PDBsum entry 1y6w

Calcium-binding protein

PDB id: 1y6w

Contents

Protein chain

145 a.a. *

Ligands

MPD

TBU

Metals

_CA ×4

Waters ×56

* Residue conservation analysis

PDB id:

1y6w

Name:

Calcium-binding protein

Title:

Trapped intermediate of calmodulin

Structure:

Calmodulin. Chain: a. Synonym: cam. Engineered: yes. Mutation: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: calm1, calm2, calm3. Expressed in: escherichia coli bl21. Expression_system_taxid: 511693.

Resolution:

2.40Å R-factor: 0.220 R-free: 0.238

Authors:

Z.Grabarek

Key ref:

Z.Grabarek (2005). Structure of a trapped intermediate of calmodulin: calcium regulation of EF-hand proteins from a new perspective. J Mol Biol, 346, 1351-1366. PubMed id: 15713486 DOI: 10.1016/j.jmb.2005.01.004

Date:

07-Dec-04 Release date: 01-Mar-05

Protein chain

P0DP23  (CALM1_HUMAN) -  Calmodulin-1 from Homo sapiens

Seq: 149 a.a.

Struc: 145 a.a.*

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

DOI no: 10.1016/j.jmb.2005.01.004

J Mol Biol 346:1351-1366 (2005)

PubMed id: 15713486

Structure of a trapped intermediate of calmodulin: calcium regulation of EF-hand proteins from a new perspective.

Z.Grabarek.

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.

Selected figure(s)

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.

Figures were selected by the author.

Author's comment

In this mutant of human calmodulin (CaM41/75) the N-terminal domain is locked in the closed (apo-like) conformation. A disulfide bond between Cys residues substituted for Gln41 in the B/C linker and Lys75 in the central helix prevents the opening of the domain, thus reducing the Ca²⁺-binding affinity and cooperativity, and rendering the protein incapable of activating the target enzymes. In the crystal structure of CaM41/75 all four EF-hands have bound Ca²⁺. In the locked domain the conformation of the Ca²⁺-binding loops is unusual. Calcium ion interacts only with the N-terminal part of the loop, while the bidentate ligand in the 12th position (Glu31 in site I and Glu67 in site II) remains distant. This structure provides evidence that the domain opening in CaM is directly coupled to the proximity of the bidentate Glu ligand in the 12th position to the Ca²⁺ immobilized by other ligands of the loop. In the structure of CaM41/75 two states that are mutually exclusive in the native CaM are captured: the closed domain conformation and the presence of Ca²⁺ at the Ca²⁺-binding loops. It is postulated that this structure corresponds to an intermediate state that occurs transiently in the native protein in the calcium binding process.
Based on analysis of the CaM41/75 and other EF-hand protein structures, a two-step Ca²⁺-binding mechanism is proposed. It is postulated that the Ca²⁺-binding and the resultant conformational response in a two EF-hand domain are governed by the central structure connecting the Ca²⁺-binding loops. This structure named EF-hand-beta-scaffold (or EFβ-scaffold) defines the position of the bound Ca²⁺, and coordinates the function of the flexible N-terminal with the rigid C-terminal parts of the Ca²⁺-binding loop. It is proposed that Ca²⁺ binds initially to the N-terminal part of the loop, thus generating a rigid link between the incoming helix and the EFβ-scaffold. In the second step, the backbone torsional flexibility of the EFβ-scaffold enables the exiting helix to change its orientation, so the bidentate C-terminal Glu ligand can move into the Ca²⁺ coordinating position. The ~2 Å shift of the bidentate Glu ligand that is required for closing the Ca²⁺-coordination sphere causes the exiting helix to move and drives the conformational change. It has been postulated that this model (referred to as the EFBS-model) is applicable to all EF-hand proteins including those containing non-canonical Ca²⁺-binding loops, and irrespective of the extent of the Ca²⁺-induced conformational change. For more detail see: Grabarek, Z., (2006), J. Mol. Biol. 359, 509-525.