Detailed characterization of the cooperative mechanism of Ca(2+) binding and catalytic activation in the Ca(2+) transport (SERCA) ATPase

Expression of heterologous SERCA1a ATPase in Cos-1 cells was optimized to yield levels that account for 10-15% of the microsomal protein, as revealed by protein staining on electrophoretic gels. This high level of expression significantly improved our characterization of mutants, including direct measurements of Ca(2+) binding by the ATPase in the absence of ATP, and measurements of various enzyme functions in the presence of ATP or P(i). Mutational analysis distinguished two groups of amino acids within the transmembrane domain: The first group includes Glu771 (M5), Thr799 (M6), Asp800 (M6), and Glu908 (M8), whose individual mutations totally inhibit binding of the two Ca(2+) required for activation of one ATPase molecule. The second group includes Glu309 (M4) and Asn796 (M6), whose individual or combined mutations inhibit binding of only one and the same Ca(2+). The effects of mutations of these amino acids were interpreted in the light of recent information on the ATPase high-resolution structure, explaining the mechanism of Ca(2+) binding and catalytic activation in terms of two cooperative sites. The Glu771, Thr799, and Asp800 side chains contribute prominently to site 1, together with less prominent contributions by Asn768 and Glu908. The Glu309, Asn796, and Asp800 side chains, as well as the Ala305 (and possibly Val304 and Ile307) carbonyl oxygen, contribute to site 2. Sequential binding begins with Ca(2+) occupancy of site 1, followed by transition to a conformation (E') sensitive to Ca(2+) inhibition of enzyme phosphorylation by P(i), but still unable to utilize ATP. The E' conformation accepts the second Ca(2+) on site 2, producing then a conformation (E' ') which is able to utilize ATP. Mutations of residues (Asp813 and Asp818) in the M6/M7 loop reduce Ca(2+) affinity and catalytic turnover, suggesting a strong influence of this loop on the correct positioning of the M6 helix. Mutation of Asp351 (at the catalytic site within the cytosolic domain) produces total inhibition of ATP utilization and enzyme phosphorylation by P(i), without a significant effect on Ca(2+) binding.     

Functional consequences of alterations to Thr247, Pro248, Glu340, Asp813, Arg819, and Arg822 at the interfaces between domain P, M3, and L6-7 of sarcoplasmic reticulum Ca2+-ATPase. Roles in Ca2+ interaction and phosphoenzyme processing

Point mutants with alterations to amino acid residues Thr(247), Pro(248), Glu(340), Asp(813), Arg(819), and Arg(822) of sarcoplasmic reticulum Ca(2+)-ATPase were analyzed by transient kinetic measurements. In the Ca(2+)-ATPase crystal structures, most of these residues participate in a hydrogen-bonding network between the phosphorylation domain (domain P), the third transmembrane helix (M3), and the cytoplasmic loop connecting the sixth and the seventh transmembrane helices (L6-7). In several of the mutants, a pronounced phosphorylation "overshoot" was observed upon reaction of the Ca(2+)-bound enzyme with ATP, because of accumulation of dephosphoenzyme at steady state. Mutations of Glu(340) and its partners, Thr(247) and Arg(822), in the bonding network markedly slowed the Ca(2+) binding transition (E2 --> E1 --> Ca(2)E1) as well as Ca(2+) dissociation from Ca(2+) site II back toward the cytosol but did not affect the apparent affinity for vanadate. These mutations may have caused a slowing, in both directions, of the conformational change associated directly with Ca(2+) interaction at Ca(2+) site II. Because mutation of Asp(813) inhibited the Ca(2+) binding transition, but not Ca(2+) dissociation, and increased the apparent affinity for vanadate, the effect on the Ca(2+) binding transition seems in this case to be exerted by slowing the E2 --> E1 conformational change. Because the rate was not significantly enhanced by a 10-fold increase of the Ca(2+) concentration, the slowing is not the consequence of reduced affinity of any pre-binding site for Ca(2+). Furthermore, the mutations interfered in specific ways with the phosphoenzyme processing steps of the transport cycle; the transition from ADP-sensitive phosphoenzyme to ADP-insensitive phosphoenzyme (Ca(2)E1P --> E2P) was accelerated by mutations perturbing the interactions mediated by Glu(340) and Asp(813) and inhibited by mutation of Pro(248), and mutations of Thr(247) induced charge-specific changes of the rate of dephosphorylation of E2P.     

Involvement of the cytoplasmic loop L6-7 in the entry mechanism for transport of Ca2+ through the sarcoplasmic reticulum Ca2+-ATPase.

