The Molecular Basis of the Modulation of the Plasma Membrane Calcium Pump by Calmodulin" ERNEST0 CARAFOLI, FELIX KESSLER, ROCCO FALCHElTO, ROGER HEIM, MANFRED0 QUADRONI, JOACHIM KREBS, EMANUEL E. STREHLER, AND THOMAS VORHERR Institute of Biochemistry Swiss Federal Institute of Technology (ETH) CH-8092 Zurich, Switzerland The plasma membrane Ca2+pump ejects CaZ+from eukaryotic cells.' In nonexcitable tissues, its role in the maintenance of the long-range gradient of CaZ+across the plasma membrane is predominant. In excitable tissues, such as heart, the role of the Ca2+pump in Ca2+ejection appears to be minor with respect to that of the larger capacity Na+/Ca2+ exchanger. The general characteristics of the pump have been discussed in recent reviews.24 The enzyme belongs to the P-class of ion-motive ATPases5s6of which it shares the essential mechanistic properties. One peculiar property of the plasma membrane Ca2+pump is the multiplicity of its modulatory mechanisms. The most important modulator is considered to be ~ a l m o d u l i n . ~ - ~ Alternative treatments include the exposure to acidic phospholipids and long chain polyunsaturated fatty acids? a controlled proteolytic treatment with a number of proteases,l0 including calpain,11J2 and a dimerization/oligomerization processI3 mediated by the calmodulin-binding domain.14 The primary sequence of several isoforms of the pump has been established in human and in rat tissues15-18:the pump is the product of a multigene family, four genes having so far been identified in humans. Additional isoform diversity is generated by alternative splicing of primary transcripts of the genes. The isoform diversity does not concern the domains of the pump that are conserved throughout the family of P-type ion-motive ATPases, such as the catalytic domain. It also does not concern the portions of the molecule that are responsible for the overall membrane architecture of the pumps of this family, such as the transmembrane domains. Rather, it concerns domains that are related to the peculiar regulation properties of the pump, such as the calmodulin-binding domain, the domains phosphorylated by protein kinases, and the putative domain(s) responsive to acidic phospholipids. The calmodulin-binding domain has been located with the help of a cross-linker coupled to c a l m ~ d u l i nnear ~ ~ the COOH-terminus of the protein. It is a positively charged segment of about 30 amino acids which tends to form an amphiphilic helix as do other calmodulin-binding domains in calmodulin-modulated proteins. The propensity to form an a-helix is particularly strong in the NH2- and COOH-terminal portions of the domain," which can therefore be subdivided into two subdomains, A aThe work described was supported by the Swiss National Science Foundation, grant numbers 31.25285.88 and 31.28772.90. 58

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and B. The domain interacts with calmodulin with a & in the nmolar range. Work with synthetic pcptides has shown that the optimal length of the domain is 28 amino acids, although a COOH-terminally abbreviated version of the domain (20 amino acids) still has adequate calmodulin affinity. The general architecture of the pump, as deduced with a variety of secondary structure prediction programs, shows that most of the mass of the molecule (about 80%) protrudes into the cytoplasm (FIG. 1 ) . Ten transmembrane domains arc presently assigned using conventional hydropathy algorithms. O n the external side, the putative transmembrane domains are connected by very short loops, whereas on

FIGURE 1. General membrane architecture of the plasma membrane Ca2+pump and location of the most important functional sites. The model is discussed in the text with cylinders indicating a-helices, arrows @-sheets. A, B are the two portions of the calmodulin-binding domain, PKA (S) is the Ser phosphorylated by protein kinase A, PKC(T) is the Thr phosphorylated by protein kinase C, and calp 1, 2 are the two sites of sequential cleavage by calpain that produce the fully activated, calmodulin-insensitive 124-kD truncated pump. T N and TC refer to the NH2- and COOH-termini of the fragments of molecular mass 90, 85, 81, and 76 kD produced by trypsin.*' The thickened portion of the first extracellular loop identifies an epitope recently recognized with the help of a monoclonal antibody.2z PL identifies the site responsive to acidic phospholipids, located in the COOH-terminal portion of the helical segment at the end of the P-stranded first protruding unit. The calmodulin-binding domain, however, also binds acidic phospholipids.23FITC is fluorescein isothiocyanate.

the internal side, three main protruding units have been postulated. The first protruding domain consists of a p-strand structure; its role in the function of the enzyme has not been assigned conclusively, but the suggestion has been made that it somehow couples the hydrolysis of ATP to the translocation of CaZ+.24The second protruding domain, which is the largest, contains the site of aspartic acid phosphorylation and that of ATP binding, which are postulated to be separated by a flexible domain (the hinge) that permits their mobility during the reaction cycle. The third protruding domain follows the tenth putative transmembrane helix and can be considered the truly distinctive structural feature of the plasma membrane Ca2+

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pumps; it extends for some 150 amino acids and contains important regulatory sites. In addition to the calmodulin-binding domain just mentioned, the region contains substrate sites for protein kinases AZ and C,Z6 and two very acidic stretches immediately preceding and following the calmodulin-binding domain. Although the role of these two acidic stretches has not been defined, it seems logical to propose that they should somehow be involved in the binding of CaZ+.The “catalytic” Ca2+ binding sites, that is, the sites involved in the channeling of Ca2+ through the membrane, are probably elsewhere in the transmembrane portion of the molecule. If the acidic sequences around the calmodulin-binding domain do indeed bind Ca2+, they probably do so for regulatory purposes.

