Ordered and Cooperative Binding of Opposing Globular Domains of Calmodulin to the Plasma Membrane Ca-ATPase
2000; Elsevier BV; Volume: 275; Issue: 3 Linguagem: Inglês
10.1074/jbc.275.3.1731
ISSN1083-351X
Autores Tópico(s)Computational Drug Discovery Methods
ResumoWe have investigated the mechanisms of activation of the plasma membrane (PM) Ca-ATPase by calmodulin (CaM), which result in enhanced calcium transport rates and the maintenance of low intracellular calcium levels. We have isolated the amino- or carboxyl-terminal domains of CaM (i.e. CaMN or CaMC), permitting an identification of their relative specificity for binding to sites on either the PM Ca-ATPase or a peptide (C28W) corresponding to the CaM-binding sequence. We find that either CaMN or CaMC alone is capable of productive interactions with the PM Ca-ATPase that induces enzyme activation. There are, however, large differences in the affinity and specificity of binding between CaMN and CaMC and either C28W or the PM Ca-ATPase. The initial binding interaction between CaMC and the PM Ca-ATPase is highly specific, having approximately 10,000-fold greater affinity in comparison with CaMN. However, following the initial association of either CaMC or CaMN, there is a 300-fold enhancement in the affinity of CaMN for the secondary binding site. Thus, while CaMC binds with a high affinity to the two CaM-binding sites within the PM Ca-ATPase in a sequential manner, CaMN binds cooperatively with a lower affinity to both binding sites. These large differences in the binding affinities and specificities of the amino- and carboxyl-terminal domains ensure that CaM binding to the PM Ca-ATPase normally involves the formation of a specific complex in which the initial high affinity association of the carboxyl-terminal domain promotes the association of the amino-terminal domain necessary for enzyme activation. We have investigated the mechanisms of activation of the plasma membrane (PM) Ca-ATPase by calmodulin (CaM), which result in enhanced calcium transport rates and the maintenance of low intracellular calcium levels. We have isolated the amino- or carboxyl-terminal domains of CaM (i.e. CaMN or CaMC), permitting an identification of their relative specificity for binding to sites on either the PM Ca-ATPase or a peptide (C28W) corresponding to the CaM-binding sequence. We find that either CaMN or CaMC alone is capable of productive interactions with the PM Ca-ATPase that induces enzyme activation. There are, however, large differences in the affinity and specificity of binding between CaMN and CaMC and either C28W or the PM Ca-ATPase. The initial binding interaction between CaMC and the PM Ca-ATPase is highly specific, having approximately 10,000-fold greater affinity in comparison with CaMN. However, following the initial association of either CaMC or CaMN, there is a 300-fold enhancement in the affinity of CaMN for the secondary binding site. Thus, while CaMC binds with a high affinity to the two CaM-binding sites within the PM Ca-ATPase in a sequential manner, CaMN binds cooperatively with a lower affinity to both binding sites. These large differences in the binding affinities and specificities of the amino- and carboxyl-terminal domains ensure that CaM binding to the PM Ca-ATPase normally involves the formation of a specific complex in which the initial high affinity association of the carboxyl-terminal domain promotes the association of the amino-terminal domain necessary for enzyme activation. calmodulin N-terminal domain of calmodulin containing amino acids 1–77 N-terminal domain of calmodulin containing amino acids 1–75 C-terminal domain of calmodulin containing amino acids 78–148 plasma membrane a peptide identical to the CaM-binding