From the
Department of Biochemistry and Molecular Biology, University of Maryland
School of Medicine, Baltimore, Maryland 21201 and
Institute of Molecular and Cellular Biosciences,
University of Tokyo, Bunkyo-ku, Tokyo 113, Japan
Received for publication, April 18, 2003 , and in revised form, May 12, 2003.
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ABSTRACT |
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INTRODUCTION |
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The ATPase molecular structure includes a transmembrane region made of 10 clustered helical segments including the Ca2+ binding domain and a cytosolic headpiece including the nucleotide binding (N), phosphorylation (P), and actuator (A) domains. Structural studies have shown that if crystallization is performed in the presence of Ca2+ the three headpiece domains are distinctly separate in an open configuration (2). On the other hand, in the absence of Ca2+, those domains gather to form a compact headpiece (3, 4). This finding suggests that in solution the three domains undergo fluctuations, yielding different enzyme conformations depending on the presence of specific ligands. Taking advantage of a selective ATPase cleavage (5), it was then shown that the addition of nucleotides in the presence of Mg2+ and Ca2+ protects the ATPase from digestion by proteinase K (1). Protection was attributed to a change in the positions of the headpiece domains with respect to the loop connecting the A domain to the transmembrane helices. These movements are directly related to gate opening and closing in the Ca2+ pathway. This is a very important finding, suggesting that the open headpiece of the Ca2+-activated enzyme acquires a compact conformation upon substrate binding.
We describe here a series of experiments on the interference of various mutations with the protective effect of nucleotide. We demonstrate that occurrence of the nucleotide-dependent conformational change requires participation of amino acid residues in both N and P domains and that nucleotide-dependent approximation of the headpiece domains occurs only when both Ca2+ sites are filled.
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MATERIALS AND METHODS |
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Experiments on limited digestion of ATPase with proteinase K and protection
by nucleotides or Ca2+ were performed in media
containing 50 mM MOPS, pH 7.0, and 50 mM NaCl in the
presence or in the absence (CDTA or EGTA present) of 5 mM
MgCl2 and/or 0.1 mM CaCl2. In experiments
with native SR vesicles, the protein concentrations were 0.3 mg of SR and 0.01
mg of proteinase K/ml. In experiments with recombinant ATPase, the protein
concentrations were 1.2 mg of microsomal protein and 0.04 mg of proteinase
K/ml. The concentration of recombinant ATPase was adjusted to the same level
in all of the experiments based on ATPase estimates by Western blotting and
compensation with empty microsomes. The reaction was quenched at serial times
by the addition of trichloroacetic acid to yield a final 2.5% concentration.
The quenched protein was then solubilized by adding sodium dodecyl sulfate
(1%), Tris (0.312 M), pH 6.8, sucrose (3.75%),
-mercaptoethanol (1.25 mM), and bromphenol blue (0.025%). The
samples were then subjected to electrophoretic analysis
(10) on 12.5% gels followed by
staining with Coomassie Blue or Western blotting. For this purpose, the
monoclonal antibody mAb CaF35C3 to the chicken SERCA-1 protein and goat
anti-mouse IgG-horseradish peroxidase-conjugated secondary antibodies were
used followed by densitometry of the bands visualized with an enhanced
chemiluminescence-linked detection system (Amersham Biosciences). Amino acid
sequencing of peptide fragments eluted from electrophoretic gels was performed
at Johns Hopkins University.
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RESULTS |
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As a preliminary to mutational analysis, we confirmed that identical results are obtained with recombinant ATPase. Because the microsomal preparations derived from COS-1 cells contain several additional proteins, the digestion products of recombinant ATPase were evidenced by Western blotting, which is favored by their reactivity to the same antibody. In fact, since unrelated protein bands are not detected by Western blotting, the results of proteinase K digestion and the protection by AMP-PCP can be demonstrated quite clearly (Fig. 2).
