immediately. For transport assays, membrane vesicles or proteoliposomes were mixed with 109Cd2+. After 3-5 min at 0°C, the reaction was initiated by the addition of 5 mM ATP and 5 mM Mg2+. Aliquots (0.1 ml) were filtered on Millipore filters (pore size, 0.22 ,m) and washed with assay buffer containing MgSO4. The radioactive samples were assayed in a scintillation counter.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviation: ORF, open reading frame. *The sequence reported in this paper is being deposited in the EMBL/GenBank data base (accession no. J04551).
3544
Qo~bAIMGDCOk;8f3
pPL_:=A~SAG_rl,'=t~~4 ~ ~ 12.5426)900
Biochemistry: Nucifora et al. Labeling of Proteins with [32P]ATP. Cell membranes were incubated in 50 mM Mops (pH 7.2)/5% (vol/vol) glycerol/5 mM MgCl2 at 40C; 10 jtM [32P]ATP (10 MiCi; 1 MCi 37 kBq)
Proc. Natl. Acad. Sci. USA 86 (1989)
determinant and the amino acid translation of the two major ORFs. The sequence starts =200 bp from the Xba I site of the physical map of plasmid pI258 (1, 2) and ends at the Bgl II site. Of the two major ORFs identified, the first one starts at position 704, after a segment of DNA containing direct and inverted repeats (indicated by arrows a through h in Fig. 1) and ends at nucleotide 1072. The resulting predicted peptide contains 122 amino acids. From deletion experiments (see below), the first 920 bp containing the repeats and coding for part of the short polypeptide appear not necessary for resistance to Cd2+. At position 1065, a second and longer ORF starts that overlaps 8 bp with the previous one. Upstream of the ATG start codon there are a reasonably good
=
added, and incubation was continued at 40C for 15 sec. After addition of cold 10% (wt/vol) trichloroacetic acid/1 mM phosphate and centrifugation, the precipitate was washed with cold H20, suspended in loading buffer, and resolved by electrophoresis on an acidic lithium dodecyl sulfate/polyacrylamide gel (13, 14). was
RESULTS
Nucleotide Sequence of the cadA Determinant. Fig. 1 shows
the nucleotide
sequence
of the 3535 bp containing the cadA
120
> X~~~~~~
h>
>
> ~ d
~
~
b-
360
A
480
600
RE35 1st (SF
e
M K K K D T
A_ S~~~~~~~~~RTOIUMAUIYM _ AD
>
c--e
_ W
_AGAAGM
720
C E I F C Y D E E K V N R I Q G D L Q T V D I S G V S Q I L GMTh:EO3YK9CAAG333U~nU~AW A= _ 1 1U=G=MkMA=
K A I A D E N R AK
I T Y A L C Q D E E
L R T L Y K Q G V V
L C V C D I A N I L
M _AG3=T=Xr~kkA~aTGGMM
G V T I A N A S H H
AG3E(rDilk PBMYWflBDUM=kTM
N F R K E G K L A L
Y S L G D E H I R Q
-35
CAAMUGAAGOOMASMMM V K L M E E E M N V
N F G A S K I D V Y
960
C A G K F E K N V K
K I P G V Q D A K V
^G N
A G A F E N L K V S
P E K L A N Q T I Q
R V K D D T K A H K E E K T P F Y K K H S T LLMMA L I PERFSNDRS A F G Y L S H FRMAA V N A03ICKWAEASMIVAT VIOMLFAI SEUGA NTBV~tAZGVICGATAIC A KYYTPOMA37=C0 V vA V P C W Y _INVIAAT L
G E D N L V T S M L M T V A V I G A T I
F V G S I V I G G Y
S L F K V G F Q N L
I R F D F D M K T L
I G K W A E A S I V
V I L F A I S E A L
E R F S M D R S R Q
T1U3MA7aG330%-
S I R S L M D I A P
7_
A
E K I A M D G I I V
K E A L V R R N G Q
A
_
S
_
..
