Structural studies of soybean calmodulin isoform 4 bound to the calmodulin-binding domain of tobacco mitogen-activated protein kinase phosphatase-1 provide insights into a sequential target binding mode

Abstract

The calcium regulatory protein calmodulin (CaM) binds in a calcium-dependent manner to numerous target proteins. The calmodulin-binding domain (CaMBD) region of Nicotiana tabacum MAPK phosphatase has an amino acid sequence that does not resemble the CaMBD of any other known Ca(2+)-CaM-binding proteins. Using a unique fusion protein strategy, we have been able to obtain a high resolution solution structure of the complex of soybean Ca(2+)-CaM4 (SCaM4) and this CaMBD. Complete isotope labeling of both parts of the complex in the fusion protein greatly facilitated the structure determination by NMR. The 12-residue CaMBD region was found to bind exclusively to the C-lobe of SCaM4. A specific Trp and Leu side chain are utilized to facilitate strong binding through a novel "double anchor" motif. Moreover, the orientation of the helical peptide on the surface of Ca(2+)-SCaM4 is distinct from other known complexes. The N-lobe of Ca(2+)-SCaM4 in the complex remains free for additional interactions and could possibly act as a calcium-dependent adapter protein. Signaling through the MAPK pathway and increases in intracellular Ca(2+) are both hallmarks of the plant stress response, and our data support the notion that coordination of these responses may occur through the formation of a unique CaM-MAPK phosphatase multiprotein complex.

FIGURE 1.

a, amino acid sequences of CaMBD from tobacco NtMKP1 (residues 438–449), Arabidopsis AtMKP1 (residues 451–462), and rice OsMKP1 (residues 456–467) are compared with various CaMBDs. The sequences are aligned at the position of the first hydrophobic anchor residue. The hydrophobic anchor residues are colored in red, while the other hydrophobic residues are shown in green. The basic residues and acidic residues are colored in cyan and pink, respectively. The residue numbers of the NtMKP1 sequence in SCaM4-NtMKP1/NtMKP1 protein are also indicated. b, schematic drawing of the two fusion proteins, SCaM4-NtMKP1 and SCaM4CT-NtMKP1.

FIGURE 2.

¹H,¹⁵N-HSQC spectrum of Ca²⁺-SCaM4CT-NtMKP1 with assignments indicated. The signals originating from the NtMKP1 domain and the poly-Gly linker region are circled by a solid and a dashed line, respectively.

FIGURE 3.

Structural data for SCaM4CT-NtMKP1. a, CSI data. The chemical shift deviations from random coil for the backbone Cα and C' atoms are added and plotted as a function of the residue number. The secondary structures obtained from the calculated structure are also shown. The boxes and arrows indicate the positions of the five α-helices and two β-strands. b, number of upper distance restraints used for the structure calculation are shown as a function of the residue number. White, gray, dark gray, and black bars indicated the number of intraresidue, sequential, medium range (–4), and long range (5+) restraints, respectively. The top of each column represents the total number of the upper distance restraints, where the length of each color segment indicates the number of upper distance restraints for each range. c, r.m.s.d. values for the backbone and all the heavy atoms are shown with a solid and dashed line, respectively.

FIGURE 4.

a, backbones of 30 structures of SCaM4CT-NtMKP1 are superimposed for the well-folded region (residue 80–163). The SCaM4CT and NtMKP1 domain are shown in navy and yellow, respectively, while the poly-Gly linker region is shown in gray. b, ribbon representations of the structure with the lowest energy. The hydrophobic side chains of Trp¹⁵⁷, Leu¹⁶⁰, and Phe¹⁶⁴ are displayed.

FIGURE 5.

a, local interactions between SCaM4CT residues and the anchor residues of the NtMKP1 domain, Trp¹⁵⁷ and Leu¹⁶⁰. The side chains of the SCaM4CT and NtMKP1 domains are colored in purple and green, respectively. b, schematic showing observed NOEs between SCaM4CT and NtMKP1 domain.

FIGURE 6.

a, superposed ¹H,¹⁵N-HSQC spectra of SCaM4CT-NtMKP1 (red) and SCaM4-NtMKP1 (black). The assignments for the well separated signals are indicated. Chemical shift differences for ¹H and ¹⁵N atoms are then plotted as the function of residue number (b). c, superposed ct-HSQC spectra of SCaM4CT-NtMKP1 (red) and SCaM4-NtMKP1 (black). d, superposed ct-HSQC spectra of SCaM4CT-NtMKP1 (red) and SCaM4 complexed with a NtMKP1p (black). In panels c and d, the signal assigned as X originates from the Met residue in the cloning artifact. In panel d, the peak volume of the black signal marked with an asterisk is almost double that of the other signals, suggesting that two signals are overlapping.

FIGURE 7.

Structural comparison of SCaM4CT-NtMKP1 with the CaM complexes with the CaMBD from RYR1 (a) and smMLCK (b). Only the C-lobes of CaM (residues 78–149) and the bound CaMBDs are displayed. In both panels, SCaM4CT-NtMKP1 is shown in navy and the other complexes are shown in yellow. The side chains of the two anchor residues of SCaM4CT-NtMKP1, Trp¹⁵⁷ and Leu¹⁶⁰, and Arg¹⁶¹ that forms a hydrogen bond to Glu⁸⁴ are shown. The residues at positions 1, 4, and 5 (see Fig. 1) are shown in green, red, and cyan, respectively, in all the complexes, while Glu⁸⁴ is colored magenta.

FIGURE 8.

¹⁵N relaxation data for SCaM4CT-NtMKP1 (open circle) and SCaM4-NtMKP1 (filled circle). The correlation time of global tumbling (τ_m) is calculated from the R₂/R₁ ratio. The positions of the secondary elements of SCaM4CT-NtMKP1 structure are shown in the same definition with Fig. 3.

FIGURE 9.

ITC experiments with SCaM4-NtMKP1 and NtMKP1 peptides. The top panels show the base line-corrected raw calorimetric traces of SCaM4-NtMKP1 with NtMKP1pA (a) and NtMKP1pB (b). The bottom panels are the derived binding isotherms for the SCaM4-NtMKP1 binding to NtMKP1pA (a) and NtMKP1pB (b). All experiments were performed at 30 °C, 100 mm KCl, 2 mm CaCl₂, 20 mm HEPES (pH 7.5).

FIGURE 10.

CSPs caused by the addition of NtMKP1pA (left) or NtMKP1pB (right) monitored by ¹H,¹⁵N-HSQC spectrum of SCaM4-NtMKP1. a, spectra of SCaM4-NtMKP1 acquired with 0.0 (black), 0.4 (red), 0.8 (green), and 1.2 (purple) molar equivalents of NtMKP1 peptides are overlaid. The separated signals with clear CSP are labeled. b, The CSP values are plotted as the function of residue number. c, the residues with CSP larger than 0.2 (red), 0.12 (orange), and 0.07 (green) are highlighted on the structure. The N- and C-lobe structures used are 2ROA and SCaM4CT-NtMKP1, respectively.

FIGURE 11.

Models for possible complex formation induced by the Ca²⁺-CaM-NtMKP1 interactions. The data obtained in this work revealed that the interactions of the N-lobe of the SCaM4-NtMKP1 fusion protein with CaMBD1 and CaMBD2 are relatively weak suggesting that the model depicted in panel d, where Ca²⁺-CaM act as a Ca²⁺-dependent adaptor protein, appears likely.