PDBsum entry 1y6w

Calcium-binding protein

PDB id: 1y6w

Contents

Protein chain

145 a.a.

Ligands

MPD

TBU

Metals

_CA ×4

Waters ×56

References listed in PDB file

Key reference

Title

Structure of a trapped intermediate of calmodulin: calcium regulation of ef-Hand proteins from a new perspective.

Author

Z.Grabarek.

Ref.

J Mol Biol, 2005, 346, 1351-1366. [DOI no: 10.1016/j.jmb.2005.01.004]

PubMed id

15713486

Abstract

Calmodulin (CaM) is a multifunctional Ca2+-binding protein that regulates the activity of many enzymes in response to changes in the intracellular Ca2+ concentration. There are two globular domains in CaM, each containing a pair of helix-loop-helix Ca2+-binding motifs called EF-hands. Ca2+-binding induces the opening of both domains thereby exposing hydrophobic pockets that provide binding sites for the target enzymes. Here, I present a 2.4 A resolution structure of a calmodulin mutant (CaM41/75) in which the N-terminal domain is locked in the closed conformation by a disulfide bond. CaM41/75 crystallized in a tetragonal lattice with the Ca2+ bound in all four EF-hands. In the closed N-terminal domain Ca ions are coordinated by the four protein ligands in positions 1, 3, 5 and 7 of the loop, and by two solvent ligands. The glutamate side-chain in the 12th position of the loop (Glu31 in site I and Glu67 in site II), which in the wild-type protein provides a bidentate Ca2+ ligand, remains in a distal position. Based on a comparison of CaM41/75 with other CaM and troponin C structures a detailed two-step mechanism of the Ca2+-binding process is proposed. Initially, the Ca2+ binds to the N-terminal part of the loop, thus generating a rigid link between the incoming helix (helix A, or helix C) and the central beta structure involving the residues in the sixth, seventh and eighth position of the loop. Then, the exiting helix (helix B or helix D) rotates causing the glutamate ligand in the 12th position to move into the vicinity of the immobilized Ca2+. An adjustment of the phi, psi backbone dihedral angles of the Ile residue in the eighth position is necessary and sufficient for the helix rotation and functions as a hinge. The model allows for a significant independence of the Ca2+-binding sites in a two-EF-hand domain.

Figure 3.
Figure 3. The overall structure of CaM41/75 compared with the wild-type CaM. The four helix-loop-helix EF- hand Ca²⁺ -binding sites are shown in different colors: site I, helices A and B, blue; site II, helices C and D, green; site III, helices E and F, magenta; and site IV, helices G and H, red. The central parts of the Ca²⁺ -binding loops that form short b-strands are shown in cyan. The yellow spheres represent the Ca ions. The linker regions (the N-terminal linker residues 3--5, the B/C linker, residues 40--44 and the F/G linker, residues 113--117) are shown in orange. Note the position of the disulfide bond in CaM41/75 connecting the central helix with the B/C linker. The PDB entry 1CLL was used for the wild-type CaM. This Figure was prepared with the POVScriptC 53 version of MOLSCRIPT 54 and rendered with POV-Ray (Persistence of Vision Raytracer Pty. Ltd).

Figure 4.
Figure 4. Structure of the Ca²⁺-binding loops in the N-terminal domain of CaM41/75; comparison with loop I of the wild-type CaM. The backbone atoms of residues 20--31 (site I) and residues 56--67 (site II) are shown. Only those side-chains that typically participate in the Ca²⁺-coordination are shown. The Ca ions are represented by the yellow spheres and the solvent molecules are shown in cyan; two water molecules in site I and an MPD molecule in site II. Parts of the helices are also shown for reference. Note the differences in the Ca²⁺ coordination geometry in CaM41/75 as compared to the wild-type protein (see the text for details).

The above figures are reprinted by permission from Elsevier: J Mol Biol (2005, 346, 1351-1366) copyright 2005.

Added reference #1^*

Title

Structural basis for diversity of the EF-hand calcium-binding proteins.

Author

Z.Grabarek.

