Asp1080 upstream of the calmodulin-binding domain is critical for autoinhibition of hPMCA4b.

The role of the plasma membrane Ca(2+) pump (PMCA) is to remove excess Ca(2+) from the cytosol to maintain low intracellular Ca(2+) levels. Asp(1080) lies within an acidic sequence between the C-terminal inhibitory region and the catalytic core of PMCAs and is part of the caspase-3 recognition site of isoform 4b. Caspase-3 cuts immediately after this residue and activates the pump by removing the inhibitory region (Pászty, K., Verma, A. K., Padányi, R., Filoteo, A. G., Penniston, J. T., and Enyedi, A. (2002) J. Biol. Chem. 277, 6822-6829). Asp(1080) had not been believed to have any other role, but here we show that it also plays a critical role in the autoinhibition and calmodulin activation of PMCA4b. Site-specific mutation of Asp(1080) to Asn, Ala, or Lys in PMCA4b resulted in a substantial increase in the basal activity in the absence of calmodulin. All Asp(1080) mutants exhibited an increased affinity for calmodulin because of an increase in the rate of activation by calmodulin. This rate was higher when the inhibition was weaker, showing that a strong inhibitory interaction slows the activation rate. In contrast, mutating the nearby Asp(1077) had no effect on basal activity or calmodulin activation. We propose that the conserved Asp(1080), even though it is neither in the regulatory domain nor in the catalytic core, plays an essential role in inhibition by stabilizing the inhibited state of the enzyme.

The plasma membrane calcium pump (PMCA) 1 is an essential element of intracellular Ca 2ϩ homeostasis. Its role is to remove excess Ca 2ϩ from the cell to maintain low cytosolic Ca 2ϩ concentrations critical for cell survival and Ca 2ϩ signaling. An increase in cellular Ca 2ϩ results in an increase in substrate Ca 2ϩ at the transport site of the pump, as well as an increase in activator Ca 2ϩ -calmodulin complex. PMCAs are encoded by four genes, and alternative splicing of the primary transcripts produces many more isoform variants (1). Two of the four PMCA gene products (PMCA1 and PMCA4) are ubiq-uitous, whereas the expression of PMCA2 and PMCA3 appears to be more cell-and tissue-specific.
PMCA4b has a low activity in the absence of calmodulin and thus a great stimulation by calmodulin (2). A series of experiments using peptides and C-terminally truncated mutants of hPMCA4b showed that the high affinity calmodulin binding region lay within a 28-residue sequence between Leu 1086 and Ser 1113 (3,4). It has been demonstrated that the same region is responsible for most of the autoinhibition, but a downstream sequence (between Ser 1113 and Asp 1157 ) also contributes. Removal of the whole C terminus from Leu 1086 to the end resulted in a constitutively active enzyme; it could not be further activated by addition of calmodulin (2).
Cross-linking experiments with the calmodulin binding synthetic peptide C28 have revealed that C28 interacts with two sites within the catalytic core of PMCA; one is located downstream of the phosphoenzyme forming aspartic acid between residues 537-544, and the other is within the small cytoplasmic loop (transduction domain or activation domain) (5)(6)(7).
Although the contribution of the sequence between residues Leu 1086 and Asp 1157 to the regulation of the enzyme has been studied extensively, the role of the sequence between transmembrane domain 10 and Leu 1086 is poorly understood. Because this region belongs neither to the autoinhibitory domain nor to the catalytic core of the enzyme, it may be considered as a connecting region or hinge. A highly acidic region is located just upstream of the calmodulin-binding/autoinhibitory sequence. This acidic region is rich in proline, aspartic/glutamic acid, and serine/threonine and has been suggested to play a role in calpain recognition (8); however, experiments using peptides from isoform 1 did not support this idea (9). Also, it has been suggested that this acidic region contains a high affinity Ca 2ϩ binding site, but its role in the regulation of the pump has not been addressed (10). In addition, part of the upstream region (between Glu 1067 and Arg 1087 ) was suggested to encode a "masked" signal for endoplasmic reticulum retention that was functional only when the C terminus downstream of it was removed (11).
In more recent experiments, we have demonstrated that Asp 1080 , located in this hinge region five residues upstream of the calmodulin binding domain of hPMCA4b, is a target for caspase-3 cleavage. hPMCA4b is cut by caspase-3 downstream of Asp 1080 specifically upon the early phase of apoptosis, producing a single 120-kDa fragment (12). This fragment showed the same characteristics as the constitutively active ct120, which is truncated at Glu 1085; it was fully active in the absence of calmodulin.
