Calcium-induced folding of a fragment of calmodulin composed of EF-hands 2 and 3

Abstract

Calmodulin (CaM) is an EF-hand protein composed of two calcium (Ca(2+))-binding EF-hand motifs in its N-domain (EF-1 and EF-2) and two in its C-domain (EF-3 and EF-4). In this study, we examined the structure, dynamics, and Ca(2+)-binding properties of a fragment of CaM containing only EF-2 and EF-3 and the intervening linker sequence (CaM2/3). Based on NMR spectroscopic analyses, Ca(2+)-free CaM2/3 is predominantly unfolded, but upon binding Ca(2+), adopts a monomeric structure composed of two EF-hand motifs bridged by a short antiparallel beta-sheet. Despite having an "even-odd" pairing of EF-hands, the tertiary structure of CaM2/3 is similar to both the "odd-even" paired N- and C-domains of Ca(2+)-ligated CaM, with the conformationally flexible linker sequence adopting the role of an inter-EF-hand loop. However, unlike either CaM domain, CaM2/3 exhibits stepwise Ca(2+) binding with a K (d1) = 30 +/- 5 microM to EF-3, and a K (d2) > 1000 microM to EF-2. Binding of the first equivalent of Ca(2+) induces the cooperative folding of CaM2/3. In the case of native CaM, stacking interactions between four conserved aromatic residues help to hold the first and fourth helices of each EF-hand domain together, while the loop between EF-hands covalently tethers the second and third helices. In contrast, these aromatic residues lie along the second and third helices of CaM2/3, and thus are positioned adjacent to the loop between its "even-odd" paired EF-hands. This nonnative hydrophobic core packing may contribute to the weak Ca(2+) affinity exhibited by EF-2 in the context of CaM2/3.

Figure 1.

The sequence and NMR-derived secondary structure of CaM2/3. (A) The amino acid sequence of CaM2/3 (residues 46–113) is highlighted in light gray within the sequence of full-length human CaM. The sequences of EF-3 and EF-2 are also aligned with those of EF-1 and EF-4, respectively, using the 12-residue Ca²⁺-binding segments (red type). Conserved aromatic residues important for forming part of the hydrophobic core of each domain are highlighted in green. Residues corresponding to the linker between the N- and C-domains of native CaM are double underlined. Residues corresponding to the loops between EF-hands in each domain are single underlined. (B) The amino acid sequence of CaM2/3 with the positions of (h) helices (cylinder) and (b) β-strands (arrows) identified by PROMOTIF (Hutchinson and Thornton 1996) within the ensemble of NMR-derived structures for CaM2/3.

Figure 2.

Ca²⁺ titration of CaM2/3 monitored by CD spectropolarimetry. The mean ellipticity at 222 nm with standard deviations from four replicates (triangles), is plotted against total Ca²⁺ concentration and the fit line generated with the CaLigator software (Andre and Linse 2002). The plotted ellipticity data were not corrected for dilution. The total CaM2/3 concentration was 40 μM, and the apparent, stepwise Ca²⁺ dissociation constants are (mean and standard error) K _d1 = 30 ± 5 μM and K _d2 > 1000 μM (25°C, pH 7.4). Binding of the first Ca²⁺ accounts for ∼90% of the total induced CD change. (Inset) The far-UV CD spectra of Ca²⁺-free (dashed line) and fully Ca²⁺-ligated (solid line) CaM2/3.

Figure 3.

The ¹⁵N-HSQC spectra of ¹⁵N-CaM2/3. (A) The unassigned spectrum of Ca²⁺-free CaM2/3 at 25°C and pH 7.4. The poor ¹H_N dispersion indicates a lack of a predominant tertiary structure. The ¹⁵N HSQC spectra of 0.37 mM ¹⁵N-CaM2/3 in the presence of (B) 0.74 mM Ca²⁺ and (C) 1.8 mM Ca²⁺. Assigned peaks are numbered as in native CaM, with horizontal lines connecting resonances from ¹⁵NH₂ groups. The insets with dashed lines correspond to the crowded regions near the center of the spectra. The insets bordered with a solid line show the downfield-shifted signals from G98 and G61. The peak for A57 is aliased from 130.9 ppm, and signals corresponding to residues A46 and E47 were not detected. Consistent with the K _d values from CD-monitored titrations, the spectrum of CaM2/3 with equimolar Ca²⁺ was very similar to that of B, whereas signals from the amides of G61 and I100 were only observed in the presence of significantly higher concentrations of Ca²⁺ (C). Note that while 1.8 mM Ca²⁺ produces ∼65% occupancy of EF-2, the ¹⁵N-HSQC spectrum of CaM2/3 does not significantly change in the presence of 10 mM Ca²⁺, as used for subsequent structural studies to ensure >90% occupancy.

Figure 4.

