Conformational changes in the metal-binding sites of cardiac troponin C induced by calcium binding

Abstract

Isotope labeling of recombinant normal cardiac troponin C (cTnC3) with 15N-enriched amino acids and multidimensional NMR were used to assign the downfield-shifted amide protons of Gly residues at position 6 in Ca(2+)-binding loops II, III, and IV, as well as tightly hydrogen-bonded amides within the short antiparallel beta-sheets between pairs of Ca(2+)-binding loops. The amide protons of Gly70, Gly110, and Gly146 were found to be shifted significantly downfield from the remaining amide proton resonances in Ca(2+)-saturated cTnC3. No downfield-shifted Gly resonance was observed from the naturally inactive site I. Comparison of downfield-shifted amide protons in the Ca(2+)-saturated forms 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 Ca(2+)-binding loop appears crucial for maintaining the integrity of the helix-loop-helix Ca(2+)-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 II, the amide proton of Gly70 is strongly hydrogen bonded to the side-chain carboxylate of Asp65. The amide protons of Ile112 and Ile148 in the C-terminal domain and Ile36 in the N-terminal domain data-sheets exhibit chemical shifts consistent with hydrogen-bond formation between the pair of Ca(2+)-binding loops in each domain of Ca(2+)-saturated cTnC3.(ABSTRACT TRUNCATED AT 250 WORDS)

The Calmodulin-related Calcium Sensor CML42 Plays a Role in Trichome Branching

“…The large number of resonances located in the central region of the spectrum (7.5-8.5 ppm) suggests a protein fold comprising substantial random coil and ␣-helical structure; an observation commensurate with the CD data described above. (32)(33)(34)(35). Although the identity of these resonances has yet to be confirmed, the presence of two backbone H N resonances in this region of the Ca 2ϩ -free spectrum (Fig.…”

Disease-causing Mutations in Cartilage Oligomeric Matrix Protein Cause an Unstructured Ca2+ Binding Domain

“…In the presence of Ca 2ϩ , the amide proton of this Gly residue forms a strong hydrogen bond with the carboxyl oxygen of the acidic residue at loop position 1. This confers an unusually large down field chemical shift (8.9 -11.0 ppm) on the H N of this Gly in the EF hands of cardiac troponin C (19,20), skeletal troponin C (21), and calmodulin (22) that can be diagnostic for Ca 2ϩ binding. The circled cross-peaks centered on 10 ppm in Fig.…”

Interaction of cardiac troponin with cardiotonic drugs: A structural perspective

“…This indicates that the pathways involved in initiating skeletal and cardiac muscle contraction are structurally very similar; however, the kinetics and thermodynamics of the pathways must differ for the two systems to account for the different physiological behavior of the two muscle types [15]. NMR studies of TnC with various TnI peptides have yielded detailed structural information on the structure of TnC when bound to TnI [16][17][18][19], on the structure of TnI inhibitory peptide [20,21], and on the overall topology of TnC-TnI arrangement [22][23][24][25][26][27][28][29][30][31][32]. The high-resolution structures of TnC-TnI available are the X-ray structure of sTnC·2Ca 2+ ·sTnI [33], the NMR structures of cNTnC·Ca 2+ ·cTnI 147-163 [13], sNTnC(rhodamine)·2Ca 2+ ·sTnI 115-131 [14], and cCTnC·2Ca 2+ ·cTnI 128-147 [34], the X-ray structure of the core domain cardiac troponin complex, cTnC·3Ca 2+ ·cTnI ·cTnT2 182-288 [35], and the X-ray structures of skeletal troponin complex in both the apo and Ca 2+ -state, sTnC·apo·sTnI ·sTnT 156-262 and sTnC·4Ca 2+ ·sTnI ·sTnT [36].…”