THE CALMODULIN-BINDING DOMAIN AS AN INTERNAL INHIBITOR OF THE PUMP

The original suggestion that the calmodulin-binding domain acts as an internal repressor of the activity of calmodulin-modulated enzymesz7was validated by trypsin proteolysis experiments on the plasma membrane Caz+ pump.10,21,z8 More recent work using photoactivatable versions of the synthetic calmodulin-binding domain of the pump have added structural information to the proposal, that is, they have identified the binding site for the domain in the pump molecule.29The 28-amino acid human erythrocyte version of the domain:

LRRGQILWRGLNRIQTQIKVVNAFSSS has been synthesized with Phe in position 9 coupled to a diazirine derivative that would produce a carbene upon photoa~tivation.2~ The synthetic domain was labeled with 3H-acetic anydride at the NHz-terminus. In separate experiments, purified preparations of the erythrocyte pump10 were treated with calpain, which has been shown” to remove the calmodulin-binding domain and the portion of the pump COOH-terminal to it, producing an essentially pure, fully active fragment of molecular mass 124-kD. The synthetic calmodulin-binding domain was incubated with the 124-kD fragment and found to indeed inhibit its Ca2+-dependentATPase activity.z9 The derivatized peptide was then cross-linked to the truncated pump, and the complex was proteolyzed using the protease from strain V8 of Staphylococcus aureus, Lys-C protease, and Asp-N protease. The proteolyzed mixtures were then fractionated by HPLC, and the labeled fragment was subjected to sequence analysis. The results showed that the domain of the pump that became labeled (CALL GFVT) was located in the main cytoplasmic protruding unit between the sites of aspartylphosphate formation and ATP (FITC) binding (FIG.2). This, however, was not the only site of interaction of the calmodulin-binding domain with the main body of the pump. In a second series of experiments,30 the synthetic, radioactively labeled calmodulin-binding domain was derivatized with the diazirine derivative on Phe 25 instead of Phe 9, that is, the label was introduced in the COOH-terminal portion of the calmodulin-binding domain. Cross-linking experiments followed by protease treatment of the complex and by fractionation of the proteolysis mixtures, analogous to those described above, were then performed.30 Sequence analysis showed that the COOH-terminal portion of the calmodulin-binding domain did not interact with the first main protruding unit of the pump. The domain labeled was in this case a longer sequence of amino acids than that in the case of the labeled site on the second protruding unit, but it was clearly located in the first cytoplasmic protruding unit of the pump (FIG.3). The picture that emerges thus shows that the calmodulin-binding

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domain in the resting pump bridges its first and second cytoplasmic units; it is easy to imagine how this could hinder the free access of the substrates to the active site causing inhibition.

INTERACTION OF THE CALMODULIN-BINDING DOMAIN WITH CALMODULIN The 28-amino acid calmodulin-binding domain interacts with calmodulin with high affinity (& in the low n molar range, 20). Work with synthetic versions of the

FIGURE 2. The interacting site for the calmodulin-binding domain in the main cytoplasmic protruding unit of the plasma membrane Ca2+ pump. The scheme shows the calrnodulinbinding domain protruding from the tenth putative transmembrane helix, Phe 9 of the domain had been modified with the diazirine derivative mentioned in the text. The scheme shows the &amino acid interacting site for the domain between the site of aspartyl-phosphate formation (P) and the site of ATP (FITC) binding (K). S 1178 is the ser residue phosphorylated by protein kinase A.

domain has shown that the Trp in position 8 is crucial to the interaction with calmodulin,20and experiments on the trypsin-produced purified NH2- and COOHterminal halves of calmodulin have shown that the domain can “bridge” them3’; it has now become clear that the central helix of calmodulin is flexible and bends to bring the NH2- and COOH-terminal CaZ+binding lobes closer to each 0ther3~.’) upon interaction with target domains, including the Ca2+ ATPase of the plasma membrane.34 The synthetic 28-amino acid, calmodulin-binding domain, derivatized with the diazirine carbene precursor on either Phe 9 (peptide C28W*) or Phe 25 (peptide C2SWC*) as indicated in the preceding section, has been used to investigate in more detail the interaction of calmodulin with the binding domain.35 A shortened,

