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• W1968121475 abstract "Protein kinase C phosphorylation of cardiac troponin, the Ca2+-sensing switch in muscle contraction, is capable of modulating the response of cardiac muscle to a Ca2+ ion concentration. The N-domain of cardiac troponin I contains two protein kinase C phosphorylation sites. Although the physiological consequences of phosphorylation at Ser43/Ser45 are known, the molecular mechanisms responsible for these functional changes have yet to be established. In this work, NMR was used to identify conformational and dynamic changes in cardiac troponin C upon binding a phosphomimetic troponin I, having Ser43/Ser45 mutated to Asp. Chemical shift perturbation mapping indicated that residues in helix G were most affected. Smaller chemical shift changes were observed in residues located in the Ca2+/Mg2+-binding loops. Amide hydrogen/deuterium exchange rates in the C-lobe of troponin C were compared in complexes containing either the wild-type or phosphomimetic N-domain of troponin I. In the presence of a phosphomimetic domain, exchange rates in helix G increased, whereas a decrease in exchange rates for residues mapping to Ca2+/Mg2+-binding loops III and IV was observed. Increased exchange rates are consistent with destabilization of the Thr129–Asp132 helix capping box previously characterized in helix G. The perturbation of helix G and metal binding loops III and IV suggests that phosphorylation alters metal ion affinity and inter-subunit interactions. Our studies support a novel mechanism for protein kinase C signal transduction, emphasizing the importance of C-lobe Ca2+/Mg2+-dependent troponin interactions. Protein kinase C phosphorylation of cardiac troponin, the Ca2+-sensing switch in muscle contraction, is capable of modulating the response of cardiac muscle to a Ca2+ ion concentration. The N-domain of cardiac troponin I contains two protein kinase C phosphorylation sites. Although the physiological consequences of phosphorylation at Ser43/Ser45 are known, the molecular mechanisms responsible for these functional changes have yet to be established. In this work, NMR was used to identify conformational and dynamic changes in cardiac troponin C upon binding a phosphomimetic troponin I, having Ser43/Ser45 mutated to Asp. Chemical shift perturbation mapping indicated that residues in helix G were most affected. Smaller chemical shift changes were observed in residues located in the Ca2+/Mg2+-binding loops. Amide hydrogen/deuterium exchange rates in the C-lobe of troponin C were compared in complexes containing either the wild-type or phosphomimetic N-domain of troponin I. In the presence of a phosphomimetic domain, exchange rates in helix G increased, whereas a decrease in exchange rates for residues mapping to Ca2+/Mg2+-binding loops III and IV was observed. Increased exchange rates are consistent with destabilization of the Thr129–Asp132 helix capping box previously characterized in helix G. The perturbation of helix G and metal binding loops III and IV suggests that phosphorylation alters metal ion affinity and inter-subunit interactions. Our studies support a novel mechanism for protein kinase C signal transduction, emphasizing the importance of C-lobe Ca2+/Mg2+-dependent troponin interactions. Troponin and tropomyosin form the Ca2+-sensitive switch that regulates striated muscle contraction. Troponin is a ternary assembly of proteins composed of the Ca2+-binding subunit troponin C (TnC), 1The abbreviations used are: TnC, troponin C (Ca2+ binding); TnI, troponin I (inhibitory); cTnC, recombinant cardiac troponin C (desMet1-Ala2, C35S); CcTnC, cTnC corresponding to residues 81–161; cTnI, cardiac TnI; cTnI(S43D/S45D), full-length recombinant mouse cTnI with the N-terminal Met cleaved during expression and purification and Ser43 and Ser45 mutated to Asp; NcTnI, recombinant mouse cTnI corresponding to residues 33–80 with the N-terminal Met cleaved during expression and purification; NcTnI(S43D/S45D), recombinant NcTnI with Ser43 and Ser45 mutated to Asp; PKC, protein kinase C; H/D, hydrogen/deuterium; HSQC, heteronuclear single quantum coherence. the inhibitory subunit troponin I (TnI), and the tropomyosin-binding protein troponin T that anchors troponin to the thin filament. Troponin C, a member of the EF-hand family of Ca2+-binding proteins, contains two globular domains connected by a linker. Each domain of cTnC contains two EF-hand or Ca2+-binding motifs. The N-lobe contains two lower affinity Ca2+-binding motifs, sites I and II, that control muscle contraction. Site I is naturally inactive in the cardiac isoform because of several amino acid substitutions and an amino acid insertion (1van Eerd J.P. Takahashi K. Biochem. Biophys. Res. Commun. 1975; 64: 122-127Crossref PubMed Scopus (116) Google Scholar). Thus, Ca2+ binding at site II in cTnC regulates muscle contraction. The C-lobe contains two high affinity Ca2+-binding sites, III and IV, which also bind Mg2+ with lower affinity. Interactions between the C-lobe of cTnC and the N-domain of cTnI form the Ca2+/Mg2+-dependent cTnC/cTnI interaction site. In addition, the C-lobe of cTnC also interacts tightly with the C terminus of cardiac troponin T (2Takeda S. Yamashita A. Maeda K. Maeda Y. Nature. 