In the previous paper (12) we also showed that mutation of Asp 1080 to Ala in the Asp 1077 -Glu-Ile-Asp 1080 caspase recognition sequence abolished the susceptibility of hPMCA4b to caspase-3. In the present paper we have analyzed the functional consequences of that mutation. When we tested the caspase-3-resistant Asp 1080 3 Ala mutant for Ca 2ϩ -transport activity, we found that it was almost fully active in the absence of calmodulin. This was the first evidence that the hinge region referred to above may be involved in the autoinhibitory interaction. Therefore, we have studied further the role of Asp 1080 in the regulation of hPMCA4b. We have mutated Asp 1080 to Ala, to Asn, and to Lys and found that these mutations not only activated the pump substantially but also increased its affinity for calmodulin. The increased affinity was because of an increased rate of activation by calmodulin. Our data indicate that Asp 1080 plays an essential role in autoinhibition and that the rate of calmodulin activation of these mutants of hPMCA4b depends on the degree of autoinhibition.

MATERIALS AND METHODS
Chemicals-Calmodulin was obtained from Sigma. LipofectAMINE and OPTI-MEM were obtained from Invitrogen. All other chemicals used for this study were of reagent grade.
Construction of the hPMCA4b Mutants-Mutation at Asp 1080 and Asp 1077 was done by the double PCR method as described previously (12). Briefly, the first PCR product was made by amplification of an hPMCA4b sequence using a primer containing the desired mutation and a primer including a unique restriction site (BamHI) already present in the sequence. The product of this PCR was then used as primer for a second round of PCR with the other primer including the second unique site (NsiI). The PCR products were cloned using the blunt PCR cloning kit from Invitrogen and sequenced by the Mayo Molecular Biology Core Facility. The NsiI-BamHI piece was cut out and placed into the wild type hPMCA4b in pSP72. The full-length SalI-KpnI piece was then cut out of pSP72 and ligated into expression vector pMM2.
Cell Culture and Transfection-COS-7 cells were grown at 37°C, 5% CO 2 in a humidified atmosphere in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 g/ml streptomycin, and 2 mM L-glutamine. COS-7 cells were transfected as described before (13) using the LipofectAMINE reagent based on the protocol recommended by the manufacturer (Invitrogen). Briefly, cells were grown in 175-cm 2 flasks to 70 -80% confluence. Cells were incubated at 37°C with DNA-LipofectAMINE complex (formed by incubating 8 g of DNA and 100 l of LipofectAMINE in 3.6 ml of serum-free OPTI-MEM medium for 30 min) in 14.5 ml of serum-free OPTI-MEM medium. After 5 h of incubation cells were supplemented with serum; 24 h later the incubation medium was replaced with fresh tissue culture medium, and cells were harvested after an additional 24 h.
Isolation of Microsomes from COS-7 Cells-Crude microsomal membranes from COS-7 cells were prepared as described (2) with the following modifications. Cells were washed with ice-cold phosphate-buffered saline solution, pH 7.4, then harvested in the same medium containing 0.1 mM phenylmethylsulfonyl fluoride, 6 g/ml aprotinin, 2.2 g/ml leupeptin, and 1 mM EGTA. After centrifugation, cells were resuspended in an ice-cold hypotonic solution containing 10 mM Tris-HCl, pH 7.4, 1 mM MgCl 2 , 0.5 mM EGTA, 4 g/ml aprotinin, 2 g/ml leupeptin, and 4 mM dithiothreitol. After lysis, homogenization, and centrifugation, the final pellet was resuspended in a solution of 10 mM Tris-HCl, pH 7.4, 0.25 M sucrose, 0.15 M KCl, 2 mM dithiothreitol, and 20 M CaCl 2 , and the suspension was stored in liquid N 2 .