sequence of the PM Ca-ATPase with the sequence LRRGQILWFRGLNRIQTQIRVVNAFRSS high performance liquid chromatography A range of diverse metabolic activities involved in intracellular signaling is mediated by calmodulin (CaM),1 which functions as the major calcium sensor in all eukaryotes. CaM binding to a range of different target enzymes, including the plasma membrane (PM) Ca-ATPase, has been suggested to result in an increase in enzymatic function as a result of decreased contact interactions between the autoinhibitory domain and catalytic domain elements that result in enhanced rates of substrate binding or utilization (1.Crivici A. Ikura M. Annu. Rev. Biophys. Biomol. Struct. 1995; 24: 85-116Crossref PubMed Scopus (693) Google Scholar). CaM binding involves two globular domains, which are connected by an exposed α-helical element often referred to as the central helix (2.Babu Y.S. Bugg C.E. Cook W.J. J. Mol. Biol. 1988; 204: 191-204Crossref PubMed Scopus (972) Google Scholar, 3.Chattopadhyaya R. Meador W.E. Means A.R. Quiocho F.A. J. Mol. Biol. 1992; 228: 1177-1192Crossref PubMed Scopus (616) Google Scholar). Upon calcium binding, the reorientation of α-helices function to expose hydrophobic binding sites within each domain element in CaM that are surrounded by charged amino acids that lead to complex formation and activation of numerous target proteins with little sequence homology (4.Kuboniwa H. Tjandra N. Grzesiek S. Ren H. Klee C.B. Bax A. Nat. Struct. Biol. 1995; 2: 768-776Crossref PubMed Scopus (613) Google Scholar, 5.Zhang M. Tanaka T. Ikura M. Nat. Struct. Biol. 1995; 2: 758-767Crossref PubMed Scopus (645) Google Scholar, 6.Tjandra N. Bax A. Crivici A. Ikura M. Carafoli E. Klee C. Calcium in Cellular Regulation. Oxford University Press, Oxford1999: 152-170Google Scholar). Backbone folds of the amino- and carboxyl-terminal domains in CaM are structurally similar, and in many instances either domain has been shown to partially activate a range of different target proteins to varying extents (7.Newton D.L. Oldewurtel M.D. Krinks M.H. Shiloach J. Klee C.B. J. Biol. Chem. 1984; 259: 4419-4426Abstract Full Text PDF PubMed Google Scholar, 8.Newton D.L. Klee C. Woodgett J. Cohen P. Biochim. Biophys. Acta. 1985; 845: 533-539Crossref PubMed Scopus (35) Google Scholar, 9.Persechini A. McMillan K. Leakey P. J. Biol. Chem. 1994; 269: 16148-16154Abstract Full Text PDF PubMed Google Scholar). However, despite the structural homology of the individual CaM binding domains, target protein activation appears to normally involve the specific association of the individual domain elements with specific sequences within the CaM-binding sequence of individual target proteins (7.Newton D.L. Oldewurtel M.D. Krinks M.H. Shiloach J. Klee C.B. J. Biol. Chem. 1984; 259: 4419-4426Abstract Full Text PDF PubMed Google Scholar, 9.Persechini A. McMillan K. Leakey P. J. Biol. Chem. 1994; 269: 16148-16154Abstract Full Text PDF PubMed Google Scholar, 10.Meador W.E. Means A.R. Quiocho F.A. Science. 1992; 257: 1251-1254Crossref PubMed Scopus (939) Google Scholar, 11.Meador W.E. Means A.R. Quiocho F.A. Science. 1993; 262: 1718-1721Crossref PubMed Scopus (611) Google Scholar, 12.Ikura M. Clore G.M. Gronenborn A., M. Zhu G. Klee C.B. Bax A. Science. 1992; 256: 632-638Crossref PubMed Scopus (1176) Google Scholar, 13.Chapman E.R. Alexander K. Vorherr T. Carafoli E. Storm D.R. Biochemistry. 