2003; 424: 35-41Crossref PubMed Scopus (642) Google Scholar). These interactions form the core of the troponin complex, tethering all three subunits throughout the contraction cycle. A variety of effectors can modulate the frequency and intensity of myocardial contraction by charge modification upon the phosphorylation of cTnI and cardiac troponin T. In the heart, phosphorylation of cTnI appears to be of particular importance in cardiac hypertrophy and failure (3Kobayashi T. Dong W-J. Burkart E.M. Cheung H.C. Solaro R.J. Biochemistry. 2004; 43: 5996-6004Crossref PubMed Scopus (23) Google Scholar, 4Sumandea M.P. Burkart E.M. Kobayashi T. de Tombe P.P. Solaro R.J. Ann. N. Y. Acad. Sci. 2004; 1015: 39-52Crossref PubMed Scopus (72) Google Scholar, 5Montgomery D.E. Wolska B.M. Pyle W.G. Roman B.B. Dowell J.C. Buttrick P.M. Koretsky A.P. Del Nido P. Solaro R.J. Am. J. Physiol. 2002; 282: H2397-H2405Crossref PubMed Scopus (59) Google Scholar). Cardiac TnI can be phosphorylated by protein kinase A at Ser23 and Ser24 (6Mittmann K. Jaquet K. Heilmeyer Jr., L.M. FEBS Lett. 1990; 273: 41-45Crossref PubMed Scopus (63) Google Scholar). β-Adrenergic stimulation leads to protein kinase A phosphorylation at Ser23 and Ser24 of cTnI, enhancing relaxation by decreasing the Ca2+ affinity at site II (7Zhang R. Zhao J. Potter J.D. J. Biol. Chem. 1995; 270: 30773-30780Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 8Chandra M. Dong W.J. Pan B.S. Cheung H.C. Solaro R.J. Biochemistry. 1997; 36: 13305-13311Crossref PubMed Scopus (92) Google Scholar). Cardiac TnI can also be phosphorylated by PKC at Ser43, Ser45, and Thr144 (9Noland Jr., T.A. Guo X. Raynor R.L. Jideama N.M. Averyhart-Fullard V. Solaro R.J. Kuo J.F. J. Biol. Chem. 1995; 270: 25445-25454Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Phosphorylation at Ser43 and Ser45 is known to decrease maximal actomyosin MgATPase activity, Ca2+ sensitivity, and cross-bridge binding to the thin filament (4Sumandea M.P. Burkart E.M. Kobayashi T. de Tombe P.P. Solaro R.J. Ann. N. Y. Acad. Sci. 2004; 1015: 39-52Crossref PubMed Scopus (72) Google Scholar, 9Noland Jr., T.A. Guo X. Raynor R.L. Jideama N.M. Averyhart-Fullard V. Solaro R.J. Kuo J.F. J. Biol. Chem. 1995; 270: 25445-25454Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 10Huang L. Wolska B.M. Montgomery D.E. Burkart E.M. Buttrick P.M. Solaro R.J. Am. J. Physiol. 2001; 280: C1114-C1120Crossref PubMed Google Scholar, 11Burkart E.M. Sumandea M.P. Kobayashi T. Nili M. Martin A.F. Homsher E. Solaro R.J. J. Biol. Chem. 2003; 278: 11265-11275Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). These biochemical changes lead to maladaptive growth and diminished contractility, culminating in end-stage heart failure (5Montgomery D.E. Wolska B.M. Pyle W.G. Roman B.B. Dowell J.C. Buttrick P.M. Koretsky A.P. Del Nido P. Solaro R.J. Am. J. Physiol. 2002; 282: H2397-H2405Crossref PubMed Scopus (59) Google Scholar). Although it is clear that PKC phosphorylation of cardiac troponin modulates contraction in response to hemodynamic stressors such as hypertension and myocardial infarction, the molecular mechanisms of this modulation remain unknown. The effects of PKC phosphorylation at Ser43 and Ser45 of cTnI can be mimicked, both in vitro and in vivo, by the introduction of negative charge at positions 43/45 (3Kobayashi T. Dong W-J. Burkart E.M. Cheung H.C. Solaro R.J. Biochemistry. 2004; 43: 5996-6004Crossref PubMed Scopus (23) Google Scholar, 4Sumandea M.P. Burkart E.M. Kobayashi T. de Tombe P.P. Solaro R.J. Ann. N. Y. Acad. Sci. 2004; 1015: 39-52Crossref PubMed Scopus (72) Google Scholar, 11Burkart E.M. Sumandea M.P. Kobayashi T. Nili M. Martin A.F. Homsher E. Solaro R.J. J. Biol. Chem. 2003; 278: 11265-11275Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Serine residues 43/45 of cTnI are at the N terminus of an amphiphilic α-helix that binds in a hydrophobic cleft in the C-lobe of cTnC (2Takeda S. Yamashita A. Maeda K. Maeda Y. Nature. 2003; 424: 35-41Crossref PubMed Scopus (642) Google Scholar). Stability of the hydrophobic core in CcTnC is largely governed by metal binding at site III (12Brito R.M. Krudy G.A. Negele J.C. Putkey J.A. Rosevear P.R. J. Biol. Chem. 1993; 268: 20966-20973Abstract Full Text PDF PubMed Google Scholar). The exchange of Ca2+ for Mg2+ in Ca2+/Mg2+-binding sites III and IV resulted in a partial closure of the hydrophobic binding cleft around site IV, allowing the possibility that Ca2+/Mg2+ exchange can modulate contraction via Ca2+/Mg2+-dependent cTnC/cTnI interactions (13Finley N.L. Howarth J.W. Rosevear P.R. Biochemistry. 2004; 43: 11371-11379Crossref PubMed Scopus (27) Google Scholar, 14Finley N. Dvoretsky A. Rosevear P.R. J. Mol. Cell. Cardiol. 2000; 32: 1439-1446Abstract Full Text PDF PubMed Scopus (25) Google Scholar). However, even absent excess Ca2+, Mg2+ did not readily displace Ca2+ in the C-lobe of cTnC bound to NcTnI (14Finley N. Dvoretsky A. Rosevear P.R. J. Mol. Cell. Cardiol. 2000; 32: 1439-1446Abstract Full Text PDF PubMed Scopus (25) Google Scholar). To define the structural consequences resulting from cTnI phosphorylation at Ser43/Ser45 by PKC, we have utilized solution NMR to identify residues in cTnC that are important in transmission of the phosphorylation signal. Backbone resonances in Ca2+-loaded [13C,15N]CcTnC bound to NcTnI(S43D/S45D) were assigned and used for secondary structure determination. The phosphorylation mimetics did not significantly alter the secondary structural elements of the paired Ca2+-binding motifs in CcTnC. Residues in CcTnC important for transmission of the phosphorylation signal were identified using chemical shift perturbation mapping and amide proton exchange. The phosphorylation mimetics induced localized conformational/dynamic perturbations in the N terminus of helix G and Ca2+/Mg2+-binding loops III and IV. Specifically, the presence of a negative charge at Ser43/Ser45 of cTnI was found to destabilize the Thr129–Asp132 N-cap in helix G of CcTnC. Perturbation of N-cap interactions in helix G have been shown previously to decrease metal ion affinities at site III (15Trigo-Gonzalez G. Awang G. Racher K. Neden K. Borgford T. Biochemistry. 