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Calmodulin-Cardiac Troponin C Chimeras

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Calmodulin-Cardiac Troponin C Chimeras THEJOURNAL OF BIOLOGICALCHEMISTRY VOl. 268, No. 33, Issue of November 25, pp. 25213-252‘20.1993 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.SA. Calmodulin-Cardiac TroponinC Chimeras EFFECTS OF DOMAIN EXCHANGE ON CALCIUM BINDING AND ENZYME ACTIVATION* (Received for publication, April 1, 1993, and in revised form, July 14, 1993) Samuel E. George$, ZenghuaSu, Daju Fan, and AnthonyR. Means From the Departments of Medicine and Pharmacology, Duke University Medical Center, Durham, North Carolina 27710 Calmodulin (CaM) and the cardiac isoform of tro- tant to understanding how CaM accomplishes its diverse ponin C (cTnC) are close structural homologs,but cTnC regulatory functions. cannot activate most CaM target enzymes. To investi- CaM is a 16.8-kDa protein that has four helix-loop-helix gate structure-function relationships, we constructed Ca2+binding domains, termed EF-hands. Thesedomains are a series of CaM-cTnC chimeras and determined their pairedin amino- and carboxyl-terminal globular domains, ability to bind Ca2+ andactivate CaM target enzymes. separated by a flexible 8-turn central helix (2-5). Upon com- Previously, we exchanged domain1 and found that the plex formation with model binding peptides, CaM undergoes chimeras exhibited profoundly impairedactivation of a striking structural rearrangement (6, 7). The central helix smooth muscle myosin light chain kinase (smMLCK) bends 100” and twists 120”, closely associating the two glob- and had differential effects on other CaM target en- ular Ca2+binding regions about a pseudo-2-fold axis of sym- zymes (George, s. E., VanBerkum, M. F. A., Ono, T., Downloaded from Cook, R., Hanley, R. M., Putkey, J. A., and Means,A. metry and enveloping the CaM-binding peptide in a hydro- R. (1990) J. Biol. Chern. 265,9228-9235). One of the phobic tunnel. The surface of the complex is composed pri- domain 1 chimeras was a potent competitive inhibitor marily of residues from the eight helical segments and the of smMLCK. We now extend our study of CaM-cTnC interhelix linker regions. The CaM-peptide complex structure chimeras by exchangingall of the remaining functionaldoes not reveal how the surface of CaM might interact with domains of CaM and cTnC. We determined the ability other functional domains of the CaM target enzyme. of the chimeras to bind Ca2+ and activate phosphodi- Chimeras between CaM and a homologous calcium-binding www.jbc.org esterase (PDE)and smMLCK.Chimeras containing protein, cardiac troponin C (cTnC), may help to define CaM- both domains 3 and 4 of cTnC exhibited high affinity target enzyme interactions. The two proteins show remarka- Caz+ binding that was indistinguishable from cTnC, ble structural similarity, with 50% amino acid identity (2, 8, whereas chimeras containing either domain 3 or 4 of 9). Although cTnC has not been crystallized, it has a high by guest, on January 15, 2010 cTnC demonstrated Ca2+affinity that was intermedi- degree of sequence similarity with both CaM and theskeletal ate between CaM andcTnC. All of the CaMOcTnC isoform of TnC (sTnC)(10). Therefore, it is likely that cTnC chimeras showed near-maximal PDE activation but shares thedumbbell-shaped structure described for CaM and required 5-775-fold higher concentrations thanCaM sTnC (2, 8, 9). to produce half-maximal PDE activation. In contrast, Despite its structural similarity to CaM, cTnC cannot ac- all of the chimeras showed impairedability to activate tivate CaM target enzymes (11,12). By exchanging functional smMLCK, and somewere potent competitive inhibitorsdomains between the two, it should be possible to define of smMLCK activation by CaM. regions of CaM that are essential for activation and cannot be substituted by corresponding domain of the inactive hom- olog, cTnC. The same approach may also be used to investi- gate other functional differences between CaM and cTnC, Calmodulin (CaM)’ is a ubiquitous intracellular Ca2+recep- such as the higher Ca2+affinity of cTnC’s carboxyl-terminal tor in eukaryotic cells. It participates in a number of intra- domains (13-15). The information gained from a CaM. cTnC cellular Ca2+signal transduction pathways through its ability chimera study can then direct a more detailed mutagenesis to bind and activate a number of regulatory proteins and study. enzymes (reviewed in Ref. 1).Understanding the structural We used this approach to show that substitution of CaM’s basis of CaM’s interaction with its target enzymes is impor- domain I had differentialeffects on CaM target enzymes (11). *This work was supported by grants from the American Heart One CaM .cTnC chimera, TaM-BM1 (domain 1 of cTnC, Association, Texas affiliate, the MethodistHospital Foundation, domains 2-4 of CaM, with