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20-amino acid version of the domain, derivatized on Phe 9, has also been synthesized (peptide C20W*). In all versions, the domain was labeled with W at the amino terminus as indicated above. Incubation of the photoactivatable synthetic domain C28W* with calmodulin, followed by proteolytic fragmentation of the complex with the protease from the V8 strain of S. uureus and by HPLC fractionation of the digest (FIG.4) yielded three labeled peptides which were subjected to sequence analysis. They corresponded to calmodulin sequences 88-114 (peak l), 88-104 (peak 2), and 88-120 (peak 3). The photoactivatable peptide thus labeled the COOH-terminal Ca2+ binding lobe of calmodulin in a region known to contain the hydrophobic pocket of the lobe. Essentially, the same results, that is, the same labeled sequence, were obtained when using the shortened peptide C20W*. When, on the other hand, calmodulin was incubated with the synthetic calmodulin-binding domain carrying the

FIGURE 3. The interacting site for the calmodulin-bindingdomain on the first cytoplasmic protruding unit of the plasma membrane Ca2+pump. The scheme is analogous to that shown in FIGURE 2. The calmodulin-binding domain was modified with the diazirine derivative on Phe 25. The interacting site was identified in the first cytoplasmicprotruding unit (the transducing unit) in a sequence of 65 amino acids (206-271).

photoactivatable group on the COOH-terminally located Phe 25 (C28WC*), a different labeling pattern was obtained after proteolysis and fragment fractionation (FIG.5). Two labeled peaks were obtained, corresponding to sequence 68-81 (peak 1) and 55-81 (peak 2) in calmodulin. These sequences belong to the NHz-terminal half of calmodulin and are part of the hydrophobic pocket in the NHz-terminal Ca2+ binding lobe of calmodulin. The results are in line with the suggestion that hydrophobic interactions are important in the interaction of calmodulin with target-binding domains36and show that the calmodulin-binding domain, in bridging the two halves of c a l m o d ~ l i nuses , ~ ~ its NH2-terminal portion to recognize the COOH-terminal half of calmodulin and its COOH-terminal portion to recognize its NHz-terminal half. The interaction of the calmodulin-binding domain with calmodulin has also been studied, making use of the expressed COOH-terminal portion of the pump in some

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CARAFOLI et al.: CALMODULIN AND THE CALCIUM PUMP

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t (rnin) FIGURE 4. Reversed phase HPLC separation of the fragments produced by the digestion of the complex between calmodulin and peptide C28W* with the V8 Glu-C protease. Details of the methods for the preparation of calmodulin and of the 'H-labeled C28W* peptide are described in ref. 35. Preparation of the diazirine derivative of the peptide carrying the photoactivatable group on Phe 9 and the V8 protease cleavage conditions are also described in ref. 35. The peptide mixture was separated on a C18 reversed phase column (Nucleosil, 3 nm, 100 A, 80 x 4 mm),with a flow rate of 300 Flimin and detection at 210 nm (solid line). A linear gradient from 0% to 100% in 40 minutes, was run; 30-second fractions were collected and 5-pl aliquots were used for scintillation counting (dottedline).The three fractions containing most of the radioactivity (peaks 1.2, and 3 from left), indicated by arrows, were submitted to sequence analysis.

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t (rnin) FIGURE 5. Reversed phase HPLC separation of the fragments produced by the digestion of the complex between calmodulin and peptide C28WC* with the V8 Glu-C protease. The 4. The conditions of the experiment were analogous to those described in the legend for FIGURE peptide (C28WC*) carried the photoactivatable diazirine derivative on Phe 25. Chromatography was performed on a C18 column (120 A, 7 Fm, 125 x 2.1 mm),with a one-step gradient of 0-100% in 40 minutes and a flow rate of 500 pllmin. The two fractions containing most of the radioactivity (peaks 1 and 2 from left; see arrows) were submitted to sequence analysis.

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of its isoforms resulting from spliced-in inserts in its calmodulin-binding domain'5,37q38(FIG. 6). Four pump isoforms arise from gene PMCAl ( 2 4 ) through alternative mRNA splicing; a 154-bp exon, used in increasing portions, expresses amino acid inserts at the end of calmodulin-binding subdomain A.I5S7The sequence deduced from human teratoma cell DNA, (PMCAlb16) does not contain any insert, that is, the calmodulin-binding subdomain A, which contains four positively charged amino acids (4 Arg) is followed by subdomain B, which contributes 2 Arg more.

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The molecular basis of the modulation of the plasma membrane calcium pump by calmodulin.

The Molecular Basis of the Modulation of the Plasma Membrane Calcium Pump by Calmodulin" ERNEST0 CARAFOLI, FELIX KESSLER, ROCCO FALCHElTO, ROGER HEIM,...
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