1993; 32: 9826-9831Crossref PubMed Scopus (24) Google Scholar). These findings support a mechanism for PKC modulation of cardiac contractility wherein the introduction of negative charge at Ser43/Ser45 of cTnI results in altered C-lobe metal ion affinities and perturbation of Ca2+/Mg2+-dependent protein-protein interactions. Presumably, these changes are transmitted to other regulatory and switch regions within cardiac troponin. These changes establish a role for the Ca2+/Mg2+-dependent cTnC/cTnI interaction in transmitting the phosphorylation signal and modulating Ca2+ sensitivity. Recombinant Protein Expression and Purification—Isotopically enriched and unlabeled recombinant proteins were expressed, purified, and quantified as described (16Abbott M.B. Gaponenko V. Abusamhadneh E. Finley N. Li G. Dvoretsky A. Rance M. Solaro R.J. Rosevear P.R. J. Biol. Chem. 2000; 275: 20610-20617Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 17Gasmi-Seabrook G.M. Howarth J.W. Finley N. Abusamhadneh E. Gaponenko V. Brito R.M. Solaro R.J. Rosevear P.R. Biochemistry. 1999; 38: 8313-8322Crossref PubMed Scopus (50) Google Scholar). Complex Formation and Resonance Assignment—Samples of Ca2+-saturated [2H,15N]cTnC or [13C,15N]CcTnC were combined in equimolar amounts with either cTnI(S43D/S45D) or NcTnI(S43D/S45D) to form stable binary complexes as described previously (16Abbott M.B. Gaponenko V. Abusamhadneh E. Finley N. Li G. Dvoretsky A. Rance M. Solaro R.J. Rosevear P.R. J. Biol. Chem. 2000; 275: 20610-20617Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Binary complexes were judged 1:1 by native gel electrophoresis in the presence of 10 mm CaCl2 and by sensitivity-enhanced 1H-15N HSQC spectra (16Abbott M.B. Gaponenko V. Abusamhadneh E. Finley N. Li G. Dvoretsky A. Rance M. Solaro R.J. Rosevear P.R. J. Biol. Chem. 2000; 275: 20610-20617Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Samples for NMR were ∼1 mm in Ca2+-saturated protein in 20 mm Trisd11, pH 6.8, 100 mm KCl, 5 mm dithiothreitol, 10 mm CaCl2, and 10% 2H2O. Complete EDTA-free protease inhibitor mixture (Roche Applied Science) was used to prevent protein degradation during NMR analysis. NMR Methodology—Experiments were collected at 40 °C using 400, 600, and 800 MHz Varian Inova spectrometers equipped with pulse-field gradient units and triple resonance probes. Acquisition parameters for heteronuclear multidimensional NMR experiments and chemical shift referencing details can be found in Gasmi-Seabrook et al. (17Gasmi-Seabrook G.M. Howarth J.W. Finley N. Abusamhadneh E. Gaponenko V. Brito R.M. Solaro R.J. Rosevear P.R. Biochemistry. 1999; 38: 8313-8322Crossref PubMed Scopus (50) Google Scholar). Inter-residue NOEs obtained from 15N-editied nuclear Overhauser effect spectroscopy-HSQC experiments at mixing times of 70 and 150 ms were used to confirm consecutive assignments. 1H-15N transverse relaxation-optimized spectroscopy spectra of the intact binary complex [2H,15N]cTnC-cTnI(S43D/S45D) were acquired with 2048 points in the direct dimension, 64 points in the indirect dimension, and 320 scans per increment. Felix 2000 was employed to process and analyze NMR data. Amide 1H and 15N chemical shift differences were obtained by subtracting the respective chemical shifts for each residue in CcTnC-NcTnI from the amide 1H and 15N chemical shifts for the same residue in CcTnC-NcTnI(S43D/S45D). The combined 1H and 15N chemical shift difference for each residue was calculated using √[(Δ1H)2 + (Δ15N/7)2]. Hydrogena/Deuterium Exchange Experiments—Complexes consisting of ∼1 mm Ca2+-loaded [15N]CcTnC bound to NcTnI(S43D/S45D) or NcTnI were prepared in NMR buffer and lyophilized. To initiate hydrogen exchange, lyophilized samples were dissolved in 99.99% 2H2O. Amide exchange rates were measured from the decay of resonance intensity in successive 1H-15N HSQC spectra collected at 25 °C every 20 min. 1H-15N HSQC spectra were acquired with 1280 and 64 points in the direct and indirect dimensions, respectively, with eight scans per increment. Spectra were processed and analyzed using Felix. The volume of each amide cross-peak was normalized, and the ratio was fitted to a single exponential function, A = A0[exp - (-kext)]. Differences in CcTnC amide proton exchange rates, Δkex, were obtained by subtracting exchange rates for each residue in CcTnC-NcTnI from the same residue in CcTnC-NcTnI(S43D/S45D). NMR Signal Assignment and Secondary Structure—Changing a phosphorylation site to a negatively charged residue such as Asp or Glu can often be used to mimic phosphorylation and facilitate biophysical studies. Incorporation of a negative charge at Ser43/Ser45 by mutation was shown to provide functional mimetics for PKC phosphorylation of cTnI (4Sumandea M.P. Burkart E.M. Kobayashi T. de Tombe P.P. Solaro R.J. Ann. N. Y. Acad. Sci. 2004; 1015: 39-52Crossref PubMed Scopus (72) Google Scholar, 18Burkart E.M. Arteaga G.M. Sumandea M.P. Prabhakar R. Wieczorek D.F. Solaro R.J. J. Mol. Cell. Cardiol. 