1992; 31: 12819-12825Crossref PubMed Scopus (57) Google Scholar, 14.Bayley P.M. Findlay W.A. Martin S.R. Protein Sci. 1996; 5: 1215-1228Crossref PubMed Scopus (142) Google Scholar, 15.Barth A. Martin S.R. Bayley P.M. J. Biol. Chem. 1998; 273: 2174-2183Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). In the case of the PM Ca-ATPase, the carboxyl-terminal domain of CaM has been suggested to be essential for enzyme activation, whereas its amino-terminal domain has been suggested to lack the ability to activate the PM Ca-ATPase without prior association of the carboxyl-terminal domain (16.Guerini D. Krebs J. Carafoli E. J. Biol. Chem. 1984; 259: 15172-15177Abstract Full Text PDF PubMed Google Scholar, 17.Elshorst B. Hennig M. Försterling H. Diener A. Maurer M. Schulte P. Chwalbe H. Friesinger C. Krebs J. Schmid H. Vorherr T. Carafoli E. Biochemistry. 1999; 38: 12320-12332Crossref PubMed Scopus (183) Google Scholar). Thus, differences in the interactions between the amino- and carboxyl-terminal domains of CaM and their corresponding binding sites within target proteins have been suggested to play essential roles in target protein activation. In this respect, previous measurements have demonstrated that the binding preferences of individual CaM-binding domains for sites within the CaM-binding sequence of skeletal myosin light chain kinase are relatively small (i.e. less than 1 kcal/mol), suggesting the possibility of multiple conformations of bound CaM that could function to regulate the extent of target protein activation observed in the presence of saturating CaM concentrations (9.Persechini A. McMillan K. Leakey P. J. Biol. Chem. 1994; 269: 16148-16154Abstract Full Text PDF PubMed Google Scholar, 15.Barth A. Martin S.R. Bayley P.M. J. Biol. Chem. 1998; 273: 2174-2183Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). These observations suggest that differences in the maximal extent of enzyme activation for the PM Ca-ATPase and other target proteins observed in the presence of saturating CaM concentrations following a range of different post-translational modifications (e.g. phosphorylation or methionine oxidation), site-directed deletions, and substitutions of specific amino acids may all involve alterations in the binding mechanism between the opposing globular domains in CaM and target proteins (7.Newton D.L. Oldewurtel M.D. Krinks M.H. Shiloach J. Klee C.B. J. Biol. Chem. 1984; 259: 4419-4426Abstract Full Text PDF PubMed Google Scholar, 18.Vorherr T. James P. Krebs J. Enyedi A. McCormick D.J. Penniston J.T. Carafoli E. Biochemistry. 1990; 29: 355-365Crossref PubMed Scopus (111) Google Scholar, 19.Quadroni M. L'Hostis E.L. Corti C. Myagkikh I. Durussel I. Cox J. James P. Carafoli E. Biochemistry. 1998; 37: 6523-6532Crossref PubMed Scopus (50) Google Scholar, 20.Zhang M. Li M. Wang J.H. Vogel H.J. J. Biol. Chem. 1994; 269: 15546-15552Abstract Full Text PDF PubMed Google Scholar, 21.Yao Y. Yin D. Jas G. Kuczera K. Williams T.D. Schöneich C. Squier T.C. Biochemistry. 1996; 35: 2767-2787Crossref PubMed Scopus (127) Google Scholar, 22.Persechini A. Gansz K.J. Paresi R.J. J. Biol. Chem. 1996; 271: 19279-19282Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 23.Chin D. Means A.R. J. Biol. Chem. 1996; 271: 30465-30471Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 24.Chin D. Soan D.J. Quiocho F.A. Means A.R. J. Biol. Chem. 1997; 272: 5510-5513Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 25.Chin D. Winkler K.E. Means A.R. J. Biol. Chem. 1997; 272: 31235-31240Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 26.Edwards R.A. Walsh M.P. Sutherland C. Vogel H.J. Biochem. J. 1998; 331: 149-152Crossref PubMed Scopus (36) Google Scholar, 27.Gao J. Yin D. Yao Y. Williams T.D. Squier T.C. Biochemistry. 