Ca 2ϩ Transport Assay-Ca 2ϩ uptake by microsomal membrane vesicles was carried out as described previously (14,15) in a reaction mixture containing 25 mM TES-triethanolamine, pH 7.2, 100 mM KCl, 7 mM MgCl 2 , 100 M CaCl 2 (labeled with 45 Ca), 40 mM KH 2 PO 4 / K 2 HPO 4 , pH 7.2, 200 nM thapsigargin, 4 g/ml oligomycin, and enough EGTA to obtain the desired Ca 2ϩ concentration. Calmodulin was added as indicated in the figure legends. The free Ca 2ϩ is calculated as described previously (16,17). Microsomes of 20 g/ml concentration were added, and Ca 2ϩ uptake was initiated by the addition of 5 mM ATP. The reaction was terminated by rapid filtration of the microsomes using Millipore membrane filters (0.45-m pore size). Data were analyzed with GraphPad Prism (GraphPad Software Inc.).
Solubilization of PMCA for ATPase Assays-COS cell membrane preparations were solubilized and reconstituted as described (18). Briefly, COS cell membrane preparations containing 100 -200 g of protein were pelleted by centrifugation in a microfuge tube and resuspended in 80 l of solubilization buffer (60 mM TES-triethanolamine, pH 7.2, 240 mM KCl, 10 mM MgCl 2 , 400 M EGTA, 10 mM NaN 3 , 2 mM dithiothreitol, 1 mM ouabain, 8 g/ml oligomycin, 400 nM thapsigargin, 4 g/ml aprotinin, 1 g/ml leupeptin, and 0.5% Triton X-100) at 4°C. After 4 min, 320 l of dilution buffer (same as solubilization buffer except 0.5 mg/ml phosphatidylcholine replaced Triton X-100) was added to the tube and then 200 mg of Bio-Beads SM-2 were added to remove the Triton X-100. The tube was placed on a gel rocker for 1 h at 4°C. The bulk of the Bio-Beads were removed by centrifugation, and the residual beads were removed by filtration with a 0.45-M spin filter.
Pre-steady State Rate of Activation by Calmodulin-The assay medium contained 30 mM TES-triethanolamine, pH 7.2, 120 mM KCl, 5 mM MgCl 2 , 2.5 mM ATP, 5 mM NaN 3 , 1 mM dithiothreitol, 0.5 mM ouabain, 200 nM thapsigargin, 4 g/ml oligomycin, 2 g/ml aprotinin, 0.5 g/ml leupeptin, 200 M EGTA, enough CaCl 2 to obtain 0.5 M free Ca 2ϩ , Ϯ235 nM calmodulin, 0.2 mM 2-amino-6-mercapto-7-methyl purine ribonucleoside (MESG), and 1 units/ml of the coupled enzyme purine nucleoside phosphorylase. Phosphorolysis of MESG was monitored by an increase in absorbance at 360 nm at 37°C on a Beckman DU 70 spectrophotometer. Data were fitted to the following equation: where Y 0 is the absorbance at 360 nm at time zero, v 0 is the steady state slope in the absence of calmodulin, and (v 0 ϩ v c ) is the steady state slope in the presence of 235 nM calmodulin (18).
Inactivation by Calmodulin Removal-Experiments to measure the rate of inactivation upon calmodulin dissociation were conducted in the same assay medium as for calmodulin activation. The enzyme was allowed to reach steady state at 0.5 M free Ca 2ϩ and 235 nM calmodulin and then 10 M calmodulin binding peptide RRKWQKT-GHAVRAIGRLSS from chicken smooth muscle myosin light chain kinase (19) was added to the reaction. Data were fitted to the following equation (18):

RESULTS
We have shown previously (12) that mutation of the aspartate residues of the 1077 DEID 1080 caspase recognition sequence made hPMCA4b partially (Asp 1077 3 Ala) or completely (Asp 1080 3 Ala, and Asp 1077 3 Ala/Asp 1080 3 Ala) resistant to caspase cleavage. In this paper we have made additional mutants (Asp 1080 3 Asn and Asp 1080 3 Lys) and studied further the effect of mutations in the 1077 DEID 1080 sequence on the activity of the pump. The mutants made are shown in Fig. 1A. Fig. 1B shows that the mutants have the expected size and are expressed equally well in COS cells. ATP-dependent Ca 2ϩ uptake by COS cell membrane vesicles was measured at a saturating Ca 2ϩ concentration in the presence and absence of calmodulin. Fig. 2 shows that all Asp 1080 mutants had higher basal activity in the absence of calmodulin than the wild type. The Asp 1080 3 Ala and Asp 1080 3 Lys mutants were almost fully active in the absence of calmodulin. Although the Asp 1080 3 Asn mutant showed considerably lower basal activity than the alanine or lysine mutants, it still had about 2-3 times higher basal activity than the wild type enzyme. In contrast, the basal activity of the Asp 1077 3 Ala mutant did not differ substantially from the wild type and was between 20 and 30% of that of the maximum. The basal activity of the wild type hPMCA4b is typically between 15 and 20% of the maximal activity in the presence of calmodulin. Although surprising, these data pointed out that the sequence between transmembrane domain 10 and the calmodulin binding/autoinhibitory sequence can play an important role in autoinhibition and that Asp 1080 must be one of the key residues of that region that are involved in stabilizing the inhibited conformation of the enzyme in the resting state (i.e. in the absence of calmodulin).