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Effects of Site-directed MutationsConsidering that protection from proteinase K digestion is produced by functionally relevant ligands such as Ca2+ and nucleotide, we studied the effects of site-directed mutations that may interfere with binding and/or utilization of such ligands for catalytic reactions. A list of mutants produced for these experiments is shown in Table I. It is also shown in Table I that most mutants were expressed at levels nearly as high as those obtained with WT protein. Few mutants such as D703A and D707A were expressed at lower levels, perhaps because of defective folding of the nascent peptide. Generally, the digestion of mutants by proteinase K proceeded somewhat faster than digestion of WT ATPase as shown by the half-time of disappearance of the main ATPase band (Table I). This was more pronounced as a consequence of D707A and the K684A mutations and may be due to some folding destabilization by the mutations.
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Mutations of Residues Involved in Ca2+ BindingThe specific Ca2+ requirement for protection by AMP-PCP was most convincingly demonstrated by the use of the E771Q and N796A mutants. Consistent with inhibition of specific Ca2+ binding (11), we found that these mutations interfere also with AMP-PCP protection of ATPase digestion by proteinase K (Fig. 3 and Table I). It is of interest that protection by AMP-PCP is totally abolished by a mutation that interferes with the binding of only one Ca2+ (i.e. N796A) as well as by a mutation that interferes with the binding of both Ca2+ (i.e. E771Q). Because it is known that enzyme activation requires the binding of both Ca2+, it is apparent that protection by nucleotide requires specific enzyme activation by Ca2+.
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Mutations of Residues in the N DomainWe recently found that mutation of Arg-560 to Ala produces strong inhibition of ATP utilization but much less inhibition of reverse enzyme phosphorylation with Pi (12). We find now that the R560A mutation interferes completely with AMP-PCP protection of the enzyme from digestion with proteinase K (Fig. 4 and Table I). These combined observations indicate that the Arg-560 side chain is specifically involved in the stabilization of nucleotide in the N domain.
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Because of their proximity to the nucleotide binding site, we also characterized the effects of Glu-439 and Arg-489 mutations. We found partial catalytic inhibition as a consequence of their mutations, but interference with the AMP-PCP-protective effect was observed only in the E439A mutant (Table I). Considering that the bound nucleotide must reach the P domain to be utilized as a catalytic substrate and that nucleotide protection form proteinase K may be related to N and P domain approximation, we extended our mutational analysis to the P domain.
Mutations of Residues in the P DomainCa2+-dependent utilization of ATP results in phosphorylation of Asp-351 to form a phosphorylated enzyme intermediate. Therefore, mutation of this residue results in complete enzyme inactivation. On the other hand, AMP-PCP protection from proteinase K digestion is retained following mutation of Asp-351 to Asn (Table I) and is actually much improved by mutation of Asp-351 to Ala (Fig. 5) or Asn-706 to Ala (Table I).
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The behavior of the Asp-351 mutant in conjunction with the effect of the
AMP-PCP pseudo substrate demonstrates unambiguously that phosphoryl transfer
is not required for the protective effect, but simple nucleotide binding is
effective. In addition, the greater protection observed with the D351A and
N706A mutants indicates that the native side chains of these amino acids play
a role in positioning the nucleotide -phosphate and limiting the
stability of the substrate-enzyme complex. In fact, a rather weak
substrate-enzyme complex, relative to the subsequent transition state, is
required for optimal enzyme kinetics.
It was previously reported that Lys-684, a P domain residue, reacts with adenosine triphosphate pyridoxal in the presence of Ca2+, and this reaction is blocked by ATP (13). Accordingly, we found that mutation of neighboring Lys-684 to Ala produces catalytic inactivation as well as interference with the protective effect of AMP-PCP (Table I). It is then apparent that the Lys-684 side chain plays an important and direct role in stabilization of the nucleotide substrate terminal phosphate.