We previously found that mutants of conserved aspartate residues of sarcoplasmic reticulum Ca(2+)-ATPase in the cytosolic loop, connecting transmembrane segments M6 and M7 (L6-7 loop), exhibit a strongly reduced sensitivity toward Ca(2+) activation of the transport process. In this study, yeast membranes, expressing wild type and mutant Ca(2+)-ATPases, were reacted with Cr small middle dotATP and tested for their ability to occlude (45)Ca(2+) by HPLC analysis, after cation resin and C(12)E(8) treatment. We found that the D813A/D818A mutant that displays markedly low calcium affinity was capable of occluding Ca(2+) to the same extent as wild type ATPase. Using NMR and mass spectrometry we have analyzed the conformational properties of the synthetic L6-7 loop and demonstrated the formation of specific 1:1 cation complexes of the peptide with calcium and lanthanum. All three aspartate Asp(813)/Asp(815)/Asp(818) were required to coordinate the trivalent lanthanide ion. Overall these observations suggest a dual function of the loop: in addition to mediating contact between the intramembranous Ca(2+)-binding sites and the cytosolic phosphorylation site (Zhang, Z., Lewis, D., Sumbilla, C., Inesi G., and Toyoshima, C. (2001) J. Biol. Chem. 276, 15232-15239), the L6-7 loop, in a preceding step, participates in the formation of an entrance port, before subsequent high affinity binding of Ca(2+) inside the membrane.     

Roles of conserved P domain residues and Mg2+ in ATP binding in the ground and Ca2+-activated states of sarcoplasmic reticulum Ca2+-ATPase

Residues in conserved motifs (625)TGD, (676)FARXXPXXK, and (701)TGDGVND in domain P of sarcoplasmic reticulum Ca(2+)-ATPase, as well as in motifs (601)DPPR and (359)NQR(/K)MSV in the hinge segments connecting domains N and P, were examined by mutagenesis to assess their roles in nucleotide and Mg(2+) binding and stabilization of the Ca(2+)-activated transition state for phosphoryl transfer. In the absence of Mg(2+), mutations removing the charges of domain P residues Asp(627), Lys(684), Asp(703), and Asp(707) increased the affinity for ATP and 2',3'-O-(2,4,6-trinitrophenyl)-8-azidoadenosine 5'-triphosphate. These mutations, as well as Gly(626)--> Ala, were inhibitory for ATP binding in the presence of Mg(2+) and for tight binding of the beta,gamma-bidentate chromium(III) complex of ATP. The hinge mutations had pronounced, but variable, effects on ATP binding only in the presence of Mg(2+). The data demonstrate an unfavorable electrostatic environment for binding of negatively charged nucleotide in domain P and show that Mg(2+) is required to anchor the phosphoryl group of ATP at the phosphorylation site. Mutants Gly(626) --> Ala, Lys(684) --> Met, Asp(703) --> Ala/Ser/Cys, and mutants with alteration to Asp(707) exhibited very slow or negligible phosphorylation, making it possible to measure ATP binding in the pseudo-transition state attained in the presence of both Mg(2+) and Ca(2+). Under these conditions, ATP binding was almost completely blocked in Gly(626) --> Ala and occurred with 12- and 7-fold reduced affinities in Asp(703) --> Ala and Asp(707) --> Cys, respectively, relative to the situation in the presence of Mg(2+) without Ca(2+), whereas in Lys(684) --> Met and Asp(707) --> Ser/Asn the affinity was enhanced 14- and 3-5-fold, respectively. Hence, Gly(626) and Asp(703) seem particularly critical for mediating entry into the transition state for phosphoryl transfer upon Ca(2+) binding at the transport sites.     

High-affinity Ca(2+) binding inhibits autoactivation of rat trypsinogen

The recent discovery that mutation Asn21 --> Ile in the human cationic trypsinogen (Tg) is associated with hereditary pancreatitis has brought into focus the functional role of amino acid 21 in mammalian Tgs. In the present paper, the effect of mutations Thr21 --> Asn and Thr21 --> Ile on the Ca(2+) dependence of zymogen activation was investigated, using the autolysis-resistant rat Tg mutant Arg117 --> His. In the absence of Ca(2+), rat Tg exhibited low but significant basal autoactivation, which was inhibited by micromolar concentrations of Ca(2+) (IC(50) 2.6 microM). Interestingly, basal autoactivation was diminished in both mutants, and no further inhibition by micromolar Ca(2+) was detectable. Millimolar Ca(2+) concentrations markedly and comparably stimulated autoactivation of wild-type and mutant zymogens (EC(50) 1.7-2.4 mM). The results indicate that rat Tg is subject to dual regulation by Ca(2+), allowing zymogen stabilization in a low-Ca(2+) environment and efficient activation in a high-Ca(2+) milieu.     