The curves in Fig. 3 show the activity of the mutants as a function of Ca 2ϩ concentration. The activities were measured in both the presence and absence of calmodulin. In the presence of calmodulin the curves of all constructs were nearly identical as shown in Fig. 3B and indicated by the parameters shown in Table I. This suggests that mutation of Asp 1080 increased specifically the basal activity of the pump with no effect on the characteristics of the fully active form of the enzyme. In the absence of calmodulin the Asp 1080 mutants all had substantially higher maximal velocity and Ca 2ϩ affinity than the wild type (see Fig. 3A and Table I). At saturating Ca 2ϩ concentrations calmodulin increased the activity of the Asp 1080 3 Ala and Asp 1080 3 Lys mutants only by a factor of 1.2-1.3 whereas the Asp 1080 3 Asn mutant retained more autoinhibition. This latter mutant was stimulated by calmodulin about 2.5 times at saturating Ca 2ϩ concentrations. However, this is still much less than the 5-7-fold stimulation observed in the wild type. In all cases the affinity for Ca 2ϩ was further increased by calmodulin.
Then we tested the effect of mutation at Asp 1080 on the calmodulin affinity. We measured the Ca 2ϩ transport activity of the constructs as a function of calmodulin concentration at a relatively low (0.3 M) Ca 2ϩ concentration (Fig. 4). This low Ca 2ϩ concentration was chosen, because under these conditions the stimulation with calmodulin was greater than at saturating Ca 2ϩ concentrations. To reach steady state or near steady state conditions, the membranes were preincubated with calmodulin for 5-10 min at 37°C. Fig. 4 shows that each Asp 1080 mutant had higher affinity for calmodulin than the wild type. In contrast mutation at Asp 1077 did not affect calmodulin affinity. The K 0.5 s for calmodulin of the Asp 1080 3 Ala, Asp 1080 3 Lys, and Asp 1080 3 Asn mutants were 14 Ϯ 2, 17 Ϯ 3, and 29 Ϯ 6 nM, respectively, compared with 65 Ϯ 11 nM for the wild type hPMCA4b. It is apparent from Table I. that the increase in calmodulin affinity was greater for the mutants Asp 1080 3 Ala and Asp 1080 3 Lys, which had higher basal activity than for the mutant Asp 1080 3 Asn, which had lower basal activity.
An increase in steady state calmodulin affinity could be because of an increase in the rate of activation by calmodulin (the product of conformational opening of PMCA and calmodulin association) or a decrease in the rate of inactivation by calmodulin removal (the product of calmodulin dissociation and conformational closing of PMCA). We have shown previously that activation of the wild type hPMCA4b by 235 nM calmodulin in the presence of 500 nM free Ca 2ϩ has a half-time of about 40 to 60 s and that the rate of inactivation is much slower, with a half-time of about 20 min (18). To measure the rate of activation, wild type, Asp 1080 3 Ala or Asp 1080 3 Asn pumps were allowed to reach steady state ATPase activity in the absence of calmodulin at 500 nM Ca 2ϩ and then activation was initiated by the addition of 235 nM calmodulin. These Ca 2ϩ and calmodulin concentrations provide for a rate of activation that is slow enough to monitor by a conventional spectrophotometer yet yield a sufficient change in steady state activity. Fig. 5 shows a typical experiment with the Asp 1080 3 Ala mutant. The acti-  (DN)) increased the basal activity of PMCA. Ca 2ϩ transport by COS cell membrane vesicles was assayed at 8.1 M free Ca 2ϩ in the absence of calmodulin. Maximum Ca 2ϩ uptake was also determined for each construct at 235 nM calmodulin, and the activities shown were expressed as a percentage of this maximum. In the presence of calmodulin the activities of the constructs were nearly identical but varied from one expression to another between 3 and 6 nmol Ca 2ϩ /(mg of membrane protein, in min). The bars represent the means Ϯ S.E. of three independent determinations.