We also found that mutations of Lys-352, Thr-353, Asp-703, and Asp-707 produce total or partial catalytic inactivation and interfere with the protective effect of AMP-PCP (Table I). All of these residues reside in close proximity of the phosphorylation site (i.e. Asp-351). Analogous mutational analysis of the Na+K+-ATPase (14) indicates that electrostatic interactions around the phosphorylation site may play an important role in substrate positioning and utilization. We considered that their mutation may alter direct interactions with the nucleotide terminal phosphate or ligation of Mg2+ in conjunction with oxygen atoms of the ATP-terminal phosphate. On the other hand, the K352E mutation is not as effective as the K352A mutation. Furthermore, Mg2+ is not required for the nucleotide-protective effect (see below).
Divalent Cation Specificity and Nucleotide Concentration DependenceIt is shown in Fig. 6A that the protective effect of AMP-PCP is obtained to the same extent, even when Mg2+ is omitted (CDTA present), and Ca2+ is present at concentrations (20100 µM) that are much lower than the effective nucleotide concentrations (1 mM).
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We also found that irrespective of the presence or the absence of Mg2+ the protective effect occurs at 0.11.0 mM nucleotide concentrations. This range is higher than the 10 micromolar Km observed with ATP as a substrate for ATPase activity. It is of interest that the concentration dependence of the protective effect of AMP-PCP shifts to a lower range when the D351A mutant is used (Fig. 6B). A high affinity of the D351A mutant for the nucleotide substrate was previously noted by McIntosh et al. (15).
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DISCUSSION |
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Useful information on this subject was provided by studies on nucleotide-induced protection of the ATPase from digestion with proteinase K. The digestion site is at Thr-242 located on the loop connecting the A domain to the M3 helix. Thus, the susceptibility is expected to be affected by the position of the A domain. In fact, this is an isolated loop in E1 Ca2+ (open configuration) but is attached to the P domain in E2(TG) (compact configuration), reflecting the different configuration of the cytoplasmic domains. Steric hindrance by the P domain appears to be the origin of the protection of this site in the E2(TG) state. However, different degrees of protection from proteolysis are observed in the E2(TG), CaE1ATP, and E2P states (even though the headpiece resides in a compact configuration in all of these states), reflecting a graded response of the A domain to these ligand-induced transitions (1).
Our mutational analysis indicates that Arg-560 and Glu-439 (N domain) as well as Asp-351, Lys-352, Thr-353, Lys-684, Asp-703, Asn-706, and Asp-707 (P domain) are involved in the nucleotide effect. Participation of N and P residues demonstrates that approximation of the two domains does indeed occur as required to accommodate the nucleotide by means of adenosine moiety interaction with the N domain and phosphate interaction with the P domain. The A domain must also reposition as shown by the protection from proteinase K. The Ca2+ requirement for nucleotide protection indicates that even though a compact arrangement of the headpiece is favored by nucleotide binding, the transmembrane domain retains bound Ca2+. The compact headpiece conformation obtained under these conditions is not identical to that observed in the absence of Ca2+ (1) but evidently represents an additional specific state produced by nucleotide binding on the Ca2+-activated enzyme. An apparently similar approximation of nucleotide binding ("fingers") and catalytic ("palm") domains is known to occur in DNA polymerases (22).
It is of interest that the nucleotide concentration dependence of the protective effect is in the 0.11.0 mM range. This range is higher than the 10 µM Km observed with ATP as a substrate for ATPase activity, suggesting that the kinetics of catalytic ATP utilization have a significant influence on the overall ATP concentration dependence of enzyme activity. It should be pointed out that a rather weak substrate-enzyme complex, relative to the stability of the subsequent transition state, is required for effective enzyme kinetics.