E I I I H V D D I A
N G L S A V N Q A A I T G E S V P V NS UMMC r M TAAG30 IM3MICA
TUA]k337
'120
V G D I M I V R P G
1800
A V D D E V F A G T
1920
I T K Y V E D T T I TK I I H L V E E A Q G E R A P A Q A F 'TVKAA : 7CMKX~~ MU AZC:T= ICMBin AA33UGNK3~aC
V D K F A K Y Y T P
20 1440
- 22 1680
..
_ =~~~~~~~~~~AAkMrOUI3K2M
_~~~~~~~~~~~3
L N E E G L I E V K
840
I M M I A L A H K K E V K V N V * e2rKd (F -10 RE; cai AlPase M S E Q K GAGTAAGZU~fGIC -A -C M 1080
Y R V Q G F T C A N
A S V E E L E K
2040
I I M V I A A L V A V V P P L F F G G S IGE=: 7r T 17, GM 'ILUKI
W D T W V Y Q G L A GM -OMOMTWCAA
V L V V G C P C A L
V I S T P I S I V S
L
L G A I K T V A F D
K T G T L T K G V P
V V T D F E V L N D
Q V E E K E L F S I
I T A L E Y R S Q H
P L A S A I M K K A
* E Q D N I P Y S N V
~~~~~EcoRI Q V E E F T S I T G , , _ ~~~~~~2520
T Y Y I G S P R L F
K E L N V S D F S L
G F E N N V K I L Q
T E R T I L G V I A
V A D E Y R E T S K
N V I Q K L H Q L G
H V G V S D I Q S E
L M P Q D K L D Y I
MAB3=TTG33YOM~n%2rj=
A I G N A A K K G V
~~~~~~~~~~~ATIGMAffGOKU2_ =AA3M
R G I K G I V N G T
K G G C
033YAAAACA TIWOMMATT=
I K Q T I M L T G D
K K M-Q S E Y D N V
iuaWGM~
AlnG3rTjGSC~~G
D D L S K L P F A V R L S R K T L N I I C~~~~rUTO MMroa _vounAAcmxo
K A N I T F A I G I APCV11rMM~na
_ :MWGTDMDrM
A:K-XDAAAkZAMG
G W L T L W I A I L
Gumknm7mup3
S D M G A T I L V A
BII ---_-
3535
2640
32760
3120
L N S L R L M R V K
C0MU377MMT1a
OWIVkAaM0_AA
2280
2880
A M G G A G T D T A
K I I A L L L V I P D RK
A M I G D G V N D A
_IGZ4C~dIMG~rICGIGAANDAGMMM MI_1_CAAAAACITCRICAGIMK
P A L A A S T V G I
I E T A D I A L M G
X
N Q G T A N A I G T
2160
V Y L E R
GNMMWAGMPx~ h < AkANrCIU3TAG N Q G K T A M I I G
3545
AMMAMU B
3240
IkK pa
3360
3480
FIG. 1. Nucleotide sequence of the 3535 bp of the cadA determinant from plasmid p1258. The strand equivalent to the mRNA is shown. Ribosome binding site
(RBS), predicted polypeptides, and three restriction nuclease sites (Xho I, Eco RI, and Bgi II) used for cloning or mutations are indicated. Inverted repeat sequences a through f and direct repeats g and h of 10 bp or longer in the region from base pair 219 to 737 are shown by labeled arrows.
