The solution structure of the Mg2+ form of soybean calmodulin isoform 4 reveals unique features of plant calmodulins in resting cells

Abstract

Soybean calmodulin isoform 4 (sCaM4) is a plant calcium-binding protein, regulating cellular responses to the second messenger Ca(2+). We have found that the metal ion free (apo-) form of sCaM4 possesses a half unfolded structure, with the N-terminal domain unfolded and the C-terminal domain folded. This result was unexpected as the apo-forms of both soybean calmodulin isoform 1 (sCaM1) and mammalian CaM (mCaM) are fully folded. Because of the fact that free Mg(2+) ions are always present at high concentrations in cells (0.5-2 mM), we suggest that Mg(2+) should be bound to sCaM4 in nonactivated cells. CD studies revealed that in the presence of Mg(2+) the initially unfolded N-terminal domain of sCaM4 folds into an alpha-helix-rich structure, similar to the Ca(2+) form. We have used the NMR backbone residual dipolar coupling restraints (1)D(NH), (1)D(C alpha H alpha), and (1)D(C'C alpha) to determine the solution structure of the N-terminal domain of Mg(2+)-sCaM4 (Mg(2+)-sCaM4-NT). Compared with the known structure of Ca(2+)-sCaM4, the structure of the Mg(2+)-sCaM4-NT does not fully open the hydrophobic pocket, which was further confirmed by the use of the fluorescent probe ANS. Tryptophan fluorescence experiments were used to study the interactions between Mg(2+)-sCaM4 and CaM-binding peptides derived from smooth muscle myosin light chain kinase and plant glutamate decarboxylase. These results suggest that Mg(2+)-sCaM4 does not bind to Ca(2+)-CaM target peptides and therefore is functionally similar to apo-mCaM. The Mg(2+)- and apo-structures of the sCaM4-NT provide unique insights into the structure and function of some plant calmodulins in resting cells.

Figure 1

HSQC spectra of apo-sCaM4 (A) N-terminal domain (NT) and (B) C-terminal domain (CT). Both samples contain 0.5 mM ¹⁵N-labeled protein, 2 mM EDTA, 100 mM KCl, 5 mM DTT, and pH 7.0 ± 0.1.

Figure 2

Circular dichroism (CD) spectra of sCaM4-NT (A) and sCaM4-CT (B) at apo- (—), Mg- (……), and Ca- (-- -- --) solution conditions, recorded at room temperature with 10 μM protein samples.

Figure 3

Assigned HSQC spectrum of Mg²⁺-sCaM4. The nearly complete assignment of the N-lobe could be obtained. However, the backbone assignment of the C-terminal domain could only be partially obtained because of intermediate exchange on the NMR chemical shift time scale. The regions in the C-lobe that could be identified are from the helices but the two Mg²⁺-saturated EF-hand loops could not be detected. Similar problems were encountered when the structure of the Mg²⁺ form of mCaM was determined by NMR, necessitating the introduction of mutations in the calcium-binding loops (PDB code 2EQC). The peaks with circles are on the Mg²⁺-binding loop 1 and the peaks with boxes are on the Mg²⁺-binding loop 2. The peaks with asterisks are folded over in the HSQC spectrum.

Figure 4

The comparison of two Mg²⁺-sCaM4-NT structures calculated with the same restraints but from two different starting models (A), and the comparisons of the Mg²⁺-sCaM4-NT structure (PBD code 2KSZ) with the apo-mCaM-NT structure (PDB code 1F70) (B) and the Ca-sCaM4-NT structure (PDB code 2ROA) (C). In (A), the blue structure is calculated from apo-sCaM4-NT as the starting model, which is generated from the apo-mCaM-NT structure (PDB code 1F70), and the red one is derived from Ca²⁺-sCaM4-NT structure (PDB code 2ROA). The red and blue balls are the coordinated Mg²⁺ ions. The rmsd of the backbone structure for residues 6–73 between these two structures is 1.00 Å. In (B) and (C), the structures in magenta, blue, and green colors represent apo-mCaM-NT, Mg²⁺-sCaM4-NT, and Ca²⁺-sCaM4-NT structures, respectively. The superposition of residues 6–73 of Mg²⁺-sCaM4-NT and apo-mCaM-NT results in an RMSD of 2.08 Å, and the corresponding one between Mg²⁺-sCaM4-NT and Ca²⁺-sCaM4-NT is 3.58 Å.

Figure 5

Fluorescence spectra of 1-anilino-8-naphthalene sulfonate (ANS) when it interacts with sCaM4-NT (A) and sCaM4-CT (B) recorded for apo-, Mg²⁺-, and Ca²⁺-solution conditions.

Figure 6

Fluorescence spectra of two CaM-binding peptides containing a Trp residue, studying the interaction between these peptides with sCaM4-NT at apo- (—), Mg- (-- -- --), and Ca- (…▵…) solution conditions, recorded at room temperature with 10 μM peptide samples. (A) Interaction between plant glutamate decarboxylase (GAD) peptide and sCaM4; (B) interaction between smooth muscle form of myosin light chain kinase (smMLCK) and sCaM4.

Figure 7

Comparisons of Mg²⁺-structures to corresponding apo- or Ca²⁺-structure for CaBP1, mCaM, and cTnC. (A) Superposition of Mg²⁺-CaBP1 C-domain (blue, PDB code 2K7C) and Ca²⁺-CaBP1 C-domain (red, PDB code 2K7D); (B) superposition of Mg²⁺-cTnC C-domain in complex with cTnI (blue, PDB code 1SBJ) and Ca²⁺-cTnC C-domain in complex with cTnI (red, PDB code 1FI5); (C) superposition of Mg²⁺-mCaM E104D/E140D mutant C-domain (blue, PDB code 2EQC) and Ca²⁺-mCaM C-domain (red, PDB code 1J7P); (D) superposition of apo-mCaM C-domain (purple, PDB code 1F70) and Mg²⁺-mCaM E104D/E140D mutant C-domain (blue, PDB code 2EQC).