Mechanism of the Ca2+-induced enhancement of the intrinsic factor VIIa activity

The intrinsic activity of coagulation factor VIIa (FVIIa) is dependent on Ca(2+) binding to a loop (residues 210-220) in the protease domain. Structural analysis revealed that Ca(2+) may enhance the activity by attenuating electrostatic repulsion of Glu(296) and/or by facilitating interactions between the loop and Lys(161) in the N-terminal tail. In support of the first mechanism, the mutations E296V and D212N resulted in similar, about 2-fold, enhancements of the amidolytic activity. Moreover, mutation of the Lys(161)-interactive residue Asp(217) or Asp(219) to Ala reduced the amidolytic activity by 40-50%, whereas the K161A mutation resulted in 80% reduction. Hence one of these Asp residues in the Ca(2+)-binding loop appears to suffice for some residual interaction with Lys(161), whereas the more severe effect upon replacement of Lys(161) is due to abrogation of the interaction with the N-terminal tail. However, Ca(2+) attenuation of the repulsion between Asp(212) and Glu(296) keeps the activity above that of apoFVIIa. Altogether, our data suggest that repulsion involving Asp(212) in the Ca(2+)-binding loop suppresses FVIIa activity and that optimal activity requires a favorable interaction between the Ca(2+)-binding loop and the N-terminal tail. Crystal structures of tissue factor-bound FVIIa(D212N) and FVIIa(V158D/E296V/M298Q) revealed altered hydrogen bond networks, resembling those in factor Xa and thrombin, after introduction of the D212N and E296V mutations plausibly responsible for tethering the N-terminal tail to the activation domain. The charge repulsion between the Ca(2+)-binding loop and the activation domain appeared to be either relieved by charge removal and new hydrogen bonds (D212N) or abolished (E296V). We propose that Ca(2+) stimulates the intrinsic FVIIa activity by a combination of charge neutralization and loop stabilization.     

Site-directed mutagenesis of conserved charged amino acid residues in ClpB from Escherichia coli

ClpB is a member of a multichaperone system in Escherichia coli (with DnaK, DnaJ, and GrpE) that reactivates strongly aggregated proteins. The sequence of ClpB contains two ATP-binding domains, each containing Walker consensus motifs. The N- and C-terminal sequence regions of ClpB do not contain known functional motifs. In this study, we performed site-directed mutagenesis of selected charged residues within the Walker A motifs (Lys212 and Lys611) and the C-terminal region of ClpB (Asp797, Arg815, Arg819, and Glu826). We found that the mutations K212T, K611T, D797A, R815A, R819A, and E826A did not significantly affect the secondary structure of ClpB. The mutation of the N-terminal ATP-binding site (K212T), but not of the C-terminal ATP-binding site (K611T), and two mutations within the C-terminal domain (R815A and R819A) inhibited the self-association of ClpB in the absence of nucleotides. The defects in self-association of these mutants were also observed in the presence of ATP and ADP. The four mutants K212T, K611T, R815A, and R819A showed an inhibition of chaperone activity, which correlated with their low ATPase activity in the presence of casein. Our results indicate that positively charged amino acids that are located along the intersubunit interface (this includes Lys212 in the Walker A motif of the N-terminal ATP-binding domain as well as Arg815 and Arg819 in the C-terminal domain) participate in intersubunit salt bridges and stabilize the ClpB oligomer. Interestingly, we have identified a conserved residue within the C-terminal domain (Arg819) which does not participate directly in nucleotide binding but is essential for the chaperone activity of ClpB.     

CrATP-induced Ca2+ occlusion in mutants of the Ca(2+)-ATPase of sarcoplasmic reticulum.