1998; 37: 9536-9548Crossref PubMed Scopus (107) Google Scholar, 28.Yuan T. Vogel H.J. Protein Sci. 1999; 8: 113-121Crossref PubMed Scopus (32) Google Scholar). It is therefore of interest to identify the binding mechanisms of CaM that normally lead to enzymatic activation of the PM Ca-ATPase. To accomplish this, we have cloned and expressed the amino-terminal domain of CaM and isolated the carboxyl-terminal domain following trypsin digestion and HPLC purification, permitting us to determine the binding specificities of the individual domains of CaM for the CaM-binding sites within the PM Ca-ATPase and the abilities of the individual CaM domains to induce enzyme activation. These measurements take advantage of the fact that the binding affinity and conformation of CaM bound to either a peptide corresponding to the CaM-binding sequence of the PM Ca-ATPase (i.e. C28W) or to the entire PM Ca-ATPase are virtually identical (13.Chapman E.R. Alexander K. Vorherr T. Carafoli E. Storm D.R. Biochemistry. 1992; 31: 12819-12825Crossref PubMed Scopus (57) Google Scholar, 18.Vorherr T. James P. Krebs J. Enyedi A. McCormick D.J. Penniston J.T. Carafoli E. Biochemistry. 1990; 29: 355-365Crossref PubMed Scopus (111) Google Scholar, 29.Yao Y. Gao J. Squier T.C. Biochemistry. 1996; 35: 12015-12028Crossref PubMed Scopus (24) Google Scholar). We find that while occupancy of the two binding sites on the PM Ca-ATPase with either CaM domain results in complete enzyme activation, there are large differences in the affinities and specificities of the carboxyl- and amino-terminal domains for the sites within the CaM-binding sequence of the PM Ca-ATPase. Thus, while the carboxyl-terminal domain binds sequentially and independently to the two binding sites, the amino-terminal domain binds in a cooperative manner to both sites. The substantially higher affinity of the carboxyl-terminal domain relative to the amino-terminal domain suggests that activation of the PM Ca-ATPase normally involves the initial association between the carboxyl-terminal domain of CaM to a single site within the CaM-binding sequence of the PM Ca-ATPase, followed by subsequent association with the amino-terminal domain. Trypsin and soybean trypsin inhibitor (STI) were from Worthington. All other chemicals were obtained from Sigma and were of the purest grade commercially available. The peptide C28W, corresponding to the CaM-binding sequence of the PM Ca-ATPase (LRRGQILWFRGLNRIQTQIRVVNAFRSS), was synthesized and purified by Quality Control Co. (Hopkinton, MA). The entire vertebrate CaM or a CaM fragment (CaMN) corresponding to first 77 amino acids in CaM was overexpressed in Escherichia coli JM109 (DE3) cells and purified using phenyl-Sepharose CL-4B (Amersham Pharmacia Biotech) chromatography. A fragment corresponding to amino acids 78–148 (CaMC) was obtained following trypsin digestion of CaM. In all cases, CaM or its individual domain elements were purified using weak anion exchange HPLC as described previously (30.Sun H. Yin D. Squier T.C. Biochemistry. 1999; 38: 12266-12279Crossref PubMed Scopus (46) Google Scholar). Erythrocyte ghost membranes containing the PM Ca-ATPase were purified as described previously (21.Yao Y. Yin D. Jas G. Kuczera K. Williams T.D. Schöneich C. Squier T.C. Biochemistry. 1996; 35: 2767-2787Crossref PubMed Scopus (127) Google Scholar, 31.Niggli V. Penniston J.T. Carafoli E. J. Biol. Chem. 1979; 254: 9955-9958Abstract Full Text PDF PubMed Google Scholar). The CaM-dependent ATPase activity associated with the PM Ca-ATPase was determined by measuring phosphate release, essentially as described previously (21.Yao Y. Yin D. Jas G. Kuczera K. Williams T.D. Schöneich C. Squier T.C. Biochemistry. 1996; 35: 2767-2787Crossref PubMed Scopus (127) Google Scholar, 32.Lanzetta P.A. Alverez L.J. Reinsch P.S. Candia O. Anal. Biochem. 