FIG. 1. Mutations at Asp 1077 and
Asp 1080 of PMCA4b. A, a scheme of PMCA and part of the amino acid sequence of the C-terminal regulatory region to show where mutations in the sequence were made. Calmodulin binding, high affinity calmodulin binding/autoinhibitory domain; DEID, caspase-3 consensus sequence. The caspase-3 cleavage site is marked by an arrow. The other arrows indicate where the ct120 and ct92 truncated mutants of hPMCA4b terminate. The star represents Asp 1080 on the scheme. B, immunoblot of microsomes prepared from COS cells transfected with plasmid encoding wild type hPMCA4b (wt) and its mutants. 0.5 g of membrane protein was applied on each lane of a 7.5% polyacrylamide gel. Immunoblots using anti-PMCA monoclonal antibody 5F10 are shown. vation rate constants were calculated from the change in the slopes using the equation described in the legend of Fig. 5. The values for k act for the Asp 1080 3 Asn and Asp 1080 3 Ala mutants were 2 and 3 times higher, respectively, than that of the wild type hPMCA4b (Table II).
Because the 2-3-fold increase in the rate of activation by calmodulin of both mutants fully accounted for the 2-4 times higher affinity for calmodulin observed in the steady state experiments we assumed that the mutation did not affect substantially the rate of inactivation by calmodulin removal. To test this assumption, the rate of inactivation was measured for one of the mutants. Inactivation measurements were performed by incubating wild type or mutant hPMCA4b with 500 nM free calcium and 235 nM calmodulin and allowing the reaction to reach a constant ATPase activity. Then a vast excess of calmodulin binding peptide from myosin light chain kinase was added to the reaction mixture (18). The myosin light chain kinase peptide has no direct effect on PMCA but will sequester any calmodulin that dissociates from PMCA. The inactivation rate constant (k inact ) was obtained by a fit to the equation for inactivation. Inactivation experiments were done only with the Asp 1080 3 Asn mutant, because its larger activation with calmodulin allowed an accurate determination of the inactivation rate constant. As expected, mutation at Asp 1080 did not affect k inact . The rate constants of the mutant and the wild type were nearly identical within the range of the variability of the assay system (Table II). Based on the k act and k inact constants, we calculated the K d s for the mutants and the wild type. When compared with the wild type, the K d s for the mutants were where V 0 is the activity in the absence of calmodulin, V max is the activity in the presence of saturating calmodulin, and v is the activity at each given calmodulin concentration. The data were fitted with a one-site binding equation using GraphPad Prism. The parameters given by the fit are shown in Table I  2-3-fold times lower, in good correlation with the steady state assays.

DISCUSSION
In this study we analyzed the role of a conserved aspartic acid residue (Asp 1080 ) between transmembrane domain 10 and the calmodulin binding/autoinhibitory domain of PMCAs in the regulation of the pump by calmodulin. We mutated this residue in hPMCA4b, which is one of the ubiquitously expressed iso-forms of PMCA, and the effect of the mutation was tested by kinetic analysis. Because Asp 1080 is highly conserved among PMCAs, our data should provide a more general information of how PMCAs are regulated by calmodulin.
In the absence of Ca 2ϩ -calmodulin, the pump is in an inhibited or "closed" conformation. In isoform hPMCA4b the closed conformation has very low activity. It has been demonstrated that the calmodulin binding domain itself is responsible for most of the inhibition (4). Experiments using synthetic peptides have revealed that in the inhibited state the calmodulin binding domain interacts with both cytoplasmic loops (5)(6)(7). This interaction makes the catalytic sites less accessible. Activation by Ca 2ϩ -calmodulin frees the catalytic sites from the autoinhibition and increases the apparent Ca 2ϩ affinity and maximum activity of the enzyme. Truncation of hPMCA4b at the C terminus has shown that residues downstream of the calmodulin binding domain between Ser 1113 and Asp 1157 also contribute to inhibition, because removal of these contacts caused partial activation of the pump (4).