An additional finding is the lack of Mg2+ requirement
for nucleotide protection of ATPase digestion by proteinase K
(Fig. 6A). It is of
interest that in structural snapshots
(23) of the phosphoserine
phosphatase (PSPase) (its catalytic domain is analogous to the P domain of
Ca2+ATPase) reaction cycle, the initial substrate-enzyme
complex does not include Mg2+, whereas interaction with
Mg2+ occurs in concomitance with the phosphoryl transfer
and hydrolytic reactions. With regard to the Ca2+ATPase,
it is probable that a complex with Mg2+ is formed
initially (21) when ATP is the
substrate due to the cation binding property of the nucleotide. On the other
hand, our present experiments suggest that nucleotide binding (and its
conformational effect) can be obtained either in the presence or in the
absence of Mg2+, although the subsequent phosphoryl
transfer and hydrolytic reactions require Mg2+ in
analogy to PSPase. This suggestion is consistent with the random mechanism
proposed by Reinstein and Jencks
(24) for ATP and
Mg2+ binding to the Ca2+ATPase. In
analogy to the PSPase, it is likely that catalytically required
Mg2+ binding occurs in concomitance with the
phosphorylation transition state, including coordination by Asp-703, Thr-353,
and -phosphate.
Mutational analysis demonstrates direct roles of Arg-560 (N domain) and
Lys-684 in nucleotide stabilization in the N and P domains, respectively.
Arg-560 may interact with the nucleotide -phosphate. Lys-684, on the
other hand, is likely to interact with
-phosphate in analogy to
stabilization of phosphorylserine by Lys-144 in the PSPase
(23). The roles of other
residues are more complex. For instance, the absence of nucleotide protection
in the Asp-703 mutant could be attributed to a role of this residue in
Mg2+ stabilization. On the other hand, nucleotide
protection is obtained even in the absence of Mg2+.
Therefore, Asp-703 must have an additional and important role in structural
stabilization by hydrogen bonding with neighboring residues. Similar
considerations can be made regarding Asp-707, Lys-352, and Thr-353, whose side
chains are likely to establish hydrogen bonding with neighboring residues
and/or water molecules (23).
Note that the K352A mutation is much more effective than the K352E mutation,
indicating that the ability of K352 to establish stabilizing interactions in
the open or compact headpiece conformation is more important than a possible
interaction with
-phosphate oxygen atoms.
Another interesting finding is related to the higher protective effect of
nucleotide in the D351A and N706A mutants as compared with WT Asp-351 and
Asp-706 (as well as with the D351N mutant). Asp-351 is in fact the residue
undergoing phosphorylation, and Asn-706 (Asn-170 in the PSPase) is a close
neighbor that contributes its side chain nitrogen
(3,
23) for coordination of the
same -phosphate oxygen as Lys-684 (Lys-144 in the PSPase). It is then
apparent that in the WT enzyme, a number of steric and electrostatic
constraints guide the
-phosphate to an optimal position for covalent
interaction. On the other hand, even tighter binding may be obtained by
removing some of these constraints.
To understand our experimental results, an atomic model was built based on
the atomic model of the PSPase with its substrate bound
(23). This was possible
because of the close similarity between the atomic structures of the catalytic
domain of the PSPase and the P domain of the MgFx complex of the
Ca2+ATPase (considered to be an E-P
analog).2 The model
for the Ca2+ATPase was built combining that for the N
domain taken from the E2(TG) form (Protein Data Bank code 1IWO
[PDB]
)
(3) and that for the P domain
from the MgFx complex. ATP in an extended form was placed so that
the -phosphate comes exactly to the same position as that of the
phosphate in phosphoserine (Protein Data Bank code 1L7P
[PDB]
).
The ATP orientation in the model (Fig.
7) suggests that Mg2+ coordinated by the
- and
-phosphates would not come to a position suitable for
coordination by Asp-703. In fact, in the D11N mutant of the PSPase
(corresponding to the D351N mutant of the Ca2+ATPase and
used for the substrate bound state), no Mg2+ is found as
opposed to the subsequent states of the catalytic cycle in which Asp-167
(Asp-703 in the Ca2+ATPase) participates. This clearly
indicates that the initial step of substrate binding does not require
Mg2+, consistent with the observation that
Mg2+ is not required for the nucleotide-induced
protection from proteinase K digestion of the
Ca2+ATPase.
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In the D11N mutant of the PSPase, the conformation of Asn-11 is different
from other states. The conformation of Asn-11 is one of the standard rotamers
and places the side chain nitrogen atom close to the phosphate. This
conformation is stabilized by hydrogen bonding between oxygen atom of Asn-11
and main chain amide of Asp-167 (Asp-703 in Ca2+ATPase).