2003; 35: 1285-1293Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). A comparison between cTnI and cTnI(S43D/S45D) bound to Ca2+-loaded [2H,15N]cTnC was made. Chemical shift perturbation mapping was used to monitor conformational changes in cTnC induced by the introduction of a negative charge at Ser43/Ser45 of cTnI in the intact binary complex. Amide proton chemical shift differences between Ca2+-loaded [2H,15N]cTnC-cTnI(S43D/S45D) and [2H,15N]cTnC-cTnI are shown in Fig. 1A. The majority of chemical shift perturbations induced by the mutation of Ser43/Ser45 to Asp were localized to the C-lobe of cTnC, with residues in helix G experiencing the largest chemical shift changes (Fig. 1A). Small amide proton chemical shift perturbations were observed for Glu66, Gly68, and Ser69 in the N-lobe of cTnC (Fig. 1A). These residues are located in the regulatory Ca2+-binding site (site II). Titration of cTnC with a cTnI peptide phosphorylated at Ser43/Ser45 was also found to induce small amide proton chemical shift perturbations in N-lobe residues corresponding to Gly42, Val72, and Val79 (19Li M.X. Wang X. Lindhout D.A. Buscemi N. Van Eyk J.E. Sykes B.D. Biochemistry. 2003; 42: 14460-14468Crossref PubMed Scopus (26) Google Scholar). Chemical shift perturbations may reflect charge-induced changes in local electrostatic interactions or changes in protein structure. It is unlikely that N-lobe chemical shift perturbations result from local electrostatic interactions, because these residues are >15 Å from the sites of cTnI phosphorylation in the core cardiac troponin structure (2Takeda S. Yamashita A. Maeda K. Maeda Y. Nature. 2003; 424: 35-41Crossref PubMed Scopus (642) Google Scholar). To examine in more detail conformational perturbations induced in the C-lobe of cTnC by PKC phosphorylation at Ser43/Ser45 of cTnI, we have studied a model phosphomimetic complex, CcTnC-NcTnI(S43D/S45D). NMR resonance assignments and solution structures for both Mg2+- and Ca2+-loaded CcTnC in the CcTnC-NcTnI complex are available (13Finley N.L. Howarth J.W. Rosevear P.R. Biochemistry. 2004; 43: 11371-11379Crossref PubMed Scopus (27) Google Scholar). Comparison of C-lobe chemical shift perturbations in cTnC-cTnI(S43D/S45D) and CcTnC-NcTnI(S43D/S45D) demonstrate that CcTnC-NcTnI(S43D/S45D) provides a suitable model for PKC phosphorylation-induced structural changes in the Ca2+/Mg2+-dependent cTnC-cTnI interaction site (Fig. 1). This finding is consistent with the core cardiac troponin x-ray structure showing that NcTnI primarily makes contacts with the C-lobe of cTnC (2Takeda S. Yamashita A. Maeda K. Maeda Y. Nature. 2003; 424: 35-41Crossref PubMed Scopus (642) Google Scholar). Phosphorylation sites Ser43/Ser45 in cTnI are located at the N terminus of helix H1 in cTnI, corresponding to residues 43–79 (2Takeda S. Yamashita A. Maeda K. Maeda Y. Nature. 2003; 424: 35-41Crossref PubMed Scopus (642) Google Scholar). Residues 43–65 of cTnI bind to the hydrophobic cleft in CcTnC (2Takeda S. Yamashita A. Maeda K. Maeda Y. Nature. 2003; 424: 35-41Crossref PubMed Scopus (642) Google Scholar). Two dimensional 1H-15N HSQC NMR spectroscopy was used to monitor individual amide resonances of residues in the C-lobe of cTnC upon complex formation with NcTnI(S43D/S45D). Only a single set of bound CcTnC amide resonances were observed as expected for high affinity binding (Kd < 1 μm) in the slow exchange regime. These results are consistent with isothermal titration microcalorimetry showing that bisphosphorylation at Ser43/Ser45 of cTnI-(1–64) had little effect on cTnC affinity (Ka ∼ 1 × 10-2 μm) (20Ward D.G. Brewer S.M. Gallon C.E. Gao Y. Levine B.A. Trayer I.P. Biochemistry. 2004; 43: 5772-5781Crossref PubMed Scopus (24) Google Scholar). Backbone chemical shift assignments for Ca2+-loaded [13C,15N]CcTnC bound to NcTnI(S43D/S45D) were obtained using standard triple resonance assignment strategies (13Finley N.L. Howarth J.W. Rosevear P.R. Biochemistry. 2004; 43: 11371-11379Crossref PubMed Scopus (27) Google Scholar, 17Gasmi-Seabrook G.M. Howarth J.W. Finley N. Abusamhadneh E. Gaponenko V. Brito R.M. Solaro R.J. Rosevear P.R. Biochemistry. 1999; 38: 8313-8322Crossref PubMed Scopus (50) Google Scholar). The resonance assignment strategy relied primarily on (Hβ)CβCα(CO)NNH, HNCαCβ, HNCα, and HNCO triple resonance experiments. Assignments were obtained for 75 of the 81 CcTnC amino acid residues. Backbone chemical shifts for the Cα,Cβ,C′, and Hα resonances were used to determine chemical shift index values for each residue in Ca2+-loaded CcTnC bound to NcTnI(S43D/S45D) (21Wishart D.S. Sykes B.D. J. Biomol. NMR. 1994; 4: 171-180Crossref PubMed Scopus (1916) Google Scholar). The chemical shift index-determined secondary structure showed a characteristic paired EF-hand motif with four helices spanning residues 93–103 (E), 114–123 (F), 130–139 (G), and 150–158 (H), as well as two short β-strands extending from residues 111–113 and 147–149 (Fig. 1B). The overall secondary structure is analogous to that previously determined for Ca2+- and Mg2+-loaded CcTnC bound to NcTnI (13Finley N.L. Howarth J.W. Rosevear P.R. Biochemistry. 