Ref.

J Mol Biol, 2006, 359, 509-525. [DOI no: 10.1016/j.jmb.2006.03.066]

PubMed id

16678204

Abstract

Figure 1.
Figure 1. The EF-hand β-scaffold. (a) Comparison of the N-terminal domain of CaM in the Ca²⁺-bound open conformation (PDB code 1CLL) with the Ca²⁺-bound closed intermediate structure of CaM41/75 (PDB code 1Y6W). The EF-hand I of the native CaM is shown in blue and the EF-hand II in green. The structure of CaM41/75 is shown in gray. For superimposition of the two structures the backbone atoms of Thr26, Ile27, Thr62 and Ile63 were used (RMSD=0.36 Å for 16 atoms). These atoms form the structure referred to as the EF-hand β-scaffold. (b) Schematic representation of the EF-hand β-scaffold. The Ca²⁺ binding loops are connected by two hydrogen bonds between the centrally located branched hydrophobic residues R_I (Ile27 in EF-hand I of CaM) and R^'_I (Ile63 in EF-hand II of CaM). The carbonyl oxygen atoms of residues R_I−1 and ^'_I-1 are the invariant Ca²⁺-ligands. The distance between the bound Ca²⁺ is strictly defined by the bond network, as shown. Changes in the backbone , ψ angles of R_I and R^'_I in the directions shown by the arrows enable the last ligand, the glutamate side-chain in R_I+4 position to move into the Ca²⁺-coordinating position, thus closing the Ca²⁺-binding loop. The black diamond indicates an approximate 2-fold symmetry axis relating the odd and even EF-hands in the domain structure and it coincides with the Z axis of the local frame of reference used for structure comparison in Figure 3. The directions of the O- and N-axes of that frame of reference are indicated by the arrows. This Figure and the molecular graphics in Figure 2 and Figure 3 were prepared with the POVScript+^150 version of MOLSCRIPT ^151 and rendered with POV-Ray (Persistence of Vision Raytracer PTy. Ltd).

Figure 3.
Figure 3. Comparison of various EF-hand motif structures in a conformation-independent frame of reference. Shown are the structures of the EF-hand motifs in the “odd” position in: (a) calcyclin (14 residue loop); (b) osteonectin (13 residue loop); (c) calmodulin (the canonical 12 residue loop); (d) calpain (11 residue loop). The amino acid sequences are listed in Table 1, and the PDB codes and references are in the legend to Table 2. The Ca²⁺-bound structures are multicolored and the partial structures of the apo forms (where available) are shown in gray. The structures are shown in approximately the same orientation defined by the local frame of reference linked to the EFβ-scaffold. The backbone atoms of the EFβ-scaffold residues were used for the superimposition of the structures (RMSD range 0.29–0.43 for 16 atoms). The N-axis is defined by the vector connecting the nitrogen atom of the R_I residue with the nitrogen atom of the R^'_I residue of the EFβ-scaffold; similarly the O-axis is defined by the carbonyl oxygen atoms in the same residues (cf. Figure 1(b)). The Z-axis is a vector perpendicular to the plane of the EFβ-scaffold calculated by approximating the position of the N and O atoms of the R_I and R^'_I residues. The Z-axis coincides with the approximate non-crystallographic 2-fold symmetry axis of the domain. The N, O, Z coordinate system as defined above is approximately orthogonal, but depending on the exact position of the selected atoms (subject to structure refinement and the intrinsic protein dynamics) the axes may not intersect in one point. For simplicity of presentation the centroid of the EFβ-scaffold plane is used as the origin. Only a 1.5 turn short segment of each helix is shown. The Ca²⁺-ligands positioned in the equatorial plane of the pentagonal bipyramid are connected by semitransparent surface and the vector normal to that plane is shown (the broken green line). For vector calculations the program Mathematica 5.0 (Wolfram Research, Inc.) was used.

The above figures are reproduced from the cited reference with permission from Elsevier

^*Note, "added" references are those not in the PDB file but which have either been obtained from the journal or suggested by the author(s).