PMC

[¹⁵N]C1C2 NMR titration with Ca²⁺/CaM. Overlay of ¹⁵N HSQC spectra of human C1C2 before (cyan) and after the addition of Ca²⁺/CaM (magenta) at a molar ratio of 1:1 ([[¹⁵N]C1C2]/[Ca²⁺/CaM]). Inset, chemical shift changes of indole resonances of Trp¹⁹⁶, Trp³²², and Trp³⁹⁶.

¹⁵N-Labeled Ca²⁺/CaM NMR titration with C1C2. A, graph showing relative changes in peak intensity for assigned ¹⁵N-labeled Ca²⁺/CaM residues upon the addition of C1C2 at a molar ratio of 0.2:1 (C1C2 to ¹⁵N-labeled Ca²⁺/CaM). The horizontal line indicates 1 S.D. above the mean intensity change. Red bars correspond to residues that were deemed to have undergone significant intensity changes (>1 S.D.). B, annotated structure of Ca²⁺/CaM (PDB 1CLL) showing that residues that undergo significant intensity changes (>1 S.D.) upon C1C2 binding are located in the N- and C-terminal lobes (shown as red spheres). Bound calcium atoms are shown as black spheres.

SAXS profiles of Ca²⁺/CaM and Ca²⁺/CaM-Cpep complex. A, SAXS data as I(q) versus q for Ca²⁺/CaM (♢) and the Ca²⁺/CaM-Cpep complex (□) with the GNOM fits (white lines) superposed. The measured data were all placed on an absolute scale using scattering from water but are shown here multiplied on the I(q) axis for clarity. B, P(r) versus r calculated using the data in A and normalized to the ratio of the square of the molecular mass of each protein. The symbols are as for A, and error bars (not shown; corresponding to ± 1 S.D.) are smaller than the symbols. Inset, Guinier plots of the SAXS data for Ca²⁺/CaM (♢) and Ca²⁺/CaM-Cpep complex (□) and linear fits (line) to the data at qR_g < 1.3. C, shape reconstructions of Ca²⁺/CaM and Ca²⁺/CaM-Cpep overlaid with the crystal structure of Ca²⁺/CaM. Crystal structure of Ca²⁺/CaM (PDB 1CLL) is shown in a ribbon diagram, and shape reconstructions of Ca²⁺/CaM alone and Ca²⁺/CaM-Cpep are shown as a gray surface and in black dots, respectively.

Affinities (K_D) of cMyBP-C motif and peptides binding to Ca²⁺/CaM determined from tryptophan fluorescence experiments. For each titration data set, the data points and fitted curves were obtained using the same approaches described in . For clarity of representation, each titration data set has been scaled by multiplying the following constants in the (F/F₀) values: motif, 0.9 (●); motif in 50 mm NaCl, 0.7 (■); Cpep, 0.2 (▴); and Npep, 0.1 (×). Each data point is the average of eight replicate measurements after correcting for solvent; the error bars (not shown; corresponding to ± 1 S.D.) are smaller than the symbols. All titrations were performed in the standard buffer except for “motif in 50 mm NaCl” data, which used a reduced NaCl concentration (see details under “Experimental Procedures”).

Cpep-binding interfaces of Ca²⁺/CaM. A, weighted chemical shift changes Δδ_ppm (H^N, H) for assigned residues of ¹⁵N-labeled Ca²⁺/CaM following the addition of Cpep at a molar ratio of 4:1 (Cpep/[¹⁵N]CaM). The horizontal line indicates 1 S.D. above the mean chemical shift change. Red bars correspond to residues deemed to have undergone significant chemical shift changes (>1 S.D.). Cyan bars correspond to residues that underwent even larger chemical shift changes but could not be traced during the titration or did not reappear in the spectrum. B, annotated structure of Ca²⁺/CaM (PDB 1CCL) showing the locations of the perturbed residues as identified in A. Residues corresponding to cyan bars and red bars in A are shown as cyan and red spheres, respectively. Bound calcium atoms are shown as black spheres. For clarity, the left panel highlights only the cyan spheres, and the right panel highlights both cyan and red spheres. C, examples of the fitted binding curves for residues in fast exchange, including Ala⁵⁷, Val¹²¹, and Ile¹³⁰ (see for corresponding K_D values).