2004; 43: 11371-11379Crossref PubMed Scopus (27) Google Scholar). The similarity in secondary structure for CcTnC bound to NcTnI and NcTnI(S43D/S45D) justified the use of available C-lobe cTnC structures for interpreting changes in chemical shifts and H/D exchange rates upon the introduction of a negative charge at the two PKC phosphorylation sites (2Takeda S. Yamashita A. Maeda K. Maeda Y. Nature. 2003; 424: 35-41Crossref PubMed Scopus (642) Google Scholar, 13Finley N.L. Howarth J.W. Rosevear P.R. Biochemistry. 2004; 43: 11371-11379Crossref PubMed Scopus (27) Google Scholar). Chemical Shift Mapping—Chemical shifts are sensitive to the local environment and can be used to monitor subtle structural changes. We have shown previously that amide chemical shifts for cTnC are extremely sensitive to the small conformational changes that occur upon cAMP-dependent protein kinase A phosphorylation of cTnI (22Finley N. Abbott M.B. Abusamhadneh E. Gaponenko V. Dong W. Gasmi-Seabrook G. Howarth J.W. Rance M. Solaro R.J. Cheung H.C. Rosevear P.R. FEBS Lett. 1999; 453: 107-112Crossref PubMed Scopus (82) Google Scholar, 23Abbott M.B. Dong W-J. Dvoretsky A. DaGue B. Caprioli R.M. Cheung H.C. Rosevear P.R. Biochemistry. 2001; 40: 5992-6001Crossref PubMed Scopus (45) Google Scholar). Chemical shifts for the 1HN, 15NH, 13Cα, and 13Cβ resonances in CcTnC-NcTnI (17Gasmi-Seabrook G.M. Howarth J.W. Finley N. Abusamhadneh E. Gaponenko V. Brito R.M. Solaro R.J. Rosevear P.R. Biochemistry. 1999; 38: 8313-8322Crossref PubMed Scopus (50) Google Scholar) were used for a residue-by-residue comparison with the chemical shifts obtained in CcTnC-NcTnI(S43D/S45D). Perturbations in the combined 1HN/15NH and 13Cα/13Cβ chemical shifts are plotted on the structure of Ca2+-loaded CcTnC bound to NcTnI (Fig. 2). The largest 1HN/15NH chemical shift perturbations were observed in the N terminus of helix G, Ile128 and Thr129, and in helix E, Glu95 (Fig. 1B). These residues cluster around the N-cTnI-binding site (2Takeda S. Yamashita A. Maeda K. Maeda Y. Nature. 2003; 424: 35-41Crossref PubMed Scopus (642) Google Scholar). Threonine 129 is the N-cap residue in the N-terminal helix capping box of helix G in both Ca2+- and Mg2+-loaded CcTnC-NcTnI complexes (13Finley N.L. Howarth J.W. Rosevear P.R. Biochemistry. 2004; 43: 11371-11379Crossref PubMed Scopus (27) Google Scholar, 17Gasmi-Seabrook G.M. Howarth J.W. Finley N. Abusamhadneh E. Gaponenko V. Brito R.M. Solaro R.J. Rosevear P.R. Biochemistry. 1999; 38: 8313-8322Crossref PubMed Scopus (50) Google Scholar). In both complexes, the side-chain hydroxyl of Thr129 hydrogen bonds to the amide of Asp132, and the Asp side-chain, in turn, forms a hydrogen bond with the amide of Thr129 (13Finley N.L. Howarth J.W. Rosevear P.R. Biochemistry. 2004; 43: 11371-11379Crossref PubMed Scopus (27) Google Scholar, 17Gasmi-Seabrook G.M. Howarth J.W. Finley N. Abusamhadneh E. Gaponenko V. Brito R.M. Solaro R.J. Rosevear P.R. Biochemistry. 1999; 38: 8313-8322Crossref PubMed Scopus (50) Google Scholar). The upfield amide proton chemical shift for Thr129 in the phosphomimetic complex is consistent with destabilization of the hydrogen bond between the amide of Thr129 (N-cap residue) and the carboxylate side-chain of Asp132 (N3 residue). The 3JNH-Cα coupling constant for Thr129 decreased slightly from 9 Hz (17Gasmi-Seabrook G.M. Howarth J.W. Finley N. Abusamhadneh E. Gaponenko V. Brito R.M. Solaro R.J. Rosevear P.R. Biochemistry. 1999; 38: 8313-8322Crossref PubMed Scopus (50) Google Scholar) to 8.3 Hz in the phosphomimetic complex, consistent with a decrease in the average φ backbone torsion angle. Changes in the amide proton chemical shift and the 3JNH-Cα coupling constant for Thr129 are consistent with destabilization of the N-terminal helix G capping box. In addition, upfield amide nitrogen chemical shifts are also observed for Ile128 and Thr129 in the phosphomimetic complex (data not shown). Hydrophobic interactions between the N′ residue (Ile128) and the N4 residue (Ile133) stabilize the N-cap box (24Harper E.T. Rose G.D. Biochemistry. 1993; 32: 7605-7609Crossref PubMed Scopus (332) Google Scholar). Such stabilizing interactions would be expected to deshield the amide nitrogen resonances of Ile128 and Thr129. However, destabilization of the hydrogen-bonding network in the Thr129–Asp132 N-cap box would reverse this effect, shifting the amide nitrogen resonances upfield as observed in the phosphomimetic complex. Chemical shift perturbations were also observed in 13Cα and 13Cβ resonances of CcTnC residues upon the introduction of a negative charge at Ser43/Ser45 in cTnI. Residues showing the largest 13Cα/13Cβ chemical shift changes are located in helix E (Leu-97, Phe101, and Phe104), helix F (Leu117 and Leu121), and helix H (Phe153, Leu154, and Phe156) (Fig. 2B). Hydrophobic and polar residues identified by chemical shift indexing form contiguous surfaces that define the binding interface between CcTnC and NcTnI within the Ca2+/Mg2+-dependent cTnC-NcTnI interaction site (Fig. 2C). Residues 43–65 of cTnI form an amphiphilic α-helix that binds to the C-lobe hydrophobic crevice via multiple polar and Van der Waals interactions (2Takeda S. Yamashita A. Maeda K. Maeda Y. Nature. 