Use of the nonphosphorylating beta,gamma-bidentate chromium(III) complex of ATP to induce a stable Ca(2+)-occluded form of the sarcoplasmic reticulum Ca(2+)-ATPase was combined with molecular sieve high performance liquid chromatography of detergent-solubilized protein to examine the ability of the Ca(2+)-ATPase mutants Gly-233-->Glu, Gly-233-->Val, Glu-309-->Gln, Gly-310-->Pro, Pro-312-->Ala, Ile-315-->Arg, Leu-319-->Arg, Asp-703-->Ala, Gly-770-->Ala, Glu-771-->Gln, Asp-800-->Asn, and Gly-801-->Val to occlude Ca2+. This provided a new approach to identification of amino acid residues involved in Ca2+ binding and in the closure of the gates to the Ca2+ binding pocket of the Ca(2+)-ATPase. The "phosphorylation-negative" mutant Asp-703-->Ala and mutants of ADP-sensitive phosphoenzyme intermediate type were fully capable of occluding Ca2+, as was the mutant Gly-770-->Ala. Mutants in which carboxylic acid-containing residues in the putative transmembrane segments had been substituted ("Ca(2+)-site mutants") and mutant Gly-801-->Val were unable to occlude either of the two calcium ions. In addition, the mutant Gly-310-->Pro, previously classified as ADP-insensitive phosphoenzyme intermediate type (Andersen, J.P., Vilsen, B., and MacLennan, D.H. (1992). J. Biol. Chem. 267, 2767-2774), was unable to occlude Ca2+, even though Ca(2+)-activated phosphorylation from MgATP took place in this mutant.     

Critical role of Val-304 in conformational transitions that allow Ca2+ occlusion and phosphoenzyme turnover in the Ca2+ transport ATPase

Site-directed mutations were produced in the distal segments of the Ca(2+)-ATPase (SERCA) transmembrane region. Mutations of Arg-290 (M3-M4 loop), Lys-958, and Thr-960 (M9 - M10 loop) had minor effects on ATPase activity and Ca(2+) transport. On the other hand, Val-304 (M4) mutations to Ile, Thr, Lys, Ala, or Glu inhibited transport by 90-95% while reducing ATP hydrolysis by 83% (Ile, Thr, and Lys), 56% (Ala), or 45% (Glu). Val-304 participates in Ca(2+) coordination with its main-chain carbonyl oxygen, and this function is not expected to be altered by mutations of its side chain. In fact, despite turnover inhibition, the Ca(2+) concentration dependence of residual ATPase activity remained unchanged in Val-304 mutants. However, the rates (but not the final levels) of phosphoenzyme formation, as well the rates of its hydrolytic cleavage, were reduced in proportion to the ATPase activity. Furthermore, with the Val-304 --> Glu mutant, which retained the highest residual ATPase activity, it was possible to show that occlusion of bound Ca(2+) was also impaired, thereby explaining the stronger inhibition of Ca(2+) transport relative to ATPase activity. The effects of Val-304 mutations on phosphoenzyme turnover are attributed to interference with mechanical links that couple movements of transmembrane segments and headpiece domains. The effects of thermal activation energy on reaction rates are thereby reduced. Furthermore, inadequate occlusion of bound Ca(2+) following utilization of ATP in Val-304 side-chain mutations is attributed to inadequate stabilization of the Glu-309 side chain and consequent defect of its gating function.     

alpha-Tropomyosin mutations Asp(175)Asn and Glu(180)Gly affect cardiac function in transgenic rats in different ways

To study the mechanisms by which missense mutations in alpha-tropomyosin cause familial hypertrophic cardiomyopathy, we generated transgenic rats overexpressing alpha-tropomyosin with one of two disease-causing mutations, Asp(175)Asn or Glu(180)Gly, and analyzed phenotypic changes at molecular, morphological, and physiological levels. The transgenic proteins were stably integrated into the sarcomere, as shown by immunohistochemistry using a human-specific anti-alpha-tropomyosin antibody, ARG1. In transgenic rats with either alpha-tropomyosin mutation, molecular markers of cardiac hypertrophy were induced. Ca(2+) sensitivity of cardiac skinned-fiber preparations from animals with mutation Asp(175)Asn, but not Glu(180)Gly, was decreased. Furthermore, elevated frequency and amplitude of spontaneous Ca(2+) waves were detected only in cardiomyocytes from animals with mutation Asp(175)Asn, suggesting an increase in intracellular Ca(2+) concentration compensating for the reduced Ca(2+) sensitivity of isometric force generation. Accordingly, in Langendorff-perfused heart preparations, myocardial contraction and relaxation were accelerated in animals with mutation Asp(175)Asn. The results allow us to propose a hypothesis of the pathogenetic changes caused by alpha-tropomyosin mutation Asp(175)Asn in familial hypertrophic cardiomyopathy on the basis of changes in Ca(2+) handling as a sensitive mechanism to compensate for alterations in sarcomeric structure.     

