Domain swapping and different oligomeric States for the complex between calmodulin and the calmodulin-binding domain of calcineurin a.

Viivi Majava,Petri Kursula

doi:10.1371/journal.pone.0005402

Abstract

BackgroundCalmodulin (CaM) is a ubiquitously expressed calcium sensor that engages in regulatory interactions with a large number of cellular proteins. Previously, a unique mode of CaM target recognition has been observed in the crystal structure of a complex between CaM and the CaM-binding domain of calcineurin A.Methodology/Principal FindingsWe have solved a high-resolution crystal structure of a complex between CaM and the CaM-binding domain of calcineurin A in a novel crystal form, which shows a dimeric assembly of calmodulin, as observed before in the crystal state. We note that the conformation of CaM in this complex is very similar to that of unliganded CaM, and a detailed analysis revels that the CaM-binding motif in calcineurin A is of a novel ‘1-11’ type. However, using small-angle X-ray scattering (SAXS), we show that the complex is fully monomeric in solution, and a structure of a canonically collapsed CaM-peptide complex can easily be fitted into the SAXS data. This result is also supported by size exclusion chromatography, where the addition of the ligand peptide decreases the apparent size of CaM. In addition, we studied the energetics of binding by isothermal titration calorimetry and found them to closely resemble those observed previously for ligand peptides from CaM-dependent kinases.Conclusions/SignificanceOur results implicate that CaM can also form a complex with the CaM-binding domain of calcineurin in a 1∶1 stoichiometry, in addition to the previously observed 2∶2 arrangement in the crystal state. At the structural level, going from 2∶2 association to two 1∶1 complexes will require domain swapping in CaM, accompanied by the characteristic bending of the central linker helix between the two lobes of CaM.

Highlights

Calmodulin (CaM) is a calcium-dependent regulatory protein, able to interact with a plethora of target proteins having diverse functions [1]
The overall conformation of the CaM-calcineurin A (CnA)-calmodulin-binding domain (CBD) complex seen in the crystal is the same as in the earlier structures of this complex; most notably, the complex is formed by 2:2 association, such that CaM forms an X-shaped dimer (Figure 1A)
The binding mode is somewhat unexpected; while the ‘1-10’ and ‘1-14’ modes are the most common for CaM ligands, in the case of the CnA-CBD, the binding mode is ‘1-11’

Summary

Introduction

Calmodulin (CaM) is a calcium-dependent regulatory protein, able to interact with a plethora of target proteins having diverse functions [1]. Upon calcium binding to the four EF-hands of CaM, a hydrophobic pocket opens in each of the two lobes of CaM, which interact with large hydrophobic or aromatic residues from the target sequence. A growing number of non-classical modes of binding for CaM towards its target peptides have been detected [1,7,8,9,10,11]. Such modes involve differences in both the conformation of CaM, which may exist in an extended conformation in a complex, and the stoichiometry of the formed complex, not always being 1:1. A unique mode of CaM target recognition has been observed in the crystal structure of a complex between CaM and the CaM-binding domain of calcineurin A

Methods

Results

Conclusion