its inactive first domain restored Houston, Texas (to S. E. G.), and Public Health Service Grant GM- by mutagenesis)activated some CaM target enzymes but 33976 (to A. R. M.). The costs of publication of this article were proved to be apotent competitive antagonistof smooth muscle defrayed in part by the payment of page charges. This article must myosin light chain kinase (smMLCK) (11).The competitive therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. inhibitor phenotype and additional data demonstrated that $Supported by a clinical investigator award from the National TaM-BM1 bound to smMLCK but failed to activate the Heart, Lung, and Blood Institute. To whom correspondence should enzyme. Further mutagenesis defined three residues in do- be addressed: Cardiovascular Division, Box 3060, Duke University main l of CaM that were essential for full activation of Medical Center, Durham, NC 27710. Tel.: 919-681-8446; Fax: 919- smMLCK, namely Glu-14, Thr-34, and Ser-38 (16). When 684-8591. these three CaM residues were mutated together,the resulting The abbreviations used are: CaM, calmodulin; MOPS, 3-(N- morpho1ino)propanesulfonicacid; DTT, dithiothreitol; PCR, polym- CaM triple mutant (E14A,T34K,S38M) reproduced the com- erase chain reaction; cTnC, cardiac troponin C; PDE, calmodulin- petitive inhibitor phenotypeof TaM-BM1. stimulated cyclic nucleotide phosphodiesterase; smMLCK, smooth Here we extend our approach to the remaining functional muscle myosin light chain kinase; bp, base pair(s). domains of CaM. We construct and assay a new series of 25213 25214 Calmodulin- TroponinC Chimeras CaM .cTnC chimeras, exchanging calcium binding domains pCaM[SacI] with NcoI and SUI. The resulting protein product con- 2, 3, and 4 and the central helices. Again we observe that tains amino acid residues 1-47 of CaM, 56-93 of cTnC, and 81-148 domain exchange has differential effects on target enzymes of CaM. and thatsome chimeras competitively inhibit CaM activation pTnCf.2 CUM/-The expression plasmid pTaM encodes a CaM. of smMLCK. cTnC chimera in which amino acid residues 1-56 (domain 1) are derived from cTnC and 57-157 correspond to 48-148 (domains 2-4) of CaM (11).We used PCR to amplify domains 1 and 2 of pTaM, EXPERIMENTALPROCEDURES designing the reverse oligo to incorporate a SacI site at nucleotides 276-281. This PCR product is treated with proteinase K (20), agarose Construction of Chimeras gel-purified, digested with NcoI and Sad, andsubcloned into avector To exchange functional domains of CaM and cTnC, we added SacI obtained by digesting pTnCPL3 with NcoI and SacI. The resulting and SalI sites to pCaMPL and a SalI site to pTnCPL3 in the cDNA protein product contains amino acid residues 1-56 of cTnC, 48-80 of sequences that correspond to the amino acid residues indicated in CaM, and 93-161 of cTnC. Fig. 1. These restriction sites enabled us toexchange domains 3 and pCaMfCH-TnCj-To construct this chimera, we used overlap ex- 4. For the central helix exchange, addition of restriction sites was tension PCR (17). In first stage PCR 1, pCaM[SacI] is the template, impractical because of objectionable mutations.Instead, we used the forward oligo is ACC ACA CCT ATG GTG TAT GC (oligo 3; lies overlap extension PCR (17). The details of construction are in the 70-bp 5’ to the NcoI site/initiation codons of pCaMPL and pTn- following paragraphs. CPL3), andthe reverse oligo is CCT TTG CTG TCA TCT pCaMf3,4 TnC], pTnCf3,4 CUM], and pTnCf3,4 CaMIfBMll- TTC ATT TTC CTG GCC ATC ATG GTC. In first stage PCR 2, The bacterial expression plasmid pTnCPL3 (chicken cTnC) (18, 19) pTnC[3,4 CaM] is the template, the forward oligo is G ACC ATG has a SocI site (GAG CTC). A SacI site was engineered into the ATG GCC AGGAAA ATG AAA GAT GACAG, and the reverse corresponding position of pCaMPL. This mutates Ile-85 of CaM oligo is oligo 2. The two first stage products are combined with oligos to Leu, which has no effect on Ca2+ binding or activation of the 2 and 3 in a second stage PCR. The second stage product is subcloned target enzymes presented here (data not shown). We call this as described for pCaM[2 TnC]. This construction yields a protein mutant pCaM[Saclj. TheSad-NdeI restriction fragments were product that is identical to CaM, except that residues 76-85 are then exchanged between pCaM[SacI] andpTnCPL3 to produce replaced by residues 85-97 of cTnC (see Fig. 1; the exchange runs Downloaded from pCaM[3,4 TnC]and pTnC[3,4 CaM]. To make pTnC[3,4 CaM] from the overlap extension splice point to the SacI site). [BMl], the NcoI-Sac1 fragment of pCBMl (18) was subcloned into pCaMf4 TnC] and pTnCf4 CaM]-Using overlap extension, a the corresponding site of pCaM[SacI]. unique Sal1 site was engineered between bp 364-370 of pCaM(Sac1) pCaMf2 TnC]-The expression plasmid pCaT encodes a CaM. and between bp 398 and 403 of pTnCPL3.
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