Previous studies showed that mutations and truncations in the calmodulin binding domain (4,20), downstream inhibitory region (15), and catalytic core (21) all affected the basal activity in the absence of calmodulin. Here we have shown that mutation of a residue outside of the three regions described previously also causes such a change. We have demonstrated that mutation of a single aspartate residue upstream of the conventional calmodulin binding/autoinhibitory domain of hPMCA4b increased the basal activity (i.e. the activity in the absence of calmodulin) substantially but did not affect the overall catalytic function of the enzyme. Calmodulin was still able to increase the activity of the mutants, however, to a much smaller extent at saturating Ca 2ϩ concentrations 1.2-1.3 times in the case of the alanine and lysine mutants and about 2-2.5 times for the asparagine mutant as compared with a 5-7-times stimulation of the wild type. It is very unlikely that the higher basal activities of the mutants were because of a higher calmodulin content of the membrane, because 1) the microsomes were washed extensively with Ca 2ϩ chelators, and 2) the mutation did not affect the rate of calmodulin removal. All these data suggest that these mutants have their conformational equilibria shifted more toward the open, active conformation. The role of Asp 1080 appears to be rather specific, because mutation of another aspartate (Asp 1077 ) in close proximity to Asp 1080 did not have any effect.
The change in the basal activity was greatest when the acidic aspartate side chain was replaced by a non-polar alanine or a positively charged lysine side chain. These mutations affected the activity equally. The more conservative substitution of an amido group for the carboxyl group had less effect. These data indicate that the aspartate side chain plays an essential role in autoinhibition by stabilizing the autoinhibited closed conformation of the pump. Mutation at Asp 1080 destabilized this conformation and caused activation of the protein as each mutant showed an increased basal activity and affinity for Ca 2ϩ activation. Because no structural data are available for PMCAs, the nature of the interactions remains unclear. We   may hypothesize that electrostatic interactions are involved; however, the greater inhibition retained in the asparagine mutant suggests that the oxygen atoms of the carboxylate group may form hydrogen bonds that are only partially perturbed in the asparagine mutant.
In addition to the change in the basal activity, mutation at Asp 1080 increased the apparent affinity of the pump for calmodulin. The increase in affinity was because of an increased rate of activation by calmodulin. The rate of inactivation by calmodulin removal was not affected by the mutation. This change in calmodulin affinity was greatest for the mutants that had the highest basal activity (alanine and lysine). Thus, the increase in calmodulin affinity was proportional to the increase observed in the basal activity of the mutants.
A general property of calmodulin is that it is able to induce secondary structure in a peptide that is unstructured prior to binding (22,23). Thus, the Asp 1080 mutation, which is outside of the calmodulin binding domain, should not affect the structure of the final calmodulin-bound state of the pump. This is consistent with the observation that mutation at Asp 1080 did not affect the rate of inactivation by calmodulin removal, i.e. dissociation of calmodulin from the calmodulin binding domain. Rather mutation at this residue seems to have an indirect effect on the interaction of the calmodulin binding/autoinhibitory domain with the catalytic core. We suggest that calmodulin activates the Asp 1080 mutants faster because of a looser competing interaction of the calmodulin binding domain with the catalytic core of PMCA that is reflected in a higher basal activity. This appears to be a general property of PMCAs, because all mutations and truncations made so far that increased the basal activity of the pump (18,20,21) also increased the rate of activation by calmodulin.
It had been demonstrated that the free calmodulin binding peptide C28 representing the calmodulin binding domain of PMCA4b had a much higher affinity for calmodulin than intact hPMCA4b (3). More recent experiments 2 showed that the rate of activation by calmodulin of the truncated mutant ct120 with bound C28 peptide was much slower than binding of calmodulin to the C28 peptide in solution. This indicates that association of the calmodulin binding domain with the cytoplasmic loops slows down the reaction with calmodulin. Moreover, the rate of activation of the ct120-C28 complex was about three times faster than the rate of activation of ct92 in which the C28 sequence is tethered to the enzyme. This is consistent with our present finding, which shows that an activating mutation in the hinge region also increases the rate of activation by calmodulin three times.
In summary, our data suggest that Asp 1080 serves as a contact point in the connecting region or hinge upstream of the calmodulin binding domain that assists in orienting the calmodulin binding domain to the catalytic sites to form stable autoinhibited conformation of the enzyme. Other contact points could be within the downstream inhibitory region, because ct92 still has higher basal activity than the wild type protein. We also conclude that weaker autoinhibitory interactions result in an increased rate of activation by calmodulin.