Biochemistry zyxwvu 1992, 31, zyxwvut 1595-1602 1595 Conformational Changes in the Metal-Binding Sites of Cardiac Troponin C Induced by Calcium Binding? George A. Krudy, Rui M. M. Brito, John A. Putkey, and Paul R. Rosevear* Department of Biochemistry and Molecular Biology, University of Texas Medical School at Houston, Houston, Texas zy 77225 Received August 20, 1991; Revised Manuscript Received November 4, 1991 ABSTRACT: Isotope labeling of recombinant normal cardiac troponin C (cTnC3) with 15N-enriched amino acids and multidimensional N M R were used to assign the downfield-shifted amide protons of Gly residues at position 6 in Ca2+-binding loops 11, 111, and IV, as well as tightly hydrogen-bonded amides within the short antiparallel @-sheets between pairs of Ca2+-bindingloops. The amide protons of Gly70, Glyl 10, and Gly146 were found to be shifted significantly downfield from the remaining amide proton resonances in Ca2+-saturated cTnC3. No downfield-shifted Gly resonance was observed from the naturally inactive site I. Comparison of downfield-shifted amide protons in the Ca2+-saturatedforms of cTnC3 and CBM-IIA, a mutant having Asp65 replaced by Ala, demonstrated that Gly70 is hydrogen bonded to the carboxylate side chain of Asp65 Thus, the hydrogen bond between Gly and Asp in positions 6 and 1, respectively, of the Ca2+-binding loop appears crucial for maintaining the integrity of the helix-loop-helix Ca2+-binding sites. In the apo- form of cTnC3, only Gly70 was found to be shifted significantly downfield with respect to the remaining amide proton resonances. Thus, even in the absence of Ca2+at binding site 11, the amide proton of Gly70 is strongly hydrogen bonded to the side-chain carboxylate of Asp65. The amide protons of Ilell2 and Ile148 in the C-terminal domain and Ile36 in the N-terminal domain @-sheets exhibit chemical shifts consistent with hydrogen-bond formation between the pair of Ca2+-binding loops in each domain of Ca2+-saturatedcTnC3. In the absence of Ca2+,no strong hydrogen bonds were detected between the @-strands in the N-terminal domain of cTnC3. Thus, Ca2+ binding at site I1 results in a tightening of the Ca2+-binding loop and formation of one strong hydrogen bond between @-strands in the N-terminal domain. These changes may initiate movement of helices in the N-terminal domain responsible for the interaction of TnC with troponin I. Toponin C (TnC)' is the Ca2+ regulatory protein of the troponin complex responsible for initiating contraction in striated muscle. The binding of CaZ+ to TnC induces a con- formational change which is transmitted to the other com- ponents of the troponin complex and results in the release of inhibition of the myosin ATPase activity which leads to muscle contraction (Zot zyxwvutsrqp & Potter, 1987; Brenner, 1989). There are two isoforms of TnC: one isoform is found in fast skeletal muscle (sTnC) and the other is found in cardiac and slow skeletal muscle (cTnC). Although both isoforms have four potential Ca2+-binding sites, sites I-IV, site I in cTnC is in- active (Van Eerd & Takahashi, 1975, 1976; Collins et al., 1977). Recently, the cDNA for chicken cTnC was incorpo- rated into a bacterial expression system, and the bacterially produced protein (cTnC3) was found to be functionally identical to tissue-derived protein (Putkey et al., 1989; Sweeney et al., 1990). A functionally inactive mutant (CBM-IIA), which is unable to bind calcium at site I1 as a result of the conversion of Asp65 to Ala, directly demonstrated that Ca2+ binding to site I1 is responsible for initiating muscle contraction (Putkey et al., 1989). NMR studies have shown that the inability of CBM-IIA to trigger muscle contraction is due to inactivation of site I1 and not the effect of mutation on the conformation or stability of the protein (Brito et al., 1991). The crystal structures of turkey and chicken sTnC showed this isoform to consist of two globular domains separated by This work was supported in part by National Institutes of Health Grants GM41232 to P.R.R. and AR39218 to J.A.P., by a grant from the Robert Welch Foundation to J.A.P., and by Robert Welch foundation Grant (2-1041 to Frederick B. Rudolph for support of R.M.M.B. J.A.P. is the recipient of a Career Research Development Award from the NIH. *Author to whom correspondence should be addressed. 0006-2960/92/043 1-1595%03.00/0 a 31-residue a-helix (Herzberg & James, 1985, 1988; Herz- berg et al., 1986; Sundaralingam et al., 1985; Satyshur et al., 1988). Both the N- and C-terminal domains contain a pair of helix-loophelix Ca2+-binding motifs. Calcium-bindingsites I11 and IV, located in the C-terminal domain, have high af- finity for Ca2+ and also bind Mg*+ but with lower affinity (Van Eerd et al., 1975; Potter & Gergely, 1977; Collins et al., 1977). Calcium-binding site I1 in cTnC and sites I and I1 in sTnC bind Ca2+specifically, but with lower affinity than sites I11 and IV (Potter & Gergely, 1977; Holroyde et al., 1980). Calcium-binding site I of cTnC is inactive due to an amino acid insertion and several critical amino acid substitutions within the calcium-binding loop. The consensus Ca2+-binding loop of helix-loophelix cal- cium-binding proteins consists of 12 sequential residues (Strynadka & James, 1989). Residues at positions 1, 3, zy 5, 7, and 12 of the loop coordinate the Ca2+ion, with residue 12 providing bidentate coordination through both side-chain carboxylate oxygens (Figure 1). Thus, the Ca2+ ion is co- ordinated by seven ligands in a pentagonal bipyramidal ge- ometry. Positions l, 6, and 12 of the loop are invariably Asp, Gly, and Glu, respectively (Strynadka & James, 1989). In I Abbreviations: TnC, cardiac or fast skeletal troponin C; cTnC, cardiac TnC; sTnC, skeletal TnC; cTnC3, bacterially synthesized cTnC(desM 1 ,D2A); CBM-IIA, cTnC3(D65A); EDTA, ethylenedi- aminetetraacetic acid; CDTA, trans- zyxwv 1,2-diaminocyclohexane-N,N,N',- "-tetraacetic acid; DTT, 1,4-dithiothreitol; NMR, nuclear magnetic resonance; NOESY, two-dimensional proton nuclear Overhauser en- hancement spectroscopy; NOE, nuclear Overhauser effect; HSMQC, heteronuclear single- and multiple-quantum shift correlation spectros- copy; NOESY-HMQC, three-dimensional nuclear Overhauser en- hancement I5N-lH multiple-quantum coherence; 2D, two dimensional; 3D. three dimensional. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPON 0 1992 American Chemical Society