2003; 424: 35-41Crossref PubMed Scopus (642) Google Scholar), similar to the interactions observed in the skeletal sTnC-sTnI (1–47) complex (25Vassylyev D.G. Takeda S. Wakatsuki S. Maeda K. Maeda Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4847-4852Crossref PubMed Scopus (193) Google Scholar). Hydrogen/Deuterium Exchange—Changes in local dynamic behavior have also been utilized to identify residues that undergo conformational change accompanying protein binding. Hydrogen/deuterium exchange allows characterization of changes in global thermodynamic stability and local conformational motion with exchange time constants on the order of minutes to days. Whereas chemical shifts report on the magnetic environment of nuclei, H/D exchange kinetics provides information about backbone dynamics, conformation, and proton-solvent interactions. Comparison of H/D exchange rates provides a mechanism to assess the effects of the PKC phosphorylation mimetics on conformational fluctuations in CcTnC. To this end, amide proton H/D exchange rates in [15N]CcTnC bound to either NcTnI or NcTnI(S43D/S45D) were measured at 25 °C. At temperatures >25 °C, many of the exchange rates were too fast to reliably measure, whereas temperatures <25 °C resulted in the broadening of 1H-15N correlations. Hydrogen/deuterium exchange rates could be monitored for 59 of the 75 assigned 1H-15N correlations in the HSQC spectrum of CcTnC. The remaining resides were excluded from analysis because of weak signal intensity and/or resonance overlap. The amide proton exchange kinetics could be classified into five categories, namely rapidly exchanging (within the first 20 min), fast (kex > 8.5 × 10-2 min-1), moderately fast (8.5 × 10-3 min-1 < kex < 8.5 × 10-2 min-1), slow (2.5 × 10-3 min-1 < kex < 8.5 × 10-3 min-1), and very slow (kex < 2.5 × 10-3 min-1) (Table I). Rapidly exchanging amide correlations, disappearing within the first 20 min of exchange in both complexes, were assigned to residues Asp87, Asp88, Ser89, Lys90, Gly91, Lys92, Thr93, Glu94, Ser98, Asp99, Arg102, Met120, Thr124, Gly125, Glu126, Thr127, Glu130, Asn144, Glu152, and Gly159. Most of these correspond to residues that are located in unstructured or mobile loop regions (13Finley N.L. Howarth J.W. Rosevear P.R. Biochemistry. 2004; 43: 11371-11379Crossref PubMed Scopus (27) Google Scholar). The time course of H/D exchange for 28 residues could be followed in both complexes and used to calculate exchange rates (Table I). The introduction of a negative charge mimicking PKC phosphorylation in NcTnI was found to both increase and decrease the amide proton H/D exchange rates of selective residues in CcTnC (Table I and Fig. 3). A comparison of 1H-15N correlation spectra of CcTnC bound to NcTnI(S43D/S45D) and NcTnI after 330 min of exchange into 2H2O shows that the amide proton of Ile133 exchanges faster in the phosphomimetic complex (Fig. 3). Isoleucine 133 is located in helix G immediately following the Thr129–Asp132 N-cap box. The H/D exchange rates for Thr129, Asp132, Ile133, and Glu135, located in helix G of CcTnC, were also increased in the phosphomimetic complex (Table I and Fig. 4). These residues are involved both in stabilizing N-terminal helix capping interactions and in the (i) to (i + 3) hydrogen bonds typical of α-helices. In addition, Asp132 and Glu135 participate in interresidue polar interactions with Arg45 and Lys46 of cTnI (2Takeda S. Yamashita A. Maeda K. Maeda Y. Nature. 2003; 424: 35-41Crossref PubMed Scopus (642) Google Scholar). Thus, the introduction of proximal negative charge in cTnI weakens the substantial hydrogen-bonding lattice in helix G, altering Ca2+/Mg2+-dependent interactions between cTnI and the C-lobe of cTnC.Table IAmide H/D exchange rates for CcTnC bound to NcTnI and the PKC phosphorylation mimetic NcTnI(S43D/S45D)CcTnC residueaResonance assignments for residues 81–86 could not be confirmed. Residues Glu96, Leu97, Leu100, Phe101, Asp109, Leu114, Glu116, Lys118, Ile119, Leu121, Ala123, Asp131, Leu136, and Tyr150 were excluded from analysis due to peak overlap in the 1H-15N correlation spectra in one or both complexes, whereas Asp105, Lys106, Ile112, Leu117, Ile128, Asp145, Ile148, Asp149, Asp151, Phe153, Leu154, Phe156, and Val160 could not be reliably measuredNcTnI kexbExchange rates were measured by fitting the relative 1H-15N cross-peak volume versus exchange time with a single first-order exponentialNcTnI(S43D/S45D) kexbExchange rates were measured by fitting the relative 1H-15N cross-peak volume versus exchange time with a single first-order exponentialΔkexcCalculated by subtracting the measured H/D exchange rate for each residue in CcTnC-NcTnI from the measured H/D exchange rate for the same residue in CcTnC-NcTnI(S43/45D). A negative value indicates H/D exchange decreased in the phosphomimetic complex. A positive value indicates that H/D exchange increased in the phosphomimetic complex10-3 min-110-3 min-110-3 min-1Glu953.0 ± 0.67.2 ± 1.24.2 ± 1.3Met1031.2 ± 1.25.4 ± 0.2-4.2 ± 1.2Phe10468.4 ± 18.682.2 ± 0.613.8 ± 18.6Asn10719.8 ± 1.87.2 ± 0.4-12.6 ± 1.8Ala10820.4 ± 0.643.8 ± 0.6-23.4 ± 0.8Gly1106.0 ± 1.22.4 ± 0.5-3.6 ± 1.3Tyr111105.0 ± 11.4141.0 ± 22.836.0 ± 25.5Asp113135.0 ± 29.453.4 ± 7.8-81.6 ± 30.4Glu11556.4 ± 6.642.6 ± 1.8-13.8 ± 6.8Gln12215.0 ± 0.612.6 ± 0.3-2.4 ± 0.7Thr1296.6 ± 8.441.4 ± 3.034.8 ± 8.9Asp1329.0 ± 0.516.2 ± 0.67.2 ± 0.7Ile1334.2 ± 0.315.6 ± 0.611.4 ± 0.7Glu1343.6 ± 0.36.0 ± 1.22.4 ± 1.2Glu1354.8 ± 1.28.4 ± 0.53.6 ± 1.3Met1379.6 ± 2.434.8 ± 4.825.2 ± 5.4Lys1382.4 ± 1.83.6 ± 0.61.2 ± 1.9Asp13934.2 ± 3.034.8 ± 2.40.6 ± 3.8Gly14015.0 ± 1.86.6 ± 0.5-8.4 ± 1.9Asp1411.8 ± 0.63.0 ± 0.21.2 ± 0.6Lys1420.6 ± 0.60.1 ± 0.2-0.5 ± 0.6Asn14318.0 ± 0.67.2 ± 0.2-10.8 ± 0.6Gly14610.8 ± 0.64.8 ± 0.2-6.0 ± 0.6Arg1471.8 ± 0.51.8 ± 0.60Glu1554.2 ± 0.42.4 ± 0.2-1.8 ± 0.4Met1576.0 ± 0.42.4 ± 