1979; 100: 95-97Crossref PubMed Scopus (1810) Google Scholar). The ghost membrane protein concentration was determined by the Biuret method (33.Gornal A. Bardawill C. David M. J. Biol. Chem. 1949; 177: 751-766Abstract Full Text PDF PubMed Google Scholar), using bovine serum albumin as the standard. CaM concentration was determined using the Micro-BCA assay (Pierce), where a stock solution of desalted CaM was used as a protein standard (ε277 = 3029 m–1cm–1) (7.Newton D.L. Oldewurtel M.D. Krinks M.H. Shiloach J. Klee C.B. J. Biol. Chem. 1984; 259: 4419-4426Abstract Full Text PDF PubMed Google Scholar). ATPase activity was measured at 37 °C in a solution containing approximately 16 nm Ca-ATPase (i.e. 0.4 mg ml−1 porcine erythrocyte ghost membranes) in 100 mm HEPES (pH 7.5), 0.1 m KCl, 5 mm MgCl2, 0.1 mm EGTA, 0.44 mm CaCl2, 5 mm ATP, and 4 μm A23187. The free calcium concentration was calculated to be 100 μm (34.Fabiato A. Methods Enzymol. 1988; 157: 378-417Crossref PubMed Scopus (973) Google Scholar). Major tryptic fragments of CaM corresponding to amino acids 1–75 (CaMN′) and 78–148 (CaMC) were purified using HPLC following proteolytic digestion, essentially as described previously (7.Newton D.L. Oldewurtel M.D. Krinks M.H. Shiloach J. Klee C.B. J. Biol. Chem. 1984; 259: 4419-4426Abstract Full Text PDF PubMed Google Scholar, 9.Persechini A. McMillan K. Leakey P. J. Biol. Chem. 1994; 269: 16148-16154Abstract Full Text PDF PubMed Google Scholar). Briefly, 0.5 mg of CaM in 0.05m NaCl, 40 mm NH4HCO3, and 1 mm CaCl2 was incubated with 20 μg ml−1 trypsin at 30 °C for 60 min. The reaction was quenched by adding 100 μg mg−1 soybean trypsin inhibitor, and the digest was directly loaded onto a J. T. Baker (Phillipsburg, NJ) weak anion exchange HPLC column (WP PEI resin) equilibrated with 25 mm Tris (pH 7.0) (buffer A) at room temperature. The gradient was developed between buffer A and buffer B (25 mm Tris (pH 7.0), 1 m(NH4)2SO4). Buffer A was decreased in 6 min from 99 to 88%, in 24 min from 88 to 56%, and in 2 min from 56 to 10%. The fractions of soybean trypsin inhibitor and trypsin were identified by loading authentic standards separately injected onto the column. Other fractions were collected and dialyzed exhaustively against 10 mm NH4HCO3, lyophilized, and identified using electrospray ionization mass spectrometry essentially as described previously (35.Gao J. Yin D.H. Yao Y. Sun H. Qin Z. Schöneich C. Williams T.D. Squier T.C. Biophys. J. 1998; 74: 1115-1134Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Final protein concentrations were determined using published extinction coefficients, where ε277(CaM) = 3029 m–1cm–1, ε259(CaMN) = 1680m–1 cm–1, and ε276(CaMC) = 3400 m–1cm–1 (7.Newton D.L. Oldewurtel M.D. Krinks M.H. Shiloach J. Klee C.B. J. Biol. Chem. 1984; 259: 4419-4426Abstract Full Text PDF PubMed Google Scholar). Steady-state fluorescence spectra of 3 μm C28W in the presence of variable CaM concentrations in 0.1 m HEPES (pH 7.5), 0.1m KCl, and 0.5 mm CaCl2 (buffer C) were measured using a Fluoro Max-2 spectrofluorometer (Jobin Yvon Spex; Edison, NJ) equipped with a xenon lamp. Excitation was at 297 nm using 5-nm slit widths. When appropriate, fluorescence intensity changes associated with CaM binding were detected at 370 nm subsequent to a KV-320 long pass filter. Changes in the solvent accessibility of Trp8 in C28W were assessed through collisional quenching, where variable amounts of acrylamide (8 m stock) were added in microliter increments to 3 μm C28W in the presence of variable concentrations of CaM, CaMN, or CaMC in buffer C (V t = 2 ml). The concentration of CaM free in solution was obtained from the following relationship, [CaM] free=[CaM] total−(V−Vmin)(Vmax−Vmin)×[CaM] maxEquation 1 where V max is the maximal calmodulin-dependent ATPase activity, V is the observed ATPase activity at a defined concentration of CaM, [CaM]free is the concentration of CaM free in solution, [CaM]total is the total concentration of CaM added to the solution, and [CaM]max is the total binding capacity of the erythrocyte ghosts for CaM, which was estimated to correspond to 40 pmol of CaM bound per mg of porcine erythrocyte ghost (21.Yao Y. Yin D. Jas G. Kuczera K. Williams T.D. Schöneich C. Squier T.C. Biochemistry. 