Fast kinetic analysis of conformational changes in mutants of the Ca(2+)-ATPase of sarcoplasmic reticulum

Rapid quench experiments at 25 degrees C were carried out on selected mutants of the sarco(endo)plasmic reticulum Ca(2+)-ATPase to assess the kinetics of the conformational changes of the dephosphoenzyme associated with ATP binding/phosphoryl transfer and the binding and dissociation of Ca(2+) at the cytoplasmically facing transport sites. The mutants Gly(233) --> Glu, Gly(233) --> Val, Pro(312) --> Ala, Leu(319) --> Arg, and Lys(684) --> Arg differed conspicuously with respect to the behavior of the dephosphoenzyme, although they were previously shown to display a common block of the transformation of the phosphoenzyme from an ADP-sensitive to an ADP-insensitive form. The maximum rate of the ATP binding/phosphoryl transfer reaction was reduced 3.6-fold in mutant Gly(233) --> Glu and more than 50-fold in mutant Lys(684) --> Arg, relative to wild type. In mutant Leu(319) --> Arg, the rate of the Ca(2+)-binding transition was reduced as much as 10-30-fold depending on the presence of ATP. In mutants Gly(233) --> Glu, Gly(233) --> Val, and Pro(312) --> Ala, the rate of the Ca(2+)-binding transition was increased at least 2-3-fold at acid pH but not significantly at neutral pH, suggesting a destabilization of the protonated form. The rate of Ca(2+) dissociation was reduced 12-fold in mutant Pro(312) --> Ala and 3.5-fold in Leu(319) --> Arg, and increased at least 4-fold in a mutant in which the putative Ca(2+) liganding residue Glu(309) was replaced by aspartate. The data support a model in which Pro(312) and Leu(319) are closely associated with the cation binding pocket, Gly(233) is part of a long-range signal transmission pathway between the ion-binding sites and the catalytic site, and Lys(684) is an essential catalytic residue that may function in the same way as its counterpart in the soluble hydrolases belonging to the haloacid dehalogenase superfamily.     

Importance of stalk segment S5 for intramolecular communication in the sarcoplasmic reticulum Ca2+-ATPase

Sixteen residues in stalk segment S5 of the Ca(2+)-ATPase of sarcoplasmic reticulum were studied by site-directed mutagenesis. The rate of the Ca(2+) binding transition, determined at 0 degrees C, was enhanced relative to wild type in mutants Ile(743) --> Ala, Val(747) --> Ala, Glu(748) --> Ala, Glu(749) --> Ala, Met(757) --> Gly, and Gln(759) --> Ala and reduced in mutants Asp(737) --> Ala, Asp(738) --> Ala, Ala(752) --> Leu, and Tyr(754) --> Ala. In mutant Arg(762) --> Ile, the rate of the Ca(2+) binding transition was wild type like at 0 degrees C, whereas it was 3.5-fold reduced relative to wild type at 25 degrees C. The rate of dephosphorylation of the ADP-insensitive phosphoenzyme was increased conspicuously in mutants Ile(743) --> Ala and Tyr(754) --> Ala (close to 20-fold in the absence of K(+)) and increased to a lesser extent in Asn(739) --> Ala, Glu(749) --> Ala, Gly(750) --> Ala, Ala(752) --> Gly, Met(757) --> Gly, and Arg(762) --> Ile, whereas it was reduced in mutants Asp(737) --> Ala, Val(744) --> Gly, Val(744) --> Ala, Val(747) --> Ala, and Ala(752) --> Leu. In mutants Ile(743) --> Ala, Tyr(754) --> Ala, and Arg(762) --> Ile, the apparent affinities for vanadate were enhanced 23-, 30-, and 18-fold, respectively, relative to wild type. The rate of Ca(2+) dissociation was 11-fold increased in Gly(750) --> Ala and 2-fold reduced in Val(747) --> Ala. Mutants with alterations to Arg(751) either were not expressed at a significant level or were completely nonfunctional. The findings show that S5 plays a crucial role in mediating communication between the Ca(2+) binding pocket and the catalytic domain and that Arg(751) is important for both structural and functional integrity of the enzyme.     

Importance of conserved alpha -subunit segment 709GDGVND for Mg2+ binding, phosphorylation, and energy transduction in Na,K-ATPase