3546
Proc. Natl. Acad. Sci. USA 86
Biochemistry: Nucifora et al.
i 80
-AL-
(1989) -
Cl
100
40 4
Ce
0~
50-
0 2
0 -
LS
0
I
5
,
10 Cd2+
4
Time at 370C, min 50 100
,tM
FIG. 2. Cd2+ resistance of B. subtilis BD224 strains containing the recombinant plasmid pGN114 (o) or vector plasmid pSK265 (o). Overnight cultures were diluted 1:100 in tryptone broth containing Cd2 . Culture turbidities were measured after 7 hr of growth.
potential ribosomal binding site and a transcriptional initiation signal (marked on Fig. 1). This ORF continues for 2184 bp, corresponding to a predicted 727-amino acid polypeptide. Alignment of the 200 bp flanking the Bgl II site with the left end of the IS427 insertion sequence element of plasmid p1524 (also from S. aureus) sequenced by Barberis-Maino et al. (15) showed a perfect match of the 134 terminal bp (not shown). Cd2I Resistance Is Determined by a Single ORF. Fig. 2 shows the effect of increasing concentration of Cd2+ on the growth of B. subtilis cells carrying plasmids pSK265 (sensitive) or pGN114 (resistant; the entire 3.5-kb Bgl II-Xba I fragment). The cells with the recombinant plasmid grew in the presence of 10 times the concentration of Cd2+ that the sensitive cells would tolerate. Similar results were obtained with cells containing plasmid pGN115, where the insert had been cloned in opposite orientation (not shown). In cation transport experiments, cells containing pSK265 or pGN114 took up equivalent amounts of 54Mn2+ over the 5-min time course of an uptake experiment. However, the sensitive cells took up 2-4 times more 10Cd2+ than the resistant cells (Fig. 3). Note that the uptake of Cd2+ was 20 times more in Fig. 3 than that of Mn2+, similar to the ratio in earlier studies (16). The roles of the two major ORFs in the cloned fragment were investigated by repeating growth and 109Cd2+ uptake experiments with cells transformed by plasmids pGN116-1 and pGN116-2 (containing only the larger ORF cloned in opposite orientations in the vector) and by plasmid pGN117 (where a framshift mutation had been introduced in the large ORF 35 bp upstream of the codon for the aspartate residue believed to be critical to the activity of E1E2 ATPases; see Discussion below). In this mutant, the first ORF had therefore been left intact. The results indicate that the protein encoded by the large ORF is necessary and sufficient for Cd2+ resistance (data not shown) and for
FIG. 4. Uptake of 109Cd2+ by B. subtilis cells containing plasmid vector pSK265 (e), recombinant plasmid pGN114 (0), the cadA large ORF cloned in both orientations in pSK265 to form pGN116-1 (n) and pGN116-2 (n), and the mutant pGN117 (A) in which a frame-shift mutation has been introduced in cadA by Xho I digestion blunt-end conversion with Klenow DNA polymerase, and ligation.
reduced Cd2+ uptake (Fig. 4). Comparable results with pGN116-1 and pGN116-2 indicate that cadA is expressed by using its own promoter. Analysis of the Predicted Polypeptides. The amino acid sequences predicted from the two major ORFs were checked against protein libraries for related proteins by using a polypeptide alignment program (17). No significant homology was found for the smaller protein. However, the 727amino acid polypeptide showed significant matches with bacterial and eukaryotic E1E2 cation-transport ATPases (Table 1). Fig. 5 shows the alignment of the 727-amino acid CadA polypeptide with the K+-ATPase from Strep. faecalis (17), which is the closest to the CadA polypeptides of currently published E1E2-type ATPase sequences. In Vitro Measurements of ATPase Activity, Protein Phosphorylation, and Cd2+ Transport. Attempts were made to overproduce the 727-amino acid protein (11), to measure ATPase activity of the protein in vitro, to measure uptake of '09Cd2+ in everted membrane vesicles of B. subtilis and in proteoliposomes (12), and to measure a large polypeptide that could be labeled with [32P]ATP (13). This series of experiments has not been successful. When the T7 RNA polymerase was induced for the expression of the cadA gene under the control of the phage T7 promoter (11), cell growth stopped abruptly, and the surviving cells contained plasmids where the insert was partially or completely deleted.
