A novel trans conformation of ligand-free calmodulin

Abstract

Calmodulin (CaM) is a highly conserved eukaryotic protein that binds specifically to more than 100 target proteins in response to calcium (Ca(2+)) signal. CaM adopts a considerable degree of structural plasticity to accomplish this physiological role; however, the nature and extent of this plasticity remain to be fully understood. Here, we report the crystal structure of a novel trans conformation of ligand-free CaM where the relative disposition of two lobes of CaM is different, a conformation to-date not reported. While no major structural changes were observed in the independent N- and C-lobes as compared with previously reported structures of Ca(2+)/CaM, the central helix was tilted by ~90° at Arg75. This is the first crystal structure of CaM to show a drastic conformational change in the central helix, and reveals one of several possible conformations of CaM to engage with its binding partner.

Figure 1. A novel trans conformation of calmodulin (CaM).

A: Ribbon representation of novel conformation of ligand-free CaM. The helices, loops, Ca²⁺ and Zn²⁺ are shown in blue, pale green, green and grey, respectively. The CaM molecule adopts an extended dumbbell-shaped conformation and the two domains are well separated. The bending at Arg75 is shown in stick representation. The α-helices are numbered from I-VIII. B: 2Fo-Fc electron density map for the region 65–80 aa of CaM. This map is contoured at a level of 1σ.

Figure 2. Comparison among various calmodulin (CaM) structures.

A: The Cα superposition of the present study novel trans conformation of CaM with several extended (trans) CaM conformations: 1PRW (magenta), 2F2P (white), 2W73 (red), and 3CLN (dark salmon). B: The Cα superposition of present study novel trans conformation of CaM with several wrapped (cis) CaM conformations: 2BE6 (yellow), 2F3Y (light blue), 2O60 (pale green), 2VAY (teal), 2X0G (orange), 3BXK (deep purple), 3DVE (gray), 1CDM (olive). CaM conformations can be classified as “wrapped” and “extended”. In “wrapped” conformation, the two lobes are close to each others in cis orientation. In “extended” conformation, the two lobes are widely separated in trans orientations. In the present study, CaM adopted a novel trans conformation. The positions of metal ions in the current structure (blue) are labeled as Ca²⁺ (Green) and Zn²⁺ (grey). These structure alignments were carried out in PyMol .

Figure 3. Comparison of cis and trans conformations of Calmodulin.

A: Side-by-side comparison of the novel trans (current structure, blue) and cis (pdb code 1PRW, magenta) conformations of CaM, which show the unwinding region of central helix in both structures in cyan and yellow, respectively. B: Side-by-side comparison of the novel trans (current structure, blue) and extended trans (pdb code 3CLN, dark salmon) conformations of CaM. In the extended trans conformation of CaM, no unwinding of the central helix was observed. The positions of the metal ions in the current novel trans (blue) are labeled as Ca²⁺ (Green) and Zn²⁺ (grey). In wrapped cis (1PRW, magenta) and extended trans (3CLN, dark salmon), all sites (EF1-EF4) were occupied by Ca²⁺ (Green).

Figure 4. Coordination of Zn²⁺ ion: A Zn²⁺ ion (grey) bound to His108 and Lys95 of chain A (magenta), and Asp81 and Glu85 from chain B (blue).

A similar Zn²⁺ ion is also present in chain A.