The segment (708)TGDGVNDSPALKK(720) in the alpha-subunit P domain of Na,K-ATPase is highly conserved among cation pumps, but little is known about its role in binding of Mg(2+) or ATP and energy transduction. Here, 11 mutations of polar residues are expressed at reduced temperature in yeast with preserved capacities for high affinity binding of ouabain and ATP, whereas the Thr(708) --> Ser mutation and alterations of Asp(714) abolish all catalytic reactions. In mutations of Asp(710) and Asn(713), ATP affinity is preserved or increased, whereas Na,K-ATPase activity is severely reduced. Assay of phosphorylation from ATP in the presence of oligomycin shows that Asp(710) contributes to coordination of Mg(2+) during transfer of gamma-phosphate to Asp(369) in the high energy Mg.E(1)P[3Na] intermediate and that Asn(713) is involved in these processes. In contrast, Asp(710) and Asp(713) do not contribute to Mg(2+) binding in the E(2)P.ouabain complex. Transition to E(2)P thus involves a shift of Mg(2+) coordination away from Asp(710) and Asn(713), and the two residues become more important for hydrolysis of the acyl phosphate bond at Asp(369). The Asp(710) --> Ala mutation blocks interaction with vanadate, whereas Asn(713) --> Ala interferes with phosphorylation from P(i) of the E(2).ouabain complex, showing that the GDGVND segment is required for stabilization of the transition state and for the phosphorylation reaction. The Asp(710) --> Ala mutation also interferes with transmission of structural changes to the ouabain site and reduces the affinity for binding of Tl(+) 2- to 3-fold, suggesting a role in transmission of K(+) stimulation of phospho-enzyme hydrolysis from transmembrane segment 5 to the P domain.     

Ca2+ occlusion and gating function of Glu309 in the ADP-fluoroaluminate analog of the Ca2+-ATPase phosphoenzyme intermediate

In the absence of ATP the sarcoplasmic reticulum ATPase (SERCA) binds two Ca(2+) with high affinity. The two bound Ca(2+) rapidly undergo reverse dissociation upon addition of EGTA, but can be distinguished by isotopic exchange indicating fast exchange at a superficial site (site II), and retardation of exchange at a deeper site (site I) by occupancy of site II. Site II mutations that allow high affinity binding to site I, but only low affinity binding to site II, show that retardation of isotopic exchange requires higher Ca(2+) concentrations with the N796A mutant, and is not observed with the E309Q mutant even at millimolar Ca(2+). Fluoroaluminate forms a complex at the catalytic site yielding stable analogs of the phosphoenzyme intermediate, with properties similar to E2-P or E1-P.Ca(2). Mutational analysis indicates that Asp(351), Lys(352), Thr(353), Asp(703), Asn(706), Asp(707), Thr(625), and Lys(684) participate in stabilization of fluoroaluminate and Mg(2+) at the phosphorylation site. In the presence of fluoroaluminate and Ca(2+), ADP (or AMP-PCP) favors formation of a stable ADP.E1-P.Ca(2) analog. This produces strong occlusion of Ca(2+) bound to both sites (I and II), whereby dissociation occurs very slowly even following addition of EGTA. Occlusion by fluoraluminate and ADP is not observed with the E309Q mutant, suggesting a gating function of Glu(309) at the mouth of a binding cavity with a single path of entry. This phenomenon corresponds to the earliest step of the catalytic cycle following utilization of ATP. Experiments on limited proteolysis reveal that a long range conformational change, involving displacement of headpiece domains and transmembrane helices, plays a mechanistic role.     

Functional consequences of alterations to hydrophobic amino acids located at the M4S4 boundary of the Ca(2+)-ATPase of sarcoplasmic reticulum.

Site-specific mutagenesis was used to investigate the functional roles of amino acids in the relatively hydrophobic sequence Ile-Thr-Thr-Cys-Leu-Ala-320, located at the M4S4 boundary of the sarcomplasmic reticulum Ca(2+)-ATPase. Each of the residues was replaced with either a less hydrophogic, a polar, or a charged residue. Mutants Ile-315----Arg and Leu-319----Arg were devoid of any Ca2+ transport function or ATPase activity, while the mutant Thr-317----Asp retained about 5 and 7% of the wild-type Ca2+ transport and ATPase activities, respectively. These three mutants were able to form the ADP-sensitive phosphoenzyme intermediate (E1P) by reaction with ATP, but this intermediate decayed very slowly to the ADP-insensitive phosphoenzyme intermediate (E2P). In the mutants Ile-315----Arg and Leu-319----Arg, the level of E2P formed in the backward reaction with inorganic phosphate was extremely low, but hydrolysis of E2P occurred at a normal rate. These mutants, in addition, displayed a higher apparent affinity for Ca2+ than the wild-type enzyme. In the mutants Ile-315----Ser and Ile-315----Asp, the Ca2+ transport and ATPase activities were moderately reduced to 30-40% of the wild-type activities, but normal affinities for Ca2+, Pi, and ATP were retained, as was the low affinity modulatory effect of ATP. Mutation of Thr-316 to Asp, Thr-317 to Ala, Cys-318 to Ala and Ala-320 to Arg had little or no effect on Ca2+ transport or ATPase activities. Introduction of two negative and one positive charge by triple mutation of the Ile-Thr-Thr-317 sequence created a mutant enzyme that, although completely inactive, was inserted into the membrane, consistent with a location of these residues on the cytoplasmic side of the M4S4 interface. Our findings suggest that the amphipathic character of the S4 helix and/or the distribution of charges in S4 is important for the stability of the E2P intermediate.     