DISCUSSION The sequence homology between the cadA gene product and EjE2-type ATPases suggests that Cd2+ resistance is mediated by a Cd2+-translocating ATPase. This is consistent with the physiological results of Tynecka et al. (5), who demonstrated that Cd2+ resistance results from energy-dependent Cd2+
V E
0) 4-
0
Time at 370C, min
FIG. 3. Uptake of 109Cd2+ (A) and of 54Mn2+ (B) by sensitive B. subtilis cells containing plasmid pSK265 (e) and by resistant cells containing plasmid pGN114 (o). The cells were prepared as described in the text and incubated for 2 min at 370C; 20 ,AM 109Cd2+ or 54Mn2+ was added. Samples [0.25 mg dry weight (wt)] were filtered and washed as indicated.
Biochemistry: Nucifora et al.
Proc. Natl. Acad. Sci. USA 86 (1989)
3547
8702 polypeptides in the current library) to mercuric reductase and to the periplasmic mercury-binding protein of the mercury-resistance operons (Table 1 and Fig. 6). The region includes conserved paired cysteine residues (Cys-23 and aa (over Cys-26 in the CadA protein). The occurrence of such paired Cation Length, FASTP range cysteine residues has become a recurring motif in soft aa score ATPase source specificity indicated) metal-binding regions, and they are hypothesized to play a 727 3381 100 Cd2+ S. aureus (727) role in the initial binding of the heavy-metal ion because ofthe 703 29.8 (543) 583 K+ Strep. faecalis strong affinity of their sulfhydryl groups for the heavy-metal 462 26.2 (577) 682 K+ E. coli KdpB ions (20, 21). In several E1E2 ATPases, the amino-terminal H+ 918 179 33.1 (148) Sac. cerevisiae part of the enzyme has been postulated to provide the 920 170 29.6 (189) H+ Neurospora recognition and the initial binding site for the cation to be 1023 159 35.6 (90) Human a chain Na+/K+ transported (22, 23). The models are based on analysis of Ca2+ 1001 133 37.9 (95) Rabbit muscle enzyme functions (recognition and binding of the cation, (E. coli Hg2+ transduction, phosphorylation, and ATP binding) after lim561 99 32.8 (58) reductase) Hg2+ ited tryptic digestion. The E1E2 ATPases are primarily Results are from a search (January 22, 1989) of the European cytoplasmic globules formed by at least four interactive Molecular Biology Organization library of protein sequences for domains, connected by a narrow stalk to the hydrophobic polypeptides homologous to CadA using the FASTP alignment algomembrane-anchoring segment of the enzyme (24-26). Transrithm (16). The closest match found was to the Streptococcus membrane hairpins are postulated to form a channel through faecalis K+-ATPase sequence (18). The FASTP score (17) is given in which the cations are transported (22, 26, 27). arbitrary units after optimization allowing gaps; the percent amino There is little homology in general between eukaryotic and acid (aa) identities and the lengths of the homologous regions from prokaryotic E1E2 ATPases in the transmembrane hairpins, the FASTP matching are shown. Results from only one of the seven beyond their positions and general hydrophobicity. The current Na+/K+-ATPase sequences, one of the current two sarcolocation of these hydrophobic sequences, defined by a