0.3-3.6 ± 0.5Lys15833.0 ± 0.614.4 ± 0.4-18.6 ± 0.7Glu16141.4 ± 3.071.4 ± 2.430.0 ± 3.8a Resonance assignments for residues 81–86 could not be confirmed. Residues Glu96, Leu97, Leu100, Phe101, Asp109, Leu114, Glu116, Lys118, Ile119, Leu121, Ala123, Asp131, Leu136, and Tyr150 were excluded from analysis due to peak overlap in the 1H-15N correlation spectra in one or both complexes, whereas Asp105, Lys106, Ile112, Leu117, Ile128, Asp145, Ile148, Asp149, Asp151, Phe153, Leu154, Phe156, and Val160 could not be reliably measuredb Exchange rates were measured by fitting the relative 1H-15N cross-peak volume versus exchange time with a single first-order exponentialc Calculated by subtracting the measured H/D exchange rate for each residue in CcTnC-NcTnI from the measured H/D exchange rate for the same residue in CcTnC-NcTnI(S43/45D). A negative value indicates H/D exchange decreased in the phosphomimetic complex. A positive value indicates that H/D exchange increased in the phosphomimetic complex Open table in a new tab Fig. 4Structure of the C-lobe of Ca2+-loaded cTnC bound to NcTnI (1SCV), colored according to perturbations in H/D exchange rates induced by the introduction of a negative charge at S43D/S45D in NcTnI. Residues (expressed as single letter amino acid abbreviations coupled with position numbers) are colored red and blue to indicate exchange rates that increased or decreased, respectively, in the phosphomimetic complex. The residues for which the amide H/D exchange rates did not change, were too fast to measure, or could not be determined are colored white. NcTnI is shown in gray. The positions of Ser43/Ser45 in NcTnI are labeled in light gray.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In contrast, comparison of the Asn107 and Asn143 1H-15N cross-peaks in correlation spectra after 330 min of exchange in 2H2O show that these amide protons exchange slower in the PKC phosphomimetic complex (Fig. 3). Both Asn107 and Asn143 occupy position 3 within Ca2+/Mg2+ binding loops III and IV, respectively. The amide proton of the residue at position 3 is known to hydrogen bond with the side-chain carboxylate of Asp at position 12 (26Strynadka N.L. James M.N. Annu. Rev. Biochem. 1989; 58: 951-998Crossref PubMed Google Scholar). The decrease in amide proton H/D exchange is consistent with increased hydrogen bonding interactions between the amide protons of Asn107 and Asn143 and the carboxylate groups of Glu116 and Glu152, respectively. Decreased amide H/D exchange in the PKC phosphomimetic complex is also observed for Ala108, Gly110, Asp113, and Glu115 in site III and for Gly146 in site IV (Table I and Fig. 4). The side chain of Asp113, located at the ninth position in Ca2+/Mg2+ binding loop III, directly coordinates the bound metal ion. Amide protons of Gly110 and Gly146, located at the sixth position in Ca2+/Mg2+ binding loop III and IV, respectively, hydrogen bond to the carboxylate side chain of the conserved Asp residues at position 1 (12Brito R.M. Krudy G.A. Negele J.C. Putkey J.A. Rosevear P.R. J. Biol. Chem. 1993; 268: 20966-20973Abstract Full Text PDF PubMed Google Scholar). Decreased H/D exchange for residues within Ca2+/Mg2+ binding loops III and IV is consistent with the stabilization of intra-loop hydrogen bonding interactions and the compaction of the metal binding loops in the phosphomimetic complex. These changes suggest that phosphorylation of NcTnI alters the conformation of Ca2+/Mg2+-binding sites III and IV, possibly resulting in altered metal ion affinity and Ca2+/Mg2+ exchange. Summary—The structural consequences of PKC phosphorylation at Ser43/Ser45 of cTnI on cTnC have been examined using chemical shift mapping and H/D exchange. To facilitate structural studies, PKC phosphorylation mimetics of cTnI having Ser43/Ser45 mutated to Asp were utilized. The overall picture obtained from chemical shift mapping shows structural perturbations predominately localized to the C-lobe of cTnC, with smaller N-lobe perturbations around Ca2+-binding site II (Fig. 1). Distances between N-lobe Ca2+-binding sites and Ser43/Ser45 of cTnI in the core cardiac troponin structure (2Takeda S. Yamashita A. Maeda K. Maeda Y. Nature. 2003; 424: 35-41Crossref PubMed Scopus (642) Google Scholar) suggest that chemical shift perturbations in Ca2+-binding site II are the result of long range effects as opposed to direct binding interactions. Recently, measurement of fluorescence resonance energy transfer distance distributions from a single cTnC donor/acceptor pair in cTnC-cTnI and cTnC-cTnI(S43E/S45E, T144E) showed that the introduction of a negative charge in cTnI alters N-lobe conformational equilibria (3Kobayashi T. Dong W-J. Burkart E.M. Cheung H.C. Solaro R.J. Biochemistry. 2004; 43: 5996-6004Crossref PubMed Scopus (23) Google Scholar). Previously we showed that amide chemical shifts could be utilized to monitor conformation equilibria between open and closed N-lobe substates (16Abbott M.B. Gaponenko V. Abusamhadneh E. Finley N. Li G. Dvoretsky A. Rance M. Solaro R.J. Rosevear P.R. J. Biol. Chem. 2000; 275: 20610-20617Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). We see no evidence that the introduction of a negative charge at Ser43/Ser45 of cTnI significantly alters conformational equilibria in the N-lobe of cTnC (Fig. 1). It is likely that the N-lobe conformational change detected by fluorescence resonance energy transfer analysis (3Kobayashi T. Dong W-J. Burkart E.M. Cheung H.C. Solaro R.J. Biochemistry. 