Interaction of nucleotides with Asp(351) and the conserved phosphorylation loop of sarcoplasmic reticulum Ca(2+)-ATPase.

The nucleotide binding properties of mutants with alterations to Asp(351) and four of the other residues in the conserved phosphorylation loop, (351)DKTGTLT(357), of sarcoplasmic reticulum Ca(2+)-ATPase were investigated using an assay based on the 2', 3'-O-(2,4,6-trinitrophenyl)-8-azidoadenosine triphosphate (TNP-8N(3)-ATP) photolabeling of Lys(492) and competition with ATP. In selected cases where the competition assay showed extremely high affinity, ATP binding was also measured by a direct filtration assay. At pH 8.5 in the absence of Ca(2+), mutations removing the negative charge of Asp(351) (D351N, D351A, and D351T) produced pumps that bound MgTNP-8N(3)-ATP and MgATP with affinities 20-156-fold higher than wild type (K(D) as low as 0.006 microM), whereas the affinity of mutant D351E was comparable with wild type. Mutations K352R, K352Q, T355A, and T357A lowered the affinity for MgATP and MgTNP-8N(3)-ATP 2-1000- and 1-6-fold, respectively, and mutation L356T completely prevented photolabeling of Lys(492). In the absence of Ca(2+), mutants D351N and D351A exhibited the highest nucleotide affinities in the presence of Mg(2+) and at alkaline pH (E1 state). The affinity of mutant D351A for MgATP was extraordinarily high in the presence of Ca(2+) (K(D) = 0.001 microM), suggesting a transition state like configuration at the active site under these conditions. The mutants with reduced ATP affinity, as well as mutants D351N and D351A, exhibited reduced or zero CrATP-induced Ca(2+) occlusion due to defective CrATP binding.     

Involvement of the L6-7 loop in SERCA1a Ca2+-ATPase activation by Ca2+ (or Sr2+) and ATP

Wild-type (WT) and the double mutant D813A,D818A (ADA) of the L6-7 loop of SERCA1a were expressed in yeast, purified, and reconstituted into lipids. This allowed us to functionally study these ATPases by both kinetic and spectroscopic means, and to solve previous discrepancies in the published literature about both experimental facts and interpretation concerning the role of this loop in P-type ATPases. We show that in a solubilized state, the ADA mutant experiences a dramatic decrease of its calcium-dependent ATPase activity. On the contrary, reconstituted in a lipid environment, it displays an almost unaltered maximal calcium-dependent ATPase activity at high (millimolar) ATP, with an apparent affinity for Ca(2+) altered only moderately (3-fold). In the absence of ATP, the true affinity of ADA for Ca(2+) is, however, more significantly reduced (20-30-fold) compared with WT, as judged from intrinsic (Trp) or extrinsic (fluorescence isothiocyanate) fluorescence experiments. At low ATP, transient kinetics experiments reveal an overshoot in the ADA phosphorylation level primarily arising from the slowing down of the transition between the nonphosphorylated "E2" and "Ca(2)E1" forms of ADA. At high ATP, this slowing down is only partially compensated for, as ADA turnover remains more sensitive to orthovanadate than WT turnover. ADA ATPase also proved to have a reduced affinity for ATP in studies performed under equilibrium conditions in the absence of Ca(2+), highlighting the long range interactions between L6-7 and the nucleotide-binding site. We propose that these mutations in L6-7 could affect protonation-dependent winding and unwinding events in the nearby M6 transmembrane segment.     

Functional role of "N" (nucleotide) and "P" (phosphorylation) domain interactions in the sarcoplasmic reticulum (SERCA) ATPase