high plasmic reticulum Ca2+-ATPase sequences, and one of the five current mercuric reductase sequences are shown. The mercuric density of charged residues on either side of a hydrophobic reductases are not ATPases but scored with a significant homology segment of -20 amino acid residues, and their occurrence in for the N-terminal region (see text). Sac., Saccharomyces. closely positioned pairs suggest very similar overall structures for the enzymes. efflux. The protein predicted by the DNA sequence shows The first transmembrane hairpin segment of the putative 26-30% amino acid identities over stretches of >500 residues Cd2' ATPase is predicted to occur between residues 106 and with the K+-uptake ATPases of Strep. faecalis and E. coli 148. The transmembrane segment occurs after a highly (Table 1). stretch (residues 86 through 105) and separates hydrophilic At its amino-terminal end, the putative Cd2+-ATPase the amino-terminal region of the protein (concerned with the shares significant homology (10 SDs from the mean score for initial binding of the cation to be transported) from the Cd2+ MSVEQKVI EEMYRV1IGCANCAGKFENVKKIPGVQDAKVNFCKIDVYQ4ASV 60 transduction domain, whose primary function is the delivery of the cation to the transmembrane channel for outward Cd2+ EEEKAGAFEULKVSPKANTIQRVKMIIK PFYKKLLFATLAFYL 120 translocation. The transduction domain consists of -180 Cd2+ SHFVND VISMLF 1VI1(YSlIKVGF8LIRFDUMLMIVGIIGKWa 180 residues in the CadA protein. It is predicted to be mostly in K+ MELKQKSPAMMMLIAM1ITVAYVYSVYSFIANLISPHTIVMDFFWE 47 a-helical conformation and is highly hydrophilic (with a net Cd2+ EASIVVILFAISEALERFSMDRSR8SIR LMDjApKEA-LVRR MNGEIIIHVDIAAIGDI 239 negative charge provided by an excess of nine glutamate and G1 1 106 K+ LATIVIML-MIWI 4SD0ELPEVKR aspartate residues). Fig. 6 shows a comparison of a relatively conserved (central) section of the putative transduction Cd2+ MIVKPGEKIANGIIVNG[SAVNQRAAII1ESVPVSKAVDDEVFAUTLNEEGLIEVKITKY 299 domain of the Cd2+-ATPase with the corresponding regions K+ LIVRAGDInCrIDKGrIVDESAV19USKGVKKQVGSVIwGSINuG'rIEIT1U- 165 of other E1E2 ATPases. The homology occurs over a stretch Cd2+ VEDlTITKIIHLVEEAQGERAPlAFVDKFAKYYTPIIMVIAALVAVVPPLFFGGSWUIW 359 of about 50 residues, and it includes several conserved K+ ItEMVWKVMEMVRKAQGEQ+QLEFLSDKVAKWLFYVALW-GIIAFIAWLFLANLP13A- 223 aspartate, glutamate, and glycine residues. ~~~~~~~~~~~~~~P04 In the putative Cd2+-ATPase, a second transmembrane Cd2+ VYQGLAVLVVVPCALVISTPISIVSAIGNAAKKGVLVKGGVYLEKVUYIKVAFDK~qF 419 hairpin segment is predicted next, with the second half of the K+ LERMVTVFIIACPHALAIPLWARSTSIAAJING[LLSNNAMIEUANDIDVIMLDWKI 283
Table 1. Comparison of the Cd2+-ATPase sequence with other cation-translocating ATPases % identities
:..
::
.
.:
..
..........
...
....
...:
Cd2+
Cd2+ LTKGVPWTDFEVIDQVEEV.IEIITAIEYRSPIASAIIMKAEQIIPYSN9QVEE 479 K+ LI1GVPIEILDEAAYQEEEILYIGALEAH AIGNJLJIM1YLKI--TPYQAfE Cd2+ FSII gRIKGIVNa1TYYIGSPKLFKENVSDFSSwNFE-NNVKIKaItAIGTaEK
.K..: :O....V..... QKMLGVGETVEDKD
341
538
*IE.... ..A .... 396 VKIINEIKRIDIDPRLKNYEFQGI.SVVSD Cd2+ T1WVIAVADEVRET1SKNVIQ=iOW1KQIIMLT¶ 598 NI2IANA1GIVWVSDIQSELMP K+
....
.... ...