2004; 43: 5996-6004Crossref PubMed Scopus (23) Google Scholar) results from the additional negative charge at Thr144 of cTnI. This probability is consistent with N-lobe conformational changes detected by NMR chemical shift analysis of cTnC binding to a cTnI regulatory peptide phosphorylated at Thr144 (19Li M.X. Wang X. Lindhout D.A. Buscemi N. Van Eyk J.E. Sykes B.D. Biochemistry. 2003; 42: 14460-14468Crossref PubMed Scopus (26) Google Scholar). Taken together, these results help clarify the molecular consequences of PKC phosphorylation at Ser43, Ser45, and Thr144 in cTnI on Ca2+-loaded cTnC. Phosphorylation of Ser43/Ser45 in cTnI induces conformational perturbations in the C-lobe of cTnC containing Ca2+/Mg2+-binding sites III and IV, whereas phosphorylation at Thr144 directly alters conformational equilibria in the N-lobe of cTnC containing Ca2+-binding site II. A combination of chemical shift mapping and H/D exchange was used to examine conformational perturbations in the Ca2+/Mg2+-dependent cTnC-cTnI interaction site induced by the introduction of a negative charge at Ser43/Ser45 of cTnI. Chemical shift mapping identified structural perturbations in CcTnC residues lining the NcTnI hydrophobic binding cleft (Fig. 2). Amide hydrogen/deuterium exchange results are consistent with the destabilization of N-terminal helix G-capping interactions and the conformational perturbation of Ca2+/Mg2+-binding sites III and IV (Fig. 4). Whereas metal binding at site III is primarily responsible for stabilizing the hydrophobic core (12Brito R.M. Krudy G.A. Negele J.C. Putkey J.A. Rosevear P.R. J. Biol. Chem. 1993; 268: 20966-20973Abstract Full Text PDF PubMed Google Scholar), helix-capping interactions are known to increase domain stability and accelerate folding (28Kapp G.T. Richardson J.S. Oas T.G. Biochemistry. 2004; 43: 3814-3823Crossref PubMed Scopus (16) Google Scholar). Destabilization of N-cap interactions in helices C and G by mutation have been shown to decrease Ca2+-binding affinity in TnC (15Trigo-Gonzalez G. Awang G. Racher K. Neden K. Borgford T. Biochemistry. 1993; 32: 9826-9831Crossref PubMed Scopus (24) Google Scholar, 27Leblanc L. Bennet A. Borgford T. Arch. Biochem. Biophys. 2000; 384: 296-304Crossref PubMed Scopus (10) Google Scholar). Thus, weakening of the hydrogen-bonding lattice in helix G would be expected to alter metal ion affinity and Ca2+/Mg2+ exchange at site IV. Microcalorimetry data suggest that Ca2+/Mg2+-dependent protein-protein interactions are 8-fold stronger in the presence of Ca2+ than in the presence of Mg2+ (29Calvert M.J. Ward D.G. Trayer H.R. Trayer I.P. J. Biol. Chem. 2000; 275: 32508-32515Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Substitution of Mg2+ for Ca2+ in CcTnC bound to NcTnI is characterized by condensation of the C-terminal portion of the metal binding loops and partial closure of the cTnI hydrophobic binding cleft around site IV (13Finley N.L. Howarth J.W. Rosevear P.R. Biochemistry. 2004; 43: 11371-11379Crossref PubMed Scopus (27) Google Scholar). The close association between helix stability and metal ion affinity provides an attractive model for modulating Ca2+/Mg2+ exchange and Ca2+/Mg2+-dependent protein-protein interactions by PKC phosphorylation. A negative charge introduced at Ser43/Ser45 of cTnI, either by mutation or phosphorylation, is expected to stabilize the NcTnI helix through favorable electrostatic interactions between neighboring polar side chains and the helix backbone (30Smart J.L. McCammon J.A. Biopolymers. 1999; 49: 225-233Crossref PubMed Scopus (39) Google Scholar). Residues 43–65 in cTnI form a α-helix that binds to the C-lobe of cTnC through multiple polar and van der Waals interactions (2Takeda S. Yamashita A. Maeda K. Maeda Y. Nature. 2003; 424: 35-41Crossref PubMed Scopus (642) Google Scholar). Computational analyses of cTnI having Glu substituted at Ser43/Ser45 suggest that the incorporation of a negative charge extends the N terminus of the cTnI helix to residue 40 (4Sumandea M.P. Burkart E.M. Kobayashi T. de Tombe P.P. Solaro R.J. Ann. N. Y. Acad. Sci. 2004; 1015: 39-52Crossref PubMed Scopus (72) Google Scholar). Solution analysis of the secondary structure of NcTnI bound to CcTnC, based on experimentally determined chemical shift index values, suggests that the NcTnI helix begins at residue 46. The mutation of Ser43/Ser45 to Asp stabilizes and extends the N terminus by three residues, from residue 46 in NcTnI to residue 43 in NcTnI(S43D/S45D). 2N. L. Finley, unpublished results. Structural changes in CcTnC and NcTnI induced by the phosphorylation of Ser43/Ser45 support a mechanism for PKC modulation of cardiac contractility wherein the introduction of a negative charge results in altered C-lobe metal ion affinities and perturbation of Ca2+/Mg2+-dependent protein-protein interactions. Structural changes in NcTnI and CcTnC, forming the Ca2+/Mg2+-dependent protein-protein interaction site, can then be transmitted to other regulatory or switch regions in troponin. These studies emphasize the importance of Ca2+/Mg2+-dependent cTnC-cTnI protein-protein interactions in the modulation of cardiac contractility." @default.
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• W1968121475 title "Introduction of Negative Charge Mimicking Protein Kinase C Phosphorylation of Cardiac Troponin I" @default.
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