1996; 35: 2767-2787Crossref PubMed Scopus (127) Google Scholar). The CaM-dependent activation of the PM Ca-ATPase by CaM assumes an ordered binding mechanism of CaM with the CaM-binding sites (A-B) of the Ca-ATPase (see Scheme I) and is described by the equation, Y=[PMCA] free×K2×[CaM] free2(1+K1×[CaM] free+K2×[CaM] free2)×span+minimumEquation 2 where [PMCA] free=−1+(1+4×K2×[CaM] free2×[PMCA] total) 1/22×K2×[CaM] free2Equation 3 Span is the maximal CaM-dependent enzymatic activity in the presence of saturating CaM, minimum represents the CaM-independent enzymatic activity, and Y represents the PM Ca-ATPase activity resulting from the association of both CaM domains with the Ca-ATPase. [CaM]free is the concentration of CaM not bound to the PM Ca-ATPase. [PMCA]total and [PMCA]free, respectively, represent the total concentrations of the Ca-ATPase and the concentration of the Ca-ATPase with no CaM bound. K 1 corresponds to the equilibrium binding constant k 1 of the carboxyl-terminal domain of CaM to the Ca-ATPase, andK 2 represents the product of the association constants of both domains (i.e. k 12 × k 3b), where k 3b is the apparent association constant for the amino-terminal domain of CaM with the Ca-ATPase. An estimate of the actual association constant (k 3) for the amino-terminal domain can be obtained by taking into account the effective concentration of the amino-terminal domain around the binding site following association of the carboxyl-terminal domain, which equals the following. k3=k3b×b/cEquation 4 The bulk concentration (b) of the amino-terminal domain is assumed to correspond to 1/k 1(i.e. 10 nm). The concentration of the amino-terminal domain (c) is approximately 1.4 mm and is calculated as follows. c=1Na×VEquation 5 N a is Avogadro's number, and V is the volume available to the amino-terminal domain, where the radius corresponds to the overall length of CaM (approximately 100 Å after association with the carboxyl-terminal domain) (36.Kruegar J.K. Bishop N.A. Blumenthal D.K. Zhi G. Beckingham K. Stull J.T. Trewhella J. Biochemistry. 1998; 37: 17810-17817Crossref PubMed Scopus (34) Google Scholar). The activation of the PM Ca-ATPase by CaMC can be described by the following equation. Y=Kv×[CaMC] free21+KU[CaMC] free+KV[CaMC] free2×span+minimumEquation 6 In this case, K U and K V, respectively, correspond to k 1 andk 1 × k 2 in Scheme I and represent the intrinsic equilibrium constants associated with ligand binding to the two classes of CaM binding sites. If one assumes thatk 1 ≫ k 2, then site A is essentially completely filled prior to the titration of site B (see Scheme I), which is associated with the activation of the Ca-ATPase. Under these latter conditions, the binding affinity of site B can be described by the simple binding equation, Y=k2×[CaMC] free1+k2[CaMC] free×span+minimumEquation 7 The activation of the PM Ca-ATPase by CaMN can be described by the following equation derived from Scheme II. Y=KQ×[CaMN] free21+KP[CaMN] free+KQ[CaMN] free2×span+minimumEquation 8 In this case, K P corresponds tok 1 + k 2, andK Q corresponds to k 1 ×k 3 or k 2 ×k 4 as defined in Scheme II. Likewise, activation of the PMCa-ATPase by an equimolar mixture of CaMN and CaMC can be described by the equation, Y=Equation 9 KB×[CaMC] free2+KC×[CaMC] free×[CaMN] free1+KA×[CaMC] free+KB×[CaMC] free2+KC×[CaMC] free×[CaMN] free×span+minimumwhere K A equals k 1,K B equals k 1 ×k 2, and K C equalsk 1 × k 3. [CaMN]free and [CaMC]free are the unbound concentration of the N-terminal and C-terminal domains of CaM, respectively. CaM contains two binding sites that associate with target proteins located in the amino- and carboxyl-terminal domain (1.Crivici A. Ikura M. Annu. Rev. Biophys. Biomol. Struct. 