The NMR-derived structural ensemble of Ca²⁺-ligated CaM2/3. (A) The lowest-energy structure (left) and 20-member structural ensemble of CaM2/3 superimposed onto each other using the backbone heavy atoms (C_α, C′, and N) within the helical regions. Following the nomenclature for native CaM, the helices are labeled C through F (residues 48–55, 65–73, 82–92, 102–109). (B) The hydrophobic side chains (green) within the lowest energy structure (left) and structural ensemble of CaM2/3, superimposed on the backbone of the lowest energy structure. In panels A and B, the helices are identified in red, the Ca²⁺ binding segments blue, the β-sheet cyan, the flexible linker region yellow, the disordered regions at the N- and C-termini (residues 46–47 and 110–113) magenta, and the Ca²⁺ ions black. (C) The surface representation of the lowest energy structure of CaM2/3. The hydrophobic residues are shaded green, aspartate and glutamate residues red, arginine and lysine residues blue, and the remaining residues gray. Residues R74, M76, R106, and M109 are labeled on the surfaces to assist in orientation.

Figure 5.

CaM2/3 superimposed onto the domains of CaM. (A,B) The backbone of the N- or C-domains of Ca²⁺-ligated CaM (gray) (1CLL.pdb) superimposed on the lowest-energy structure of CaM2/3 (colored according to Fig. 4). In this figure, the view of CaM2/3 is held approximately constant, and that of CaM varied, with the EF-hands of CaM black (the EF-hands as well as the N and C termini of CaM2/3 are red in A). (A, left) The superimposition of the N-domain EF-1 and EF-2 of Ca²⁺-ligated CaM on EF-2 and EF-3 of CaM2/3, respectively. (Right) The opposite superimposition (i.e., EF-2 and EF-1 of CaM on EF-2 and EF-3 of CaM2/3, respectively). (B, left) The superimposition of the C-domain EF-3 and EF-4 of Ca²⁺-ligated CaM on EF-2 and EF-3 of CaM2/3, respectively. (Right) The opposite superimposition. (C) The superimposition from A, (right) along with the crystal structure of the N-domain of Ca²⁺-free CaM (1QX5.pdb; green) displayed as transparent C_α tubes. The aromatic residues involved in stacking interactions are shown in gray for Ca²⁺-ligated CaM, green for Ca²⁺-free CaM, and according to secondary structure as in Figure 4 for CaM2/3. All superimpositions were made using backbone atoms C_α, C′, and N over the Ca²⁺-binding segments and eight residues of the flanking helical regions. See Table 3 for the corresponding RMSDs. The disordered regions at the N and C termini (residues 45–47 and 110–113) of CaM2/3 and the Ca²⁺ ions are removed for clarity.

Figure 6.

Backbone ¹⁵N relaxation data for Ca²⁺-saturated CaM2/3. (A) The longitudinal (T ₁) and (B) transverse (T ₂) ¹⁵N relaxation times (and standard errors), and (C) the heteronuclear ¹H-{¹⁵N}NOE ratios measured for CaM2/3 at 25°C, along with (D) the fit anisotropic model free order parameters S ² and (E) the conformational exchange broadening (R _ex) terms. (F) The per-residue RMSDs of backbone heavy atoms (C_α, C′, and N) after superimposition of residues 48–71 and 82–106 for the CaM2/3 structural ensemble. Missing data correspond to P66 and residues with overlapping or very weak signals. The reduced S ² values and ¹H-{¹⁵N}NOE ratios, and the elevated RMSDs for residues near the N and C termini of CaM2/3, as well as the loop between helices D and E (corresponding to the linker region in native CaM) indicate that these regions are more conformationally dynamic, and less well structurally defined, than the remainder of the protein. A schematic of the secondary structure of CaM2/3 is also shown.

Figure 7.

Schematic representations showing the relative positions of conserved aromatic residues and the inter-EF-hand loop in (A) the C-domain of CaM, (B) an EF-hand homodimer (such as TnC3), and (C) CaM2/3. Helices are cylinders and the aromatic residues involved in aromatic stacking interactions are represented as hexagons. In the CaM domain, the aromatic residues stack on each other opposite the loop between EF-hands, and may help position the EF-hands for high-affinity, cooperative Ca²⁺ binding. In the EF-hand homodimer, the aromatic stacking interactions cannot form, whereas with CaM2/3, the aromatic stacking interactions occur, but in a position adjacent to the loop between EF-hands. This may lead to low affinity for binding the second Ca²⁺ by these nonnative EF-hand domains. An EF-hand heterodimer would look like A, but without the loop between EF-hands. (D) The superimposition of CaM2/3 (dark gray) onto the C-domain of Ca²⁺-ligated CaM (light gray). The structures are arranged such that EF-3 and EF-4 of CaM are superimposed on EF-2 and EF-3 (labeled) of CaM2/3, respectively. As a result, the loop regions overlap, whereas the aromatic residues do not. The Ca²⁺ ions of CaM2/3 are black spheres.