455
K+
KLVAVIALVIKPEAKEFIQAIKENI-IPVML¶IPQWQPAEYGINEEYYGY iEYNEYVAMIGGVNDAPALAASTVGIAKGAG-1EATADIAUGJL DDKEAIVQRYLDQGKVIM2CINAAPSLARAT1IGAI-GA1IAIDSADVVLISsDP
Cd2+
SKLPFAVRLSRKIMNIIKANITFAIGIKIIALLLVIPGWLTLWIAILSDMATILVALNS
718
Kt
KDIUH
573
K+
Cd2+ QDK0DYIK
658 514
Cd2+ K
-LAI0MI(*ILAGNIIAIPLA-ACILAJPIGLLSPAVGALMSLSr P
UIRVKDK
727
VVVAUNALTLK
FIG. 5. versus the
583
Alignment of the putative cadA amino acid sequence K+-ATPase from Strep. faecalis. Dashes indicate gaps introduced to optimize alignment, semicolons indicate identities, and dots indicate conservative amino acid replacements. The marked proline (*) and aspartate (P04) residues are considered in the Discussion.
hairpin containing a crucial proline residue. This proline (Pro-372, Fig. 5), is located in all E1E2 ATPases 43 residues before the aspartate residue (Asp-415, Fig. 5) that is phosphorylated in E1E2 ATPases. The position of the second transmembrane hairpin as well as the location of the proline next to the junction between transmembrane and cytoplasmic regions are in agreement with the structural model developed for other E1E2 ATPases (22, 24, 26, 28). Asp-415 in the CadA protein is the first of a string of 7 amino acids [AspLys-Thr-Gly-Thr-Leu (or Ile)-Thr] that are conserved in E1E2 ATPases (from different species and with different cationtransport specificities; Fig. 6C) and are flanked by conservative replacements. By analogy to other E1E2 ATPases, Asp-415 of the CadA protein should be the aspartate residue that undergoes phosphorylation (23, 28). The next and most extended region of homology and conservative replacements between the CadA protein and other E1E2 ATPases starts around residue 600 and continues for approximately 50 residues. This region is centered around two aspartate residues (Asp-620 and Asp-624 in the cadA gene product; Fig. 6D). By comparing the sequences in
Biochemistry: Nucifora et al.
3548 A
region Hypothesized cation binding N-terminal * * **
*
**
18 VQGF'1ANCAGKFEKIPGV 40 6 ITGICADSCAVHVIAEKVPGV 28 28 VPM!rANPITVKKALSKVEGV 50
Cd2+ ATPase S.
aureus Mercuric reduc ase E. coli Periplasmic Hg +-BP E. coli
B
Proc. Natl. Acad Sci. USA 86 (1989)
Transduction dmnain
Cd2+ ATPase S. aureus K+ ATPase E. coli K+ ATPase S. faecalis
H+ MTPase
yeast
H+ ATPase Neurospora Na+/K+ ATPase human
Na+/K+
IHIVDDIAVGDIMIVKPGEKIAMDGIIVNGL VPADQLPKGDIVLVEADIIPCDGEVIEGG 96 VSLKEVHEGDRLIVRAQ7KMPIDX r'IDKGH 191 IPANEVVPGDILQLEDI VIPJIGIVTEDC 191 IEAPEWPGDILDVEEGTIIPADGRIVIJA
SAVNQAAITIESVPVSKAVDDEVF
229 121
282
ASVDESAI¶IGESAPVIRESGGDFA 174 TIVDESAV1IIESKGVKKQVtDSVI 149
FLQIDQSAI1TESLAVDKHYGJQF 246 FQVDQSALTI3SAVKHKGDQVF 246
180 INAEEVVVGDLVEVKGGDRIPADLRIISAN GKVDNSSLIGESEPQgRSPDFrNE 234 180 INAEQVVVGDLVEVKGGDRIPADIRIISAC SRKDSLTG ESEPQSRSPEYSSE 234 140 IKARDIVPGDIVEVAVMDKVPADIRILSIKSTLIRVQSIL¶BESVSVIKHTEPVPD 196
Ca2+
ATPase eel ATPase rabbit