1995; 24: 85-116Crossref PubMed Scopus (693) Google Scholar, 6.Tjandra N. Bax A. Crivici A. Ikura M. Carafoli E. Klee C. Calcium in Cellular Regulation. Oxford University Press, Oxford1999: 152-170Google Scholar), respectively. Following mild trypsin digestion, it is possible to isolate an amino-terminal fragment containing amino acids Ala1–Lys75 (CaMN′) and Asp78 -Lys148 (CaMC) using weak anion exchange HPLC. The identity of these fragments was determined using electrospray ionization mass spectrometry; the observed average molecular masses of CaMN′ and CaMC are 8316 ± 1 and 8147 ± 1 Da, respectively, in close agreement with expected monoisotopic molecular masses of 8316.2 and 8147.8 Da for these CaM fragments. These results suggest that previous measurements in which the amino-terminal domain of CaM was reported to be unable to activate the PM Ca-ATPase probably involved CaMN′ (16.Guerini D. Krebs J. Carafoli E. J. Biol. Chem. 1984; 259: 15172-15177Abstract Full Text PDF PubMed Google Scholar). However, in case Met76 and Lys77 located in the linker region between the amino- and carboxyl-terminal domains might play a role in facilitating target protein binding, we have cloned and expressed the amino-terminal domain of CaM containing amino acids Ala1–Lys77(CaMN) in E. coli. CaMN was purified using a phenyl-Sepharose hydrophobic column essentially as described for intact CaM (30.Sun H. Yin D. Squier T.C. Biochemistry. 1999; 38: 12266-12279Crossref PubMed Scopus (46) Google Scholar), and was subsequently purified using weak anion exchange HPLC. Using electrospray ionization mass spectrometry, we found that CaMN has an observed average molecular mass of 8576 ± 1 Da, in close agreement with the expected average molecular mass of 8575.6 Da. CaM activates the PM Ca-ATPase; the amount of CaM necessary for half-maximal activation is 3.9 ± 0.4 nm (Fig.1). A similar level of enzymatic activation of the PM Ca-ATPase is observed in the presence of saturating concentrations of either CaMN or CaMC. Thus, in contrast to previous reports where CaMN′ was found to lack the ability to activate the Ca-ATPase (16.Guerini D. Krebs J. Carafoli E. J. Biol. Chem. 1984; 259: 15172-15177Abstract Full Text PDF PubMed Google Scholar), we found that the inclusion of two additional amino acids in CaMN provided an amino-terminal domain fully capable of activation of the PM Ca-ATPase. However, the apparent affinities of these isolated domains are dramatically lower; half-maximal activation occurs at 6 ± 1 μm CaMN and 1.7 ± 0.2 μm CaMC (Table I). The 1500- and 400-fold higher concentrations, respectively, of CaMN or CaMC necessary for enzyme activation indicate that (i) individual CaM binding domains are not equivalent and may preferentially interact with one of the two binding sites on the CaM-binding sequence and (ii) that the central helix, which functions to join the individual domains of CaM, facilitates specific association of individual domains with their target sites on the Ca-ATPase. The role of the central helix and possible differences in the binding specificity of individual CaM domains was further assessed by adding an equimolar mixture of CaMN and CaMC, which results in activation of the PM Ca-ATPase, with a half-maximal activation of enzymatic activity occurring with a concentration of both fragments of 0.35 ± 0.02 μm(Fig. 1). The requirement of 100-fold higher concentrations of CaMN and CaMC for enzyme activation relative to that required for intact CaM indicates that the central helix facilitates binding and enzyme activation, in agreement with previous observations (37.Persechini A. Kretsinger R.H. J. Biol. Chem. 1988; 263: 12175-12178Abstract Full Text PDF PubMed Google Scholar, 38.Persechini A. Jarrett H.W. Kosk-Kosicka D. Krinks M.H. Lee H.G. Biochim. Biophys. Acta. 1993; 1163: 309-314Crossref PubMed Scopus (12) Google Scholar, 39.Persechini A. Gansz K.J. Paresi R.J. Biochemistry. 