Experimental perturbations of the nucleotide site in the N domain of the SR Ca2+ ATPase were produced by chemical derivatization of Lys492 or/and Lys515, mutation of Arg560 to Ala, or addition of inactive nucleotide analogue (TNP-AMP). Selective labeling of either Lys492 or Lys515 produces strong inhibition of ATPase activity and phosphoenzyme intermediate formation by utilization of ATP, while AcP utilization and reverse ATPase phosphorylation by Pi are much less affected. Cross-linking of the two residues with DIDS, however, drastically inhibits utilization of both ATP and AcP, as well as of formation of phosphoenzyme intermediate by utilization of ATP, or reverse phosphorylation by Pi. Mutation of Arg560 to Ala produces strong inhibition of ATPase activity and enzyme phosphorylation by ATP but has a much lower effect on enzyme phosphorylation by Pi. TNP-AMP increases the ATPase activity at low concentrations (0.1-0.3 microM), but inhibits ATP, AcP, and Pi utilization at higher concentration (1-10 microM). Cross-linking with DIDS and TNP-AMP binding inhibits formation of the transition state analogue with orthovanadate. It is concluded that in addition to the binding pocket delimited by Lys 492 and Lys515, Arg560 sustains an important and direct role in nucleotide substrate stabilization. Furthermore, the effects of DIDS and TNP-AMP suggest that approximation of N (nucleotide) and P (phosphorylation) domains is required not only for delivery of nucleotide substrate, but also to favor enzyme phosphorylation by nucleotide and nonnucleotide substrates, in the presence and in the absence of Ca2+. Domain separation is then enhanced by secondary nucleotide binding to the phosphoenzyme, thereby favoring its hydrolytic cleavage.     

Characterization of the iron- and 2-oxoglutarate-binding sites of human prolyl 4-hydroxylase.

Prolyl 4-hydroxylase (EC 1.14.11.2), an alpha2beta2 tetramer, catalyzes the formation of 4-hydroxyproline in collagens. We converted 16 residues in the human alpha subunit individually to other amino acids, and expressed the mutant polypeptides together with the wild-type beta subunit in insect cells. Asp414Ala and Asp414Asn inactivated the enzyme completely, whereas Asp414Glu increased the K(m) for Fe2+ 15-fold and that for 2-oxoglutarate 5-fold. His412Glu, His483Glu and His483Arg inactivated the tetramer completely, as did Lys493Ala and Lys493His, whereas Lys493Arg increased the K(m) for 2-oxoglutarate 15-fold. His501Arg, His501Lys, His501Asn and His501Gln reduced the enzyme activity by 85-95%; all these mutations increased the K(m) for 2-oxoglutarate 2- to 3-fold and enhanced the rate of uncoupled decarboxylation of 2-oxoglutarate as a percentage of the rate of the complete reaction up to 12-fold. These and other data indicate that His412, Asp414 and His483 provide the three ligands required for the binding of Fe2+ to a catalytic site, while Lys493 provides the residue required for binding of the C-5 carboxyl group of 2-oxoglutarate. His501 is an additional critical residue at the catalytic site, probably being involved in both the binding of the C-1 carboxyl group of 2-oxoglutarate and the decarboxylation of this cosubstrate.     

Hereditary pancreatitis-associated mutation asn(21) --> ile stabilizes rat trypsinogen in vitro.

Mutations Arg(117) --> His and Asn(21) --> Ile in human trypsinogen-I have been recently associated with hereditary pancreatitis (HP). The Arg(117) --> His substitution is believed to cause pancreatitis by stabilizing trypsin against autolytic degradation, while the mechanism of action of Asn(21) --> Ile has been unknown. In an effort to understand the effect(s) of this mutation, Thr(21) in the highly homologous rat trypsinogen-II was replaced with Asn or Ile, and the recombinant zymogens and their active trypsin forms were studied. Kinetic parameters of all three trypsins were comparable, and the active enzymes suffered autolysis at similar rates, indicating that neither catalytic properties nor proteolytic stability of trypsin are influenced by mutations at position 21. When incubated at pH 8.0, 37 degrees C, pure zymogens underwent autoactivation with concomitant trypsinolytic degradation in a Ca(2+)-dependent fashion. Thus, in the presence of 5 mM Ca(2+), autoactivation and digestion of the zymogens after Arg(117) and Lys(188) were observed, while in the presence of 1 mM EDTA autoactivation and cleavage at Lys(188) were reduced, and zymogenolysis at the Arg(117) site was enhanced. Overall rates of zymogen degradation in [Asn(21)]- and [Ile(21)]trypsinogens were higher in Ca(2+) than in EDTA, while [Thr(21)]trypsinogen demonstrated inverse characteristics. Remarkably, both in the presence and absence of Ca(2+), [Ile(21)]trypsinogen exhibited significantly higher stability against autoactivation and proteolysis than zymogens with Asn(21) or Thr(21). The observations suggest that autocatalytic trypsinogen degradation may be an important defense mechanism against excessive trypsin generation in the pancreas, and trypsinogen stabilization by the Asn(21) --> Ile mutation plays a role in the pathogenesis of HP.