C
Phosphoryl-aspartate rec P04 *****
*
Cd2+ ATPase S. aureus K+ ATPase E. coli K+ ATPase S. faecalis H ATPase yeast H+ ATPase Neurospora
Na4/K4 ATPase human Na+/K+ ATPase electric eel
Ca2+ ATPase rabbit D
404 296 268 367 367 365 365 340
*
EKLGAIKTVAFDK1LMTKG 423 EAAGDVDVLlDK'rITLG 315 EQANDLDVIMLDKIUl¶lQG 287 ESLAGVEIICSDKILMLTKN ESLAGVEILCSDKI TJKN ETIGSTSTICSDK=1X LQN ETLGSTSTICSDUO IW ErL SVICSDIUrl1TIN
386 386 384 384 359
Hinge region and end of ATP-binding donain ***
Cd2 ATPase S. aureus K+ ATPase E. coli K4 ATPase S. faecalis yeast Neurospora Na4/K4 ATPase human Na+/K+ ATPase electric eel Ca2+ ATPase rabbit
ATPase H+ H+ ATPase
598 496 455 612 612 695 -694 681
**
*
*
*
A 645 I PQDKLDYIIK SEYDNAMIGDGVNDAPALAASTVGIMJGAG PEAKLALIIRYQAEGRLVAMJIYGRMIAPALAOAIVAVAM NSGI-A 542 PDDKEAIVQRYILQGKKVIMV GINDAPSLARATIGMAI GAGTDIA 501 P1HKYRVVEILNRGYLVARIEGGVNDAPSLKKAMyj;AVEGi TDAR 658
AVEGS SDAA 658 742 PQQKLIIVECCQRQGAIVAVXI'GVNDSPALKKADIGVAM3IAGSDVS 741
QHKYNVVEIIQRGYLVAM1JGVNDAPSLKKAmSI
PQKLIIVEGCQQGAIVAV'UGDGVNDSPALKKADIGVAK4IAGSDVS
FIG. 6. Conserved sequences in eukaryotic and bacterial E1E2type ATPases. Asterisks indicate identical residues in all sequences shown. (A) Putative cation binding N-terminal region is compared to the soft-metal-binding region of mercury-binding proteins. (B, C, and D) Three putative functional domains of the cation-translocating ATPases. P04 indicates the aspartate that undergoes phosphorylation. The primary references for the sequences shown can be found in a recent short review
PSHKSKIVEYIQMDEITAMI1DGVNDAPATKAEIGIAM GSGrAVA 727 (19). E., Gryczan, T. J., Baron, E. & Edelman, Murphy, R. P., 2. Novick, extrapolated: Val-Alathis region, a620consensus can be 624 I. (1979) Plasmid 2, 109-129. Met-Thr-Gly-Asp-Gly-Val-Asn-Asp-Ala-Pro-Ala-Leu. This 3. Perry, R. D. & Silver, S. (1982) J. Bacteriol. 150, 973-976. region is believed to include the end of the nucleotide-binding 4. Weiss, A. A., Silver, S. & Kinscherf, T. G. (1978) Antimicrob. Agents Chemother. 14, 856-865. domain (22) and extends into the next transmembrane 5. Tynecka, Z., Gos, Z. & Zajac, J (1981) J. Bacteriol. 147, 313-319. stretch. In all of the E1E2 ATPases (including the postulated 6. Tynecka, Z., Gos, Z. & Zajac, J. (1981) J. Bacteriol. 147, 305-312. Cd2+-ATPase), the ATPase region constitutes a single intra7. Jones, C. L. & Khan, S. A. (1986) J. Bacteriol. 166, 29-33. by uninterrupted acids, amino 250-400 of domain cellular 8. Dubnau, D., Davidoff-Abelson, R., Scher, B. & Cirigliano, C. (1973) transmembrane segments. J. Bacteriol. 114, 273-286. 9. Laddaga, R. A., Chu, L., Misra, T. K. & Silver, S. (1987) Proc. Finally, after the last putative transmembrane hairpin, Natl. Acad. Sci. USA 84, 5106-5110. predicted to be between residues 624 and 707, the CadA 10. Misra, T. K. (1987) Methods Enzymol. 