1996; 35: 224-228Crossref PubMed Scopus (46) Google Scholar). The approximately 4-fold lower concentration of CaMC necessary for half-maximal activation using a combination of CaMC and CaMN relative to that observed using CaMC alone is consistent with previous suggestions that the carboxyl- and amino-terminal domains of CaM may have different specificities for the two CaM-binding sites within the CaM-binding sequence of the PM Ca-ATPase (13.Chapman E.R. Alexander K. Vorherr T. Carafoli E. Storm D.R. Biochemistry. 1992; 31: 12819-12825Crossref PubMed Scopus (57) Google Scholar). To further understand possible differences in the individual domains of CaM in the activation mechanism of the Ca-ATPase, additional measurements of possible differences in binding specificity are necessary.Table IEquilibrium binding affinities between CaM domains and the plasma membrane Ca-ATPaseCaM sequence[CaM]1/2k 1k 2k 3mm −1m −1m −1CaM (Ala1–Lys148)3.9 × 10−91.0 × 108NAaNA, not applicable.∼1 × 105bEstimated affinity based on correction of apparent binding constant (k 3b = 5 ± 4 × 109m−1) for the effective volume available to the amino- terminal domain following association of the carboxyl-terminal domain (see Equation 4).(0.4 × 10−9)(0.1 × 108)CaMN (Ala1–Lys77)6 × 10−61 × 104cAssumes that CaMN has the same affinity for sites A and B (i.e. k 1 = k 2 andk 3 = k 4 in Scheme II).NA3.2 × 106cAssumes that CaMN has the same affinity for sites A and B (i.e. k 1 = k 2 andk 3 = k 4 in Scheme II).(1 × 10−6)(1 × 104)(0.5 × 106)CaMC (Asp78–Lys148)1.7 × 10−6 dParameter value based on binding affinity of carboxyl-terminal domain determined for CaM.0.7 × 106ek a = 1.1 ± 0.1 × 106m−1 using Equation 7.NA(0.2 × 10−6)(0.1 × 106)CaMN + CaMC0.4 × 10−6 dParameter value based on binding affinity of carboxyl-terminal domain determined for CaM. fParameter value based on activation of PM-Ca-ATPase using CaMC.2.7 × 106gk a = 2.8 ± 0.2 × 106m−1 using Equation 7.(0.1 × 10−6)(0.3 × 106)Equilibrium association constants were derived from macroscopic equilibrium constants obtained from fitting data in Fig. 1 to Equation2 (CaM), Equation 8 (CaMN), Equation 6 (CaMC), and Equation 9(CaMN + CaMC), where relationships between derived binding constants and the mechanisms of CaM association are illustrated in Schemes I and II. Values in parentheses represent the S.D. of the measurement.a NA, not applicable.b Estimated affinity based on correction of apparent binding constant (k 3b = 5 ± 4 × 109m−1) for the effective volume available to the amino- terminal domain following association of the carboxyl-terminal domain (see Equation 4).c Assumes that CaMN has the same affinity for sites A and B (i.e. k 1 = k 2 andk 3 = k 4 in Scheme II).d Parameter value based on binding affinity of carboxyl-terminal domain determined for CaM.e k a = 1.1 ± 0.1 × 106m−1 using Equation 7.f Parameter value based on activation of PM-Ca-ATPase using CaMC.g k a = 2.8 ± 0.2 × 106m−1 using Equation 7. Open table in a new tab Equilibrium association constants were derived from macroscopic equilibrium constants obtained from fitting data in Fig. 1 to Equation2 (CaM), Equation 8 (CaMN), Equation 6 (CaMC), and Equation 9(CaMN + CaMC), where relationships between derived binding
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