Thus, this is likely to be the conformation in the native enzyme when
substrates bind initially. Here phosphates are coordinated by atoms
corresponding to Lys-352 (main chain NH), Thr-353 (main chain NH), Thr-625
(O1), Gly-626 (main chain NH), Lys-684 (N
), and Asn-706
(N
2) in the Ca2+ATPase. Thus Lys-684 is the only
positively charged residue likely to be a key attractor for the
-phosphate. Hence the position of the side chain nitrogen atom must be
very important. In fact, in phosphoglucomutase
(25,
26), the C
position of
the corresponding residue is different by one residue but the position of the
nitrogen atom is exactly the same. This position appears to be controlled by
Asp-351 and Asp-707 in the Ca2+ATPase when substrates
are absent. Thus, mutations of these residues may strongly affect the affinity
for ATP and consequently its protective effect. This may explain the higher
protection observed with the D351A as compared with the D351N mutant.
The effect of the N706A mutant is particularly interesting. N706 in the MgFx complex of the Ca2+ATPase as well as the corresponding Asn-170 in the PSPase provides a nitrogen atom for coordination of the same phosphate oxygen as Lys-684. Thus, the strong nucleotide protection observed in the N706A mutant was contrary to our expectation. Although this Asn residue is not well conserved in the haloacid dehologenase superfamily, the corresponding residue (Asn-170) of the PSPase is involved in a large conformation change and appears to regulate the flap (or lid) of the substrate binding site by changing the interaction with Phe-49 in the subdomain. Asn-706 may bear a similar role and might adjust the position of the N or A domain. At least in the MgFx complex, this residue does interact with the key loop in the A domain (181TGES184) or it may have an influence on the position of Lys-684. If mutated to Ala, Lys-684 might take a position that allows stronger interaction with ATP. This may be a likely reason for the strong nucleotide protection observed in the Asn-706 mutant.
Finally, it should be pointed out that in the model
(Fig. 7) Arg-560 takes a very
strategic position. It stabilizes ATP by interacting with the
-phosphate and also stabilizes the closed configuration of the N and P
domains by making a salt bridge with Asp-627, one of the critical residues in
the P domain. In this sense, Arg-560 may have a similar role to Arg-56 in the
PSPase. Why Asp-627 is important has never been explained. This conformation
of Asp-627 is realized by an interaction with Lys-352, which appears to serve
also in positioning Thr-353, another critical residue. This may be the reason
for the profound effect of the K352A mutation.
In conclusion, our experiments indicate that nucleotide binding occurs by
collision with the N domain in the Ca2+-dependent open
conformation of the enzyme headpiece. A substrate-induced conformational fit
then takes place relative to stabilization of the headpiece domains in a
compact configuration. This allows approximation of the ATP -phosphate
to Asp-351 in the P domain, whereas the membrane-bound domain still binds two
Ca2+. The roles of several amino acid residues are
demonstrated, in some cases related to direct substrate binding and in other
cases related to short and long range interactions of protein structure.
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FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 410-706-3220; Fax:
410-706-8297; E-mail:
ginesi{at}umaryland.edu.
1 The abbreviations used are: SR, sarcoplasmic reticulum; SERCA,
sarco(endo)plasmic reticulum Ca2+ATPase; WT, wild type;
MOPS, 3-(N-morpholino)propanesulfonic acid; CDTA,
1,2-cyclohexylenedinitrilotetraacetic acid; AMP-PCP, adenosine
5'-(,
-methylenetriphosphate); TNP-AMP,
2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5';
PSPase, phosphoserine phosphatase; N domain, nucleotide binding domain; P
domain, phosphorylation domain; A domain, activator domain; E2, ATPase with no
bound Ca2+; E1, ATPase with bound
Ca2+; TG, thapsigargin.
2 C. Toyoshima, unpublished observations.
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REFERENCES |
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