155, 119-139. is region The carboxyl-terminal molecule ends with Lys-727. 11. Studier, F. W. & Moffatt, B. A. (1986) J. Mol. Biol. 189, 113-130. considerably longer in eukaryotic E1E2 ATPases, where it 12. Ambudkar, S., Lynn, A. R., Maloney, P. C. & Rosen, B. P. (1986) often includes two additional transmembrane segments (23, J. Biol. Chem. 261, 15596-15600. 13. Hugentobler, G., Heid, I. & Solioz, M. (1983) J. Biol. Chem. 258, 24, 26). 7611-7617. Despite different cellular origins and different specificities 14. Fairbanks, G. & Avruch, J. (1972) J. Supramol. Struct. 1, 66-75. of cation transport and orientation, the bacterial and eukary15. Barberis-Maino, L., Berger-Bachi, B., Weber, H., Beck, W. D. & otic ATPases are related in function and have homologies in Kayser, F. H. (1987) Gene 59, 107-113. the regions (Fig. 6) that may correspond to the common 16. Laddaga, R. A., Bessen, R. & Silver, S. (1985) J. Bacteriol. 162, 1106-1110. properties of the proteins essential for their activity as 17. Lipman, D. J. & Pearson, W. R. (1985) Science 227, 1435-1441. primary cation pumps. 18. Solioz, M., Mathews, S. & Furst, P. (1987) J. Biol. Chem. 262,7358It often has been speculated that sequence similarities for 7362. E1E2 cation-translocating ATPases must be a consequence of 19. Silver, S., Nucifora, G., Chu, L. & Misra, T. K. (1989) Trends a common ancestry. It is attractive to hypothesize that the Biochem. Sci. 14, 76-80. proteins underwent modifications evolving the capability to 20. Silver, S. & Misra, T. K. (1988) Annu. Rev. Microbiol. 42, 717-743. 21. Brown, N. L. (1985) Trends Biochem. Sci. 10, 400-403. transport different cations inwardly and outwardly through 22. Brandl, C. J., Green, N. M., Korczak, B. & MacLennan, D. H. different membranes according to the needs for the survival (1986) Cell 44, 597-607. of the cell, including the elimination of toxic cations. 23. Shull, G. E., Schwartz, A. & Lingrel, J. B. (1985) Nature (London) 316, 691-695. We thank M. Walderhaug and W. Epstein for useful discussions, 24. MacLennan, D. H., Brandl, C. J., Korczak, B. & Green, N. M. A. Lynn and B. P. Rosen for help with ATPase and transport assays, (1985) Nature (London) 316, 696-700. R. A. Laddaga for plasmid pRAL3A, S. A. Kahn for plasmid 25. Addison, R. & Scarborough, G. A. (1982) J. Biol. Chem. 257, pSK265, and F. W. Studier for E. coli stain BL21. This work was 10421-10426. 26. Addison, R. (1986) J. Biol. Chem. 261, 14896-14901. supported by Grant DMB-86-0481 from the National Science Foun27. Brandl, C. J. & Deber, C. M. (1986) Proc. Natl. Acad. Sci. USA 83, dation. 1. Witte, W., Green, L., Misra, T. K. Agents Chemother. 29, 663-669.
&
Silver, S. (1986) Antimicrob.
917-921. 28. Hesse, J. E., Wieczoreck, L., Altendorf, K., Reicin, A. S., Dorus, E. & Epstein, W. (1984) Proc. Natl. Acad. Sci. USA 81, 4746-4750.