SDS-PAGE pull-down assays indicating interactions between different cMyBP-C N-terminal fragments and CaM. The effects of Ca²⁺ on the interactions are tested by comparing results obtained in the presence of Ca²⁺ or with EGTA. Samples in lanes from left to right are as follows. Lane 1, molecular weight (M_w) standards; lane 2, CaM-agarose beads only; lanes 3 and 4, C0C1 + CaM-agarose; lanes 5 and 6, C1 + CaM-agarose; lanes 7 and 8, C1C2 + CaM-agarose; lanes 9 and 10, C0C2 + CaM-agarose. For each of the paired lanes (lanes 3 and 4, 5 and 6, 7 and 8, and 9 and 10), the left lane is with Ca²⁺, and the right is with EGTA. The calculated molecular masses for C0C2, C0C1, C1C2, and C1 are 49, 27, 33, and 11 kDa, respectively, based on their amino acid sequences. C0C1 and C1 both consistently migrate at “larger than expected” molecular masses.

N-terminal modules and the motif of cMyBP-C. A, schematic representation of the three N-terminal Ig modules (C0, C1, and C2) and their connectors: the proline/alanine-rich linker (P/A_L) and the motif (m). The approximate molecular weight (M_w) of each region is indicated, and the color coding for different regions of sequence/structure is conserved in panels A, B, and C. B, ribbon diagrams of (from left to right) the human C1 (PDB 2AVG) module, the structured region of mouse motif (residues 315–351) (PDB 2LHU), and the human C2 (PDB 1PD6) module with tryptophan residues (Trp¹⁹¹, Trp¹⁹⁶, Trp³¹⁸, and Trp³⁹⁶) shown as red sticks. Note that Trp³¹⁸ in the mouse sequence is equivalent to Trp³²² in the human sequence. In the mouse motif ribbon diagram (middle), the sequence corresponding to human Cpep used in this study (incorporating Trp³¹⁸) is shown in yellow, and the remaining sequence is orange. C, sequence alignments of human and mouse cMyBP-C motifs and of human motif peptides (Npep and Cpep). For the human motif, the three putative phosphorylation sites are marked by asterisks above the sequence, and the only tryptophan residue, Trp³²², is highlighted in red. Sequences corresponding to the structured region of mouse motif (amino acids (aa) 315–351) are highlighted in yellow and orange as in B. Note that for the human triphosphorylation mimic C1C2EEE, the three putative phosphorylation sites are mutated to glutamates (i.e. S275E/S284E/S304E).

Tryptophan fluorescence from the cMyBP-C N-terminal fragments C1C2 and C1C2EEE titrated with Ca²⁺/CaM. A, fluorescence emission spectra for the titration of C1C2 with CaM in the standard buffer and in the presence of Ca²⁺. A blue shift of the maximum emission wavelength is accompanied by a decrease in the maximum fluorescence intensity. The black line corresponds to the spectrum of the free protein before adding Ca²⁺/CaM, and the gray lines represent spectra from the sequential titration points. B, determination of binding affinities (K_D) from different titration experiments. For each titration data set, the symbols represent the experimental data of the fluorescence intensity at each titration step (F) relative to the fluorescence in the absence of Ca²⁺/CaM (F₀) at a chosen wavelength. The lines are fitted curves for determination of K_D based on a 1:1 model. For clarity of representation, each titration data set has been scaled by multiplying with the following constants in the (F/F₀) values: C1C2EEE, 1.4 (♦); C1C2, 1.0 (▴); C1C2 in 100 mm NaCl, 0.6 (△); and C1C2 in EGTA, 0.2 (○). Each data point is the average of eight replicate measurements after correcting for solvent; the error bars (not shown; corresponding to ±1 S.D.) are smaller than the symbols. Note that “C1C2 in 100 mm NaCl” data were acquired in buffer containing 100 mm NaCl, and “C1C2 in EGTA” data were acquired in buffer containing 10 mm EGTA; the rest of the titrations were performed in the standard buffer containing 350 mm NaCl (for details, see “Experimental Procedures”).