Elsevier

Chemical Physics Letters

Volumes 605–606, 17 June 2014, Pages 103-107
Chemical Physics Letters

Quantum chemical studies on the role of residues in calcium ion binding to Calmodulin

https://doi.org/10.1016/j.cplett.2014.05.017Get rights and content

Highlights

  • Coordination state molecular orbitals reveal role of ligand residues.

  • Non-ligand residues important in stabilizing coordination geometry.

  • Quantum chemical results rationalize mutational studies.

  • Coordination state molecular orbitals robust under system details.

Abstract

We perform density functional theory (DFT) based quantum chemical calculations on calcium ion coordination to an isolated loop of Calmodulin. We show that the coordination molecular orbitals in the ground state, having contributions from the valence orbitals of the metal ion and the loop atoms, bring out the roles of the coordinating and the non-coordinating residues to stabilize the coordination geometry in agreement to the mutational studies. The coordinating molecular orbitals are observed to be robust under various truncations of the binding loop and capping at the terminal residues.

Introduction

Microscopic quantum chemical (QC) calculation of bio-macromolecular binding is a formidable challenge due to large molecular sizes, comprising of thousands of atoms. The QC calculations are carried out by truncating the system: One considers the binding region alone, usually having about 10–100 atoms. Apart from large number of atoms involved in the binding process, the binding region in general possesses a variety of interactions. Since the truncation of the protein structure may affect some of these interactions, it is non-trivial to assess the effects of using such truncated systems on the results of the quantum calculations.

We address the robust features of the quantum ground state, describing the coordination of metal ion to the active sites of a protein [1]. Such metal-protein complexes are known as metalloproteins [2]. Different electronegative ligand atoms like oxygen (O), nitrogen (N) and sulfur (S) from amino acid side chain functional groups and backbone carbonyl O generally coordinate to metal ions [3]. In addition to acidic residues, there are in general polar, hydrophobic and basic residues in the binding region. When one takes into account only the binding region of the protein for QC calculations, some of these interactions are cut-off and replaced by capping at the terminal residues. We consider the levels in the ground state which remain largely invariant under a variety of truncations and terminal capping for calcium (Ca2+) ion coordination to Calmodulin (CaM), a well studied Ca2+ coordinating EF-hand [4], [5] metalloprotein, which participates in calcium sensing and signal transduction in eukaryotic cells [6].

The EF-hands exhibit a helix-loop-helix structure with conserved loop residues for a variety of metal ion binding proteins. Figure 1 shows the EF-hand loop 1 of CaM with the Ca2+ ion coordinated in pentagonal bipyramidal geometry through a number of acidic residues. The mutation of the coordinating bidentate acidic glutamate (E) leads to drastic loss in Ca2+ binding affinity [7]. Computer simulations report that the glutamate extends its side chain to grab Ca2+ and delivers it to the centre of the binding loop [8]. The reduced sensitivity for Ca2+ is also reported from the mutagenesis of the coordinating acidic aspartates (D) [4], [7]. The importance of the conserved hydrophobic isoleucine (I) in maintaining structural stability of the loop, has been reported through mutagenesis [7]. The loop has as well non-ligand basic lysines (K), polar threonines (T) and hydrophobic glycines (G). The metal ion and ligand binding has been studied by QC-DFT methods [2], [9], [10]. There have been DFT studies on the interaction of oxygen atoms in carboxylate (CO1O2) group of the acidic residues with the metal ion [2], considering only the functional group instead of the entire residues. Recent DFT calculations on isolated CaM loops [9] suggest that E31 has lower participation in charge transfer to the metal ion than the other residues, in contrast to the suggestions of the mutational studies. Earlier studies also show that the treatment of the solvent is essential to compute the energy states near the Highest Occupied Molecular Orbital (HOMO) and the Lowest Unoccupied Molecular Orbital (LUMO) with reasonable accuracy [11], [12]. Since the charge transfer is computed by taking contributions from all the molecular orbitals (MOs), the changes in the levels in the vicinity of HOMO and LUMO may affect the results quite non-trivially. Moreover, the charge transfer cannot address how the non-ligand residues contribute to the stability of the metal ion coordination, for they do not participate in transferring charge to the metal ion.

With this backdrop we perform the DFT based QC calculations for an isolated Ca2+–CaM loop 1 complex. Our primary objective is to extract the coordination picture, emphasizing the roles of both ligand and non-ligand residues as suggested by the mutational studies. We designate the energetically low-lying MOs in the ground state, having contributions from the valence orbitals of the loop atoms and the Ca2+ ion, as the coordination state MOs. These coordination state MOs yield a microscopic picture of the coordination, emphasizing the role of ligand and non-ligand residues, as suggested by the mutational studies. The coordination MOs are observed to be robust under different truncations of the binding loop and capping at the terminal residues, unlike the charge transfer. The robustness of the coordinating MOs suggests that they may be useful for microscopic understanding of bio-macromolecular binding.

Section snippets

Methods

The vacuum QC DFT calculations are done by variationally minimizing the many-body electronic Hamiltonian for fixed nuclear coordinates where the electronic correlations are approximately taken into account. We perform the minimization using the Gaussian03 package [13] with the B3LYP functional for the electron exchange–correlation in the 6-31G(d,p) basis set, used extensively in the earlier studies [2]. We calculate the total ground state energy by the Self Consistent Field method and compute

Results

Since Ca2+ coordinates to the loop via the oxygen atoms, we compute the energy levels of isolated Ca2+ ion and O atom in vacuum (see Supplementary Information, SI, Table S1). We observe that the highest occupied orbital has 3p character, while the lowest unoccupied orbital is a hybrid of the 3s, 4s and 3d orbitals in the isolated Ca2+ ion. On the other hand, the 2p orbitals of the O atom constitute its valence orbitals. The hybridization nature changes in the metal-loop complex. The

Conclusions

In summary we show that the coordination MOs emphasize the roles of both ligand and non-ligand atoms, participating in the stabilization of Ca2+ ion coordination to metal binding loop 1 of CaM. The pivotal role of E31 in coordination is evident from its contribution to the LCMO of the Ca2+-loop complex. The stabilization of the coordination geometry follows through the participation of axial D20, the planar T26 along with non-ligand I27 and then D22 and D24. The crystal water exhibits

Acknowledgments

S.S. thanks UGC, INDIA for fellowship; M.G. and J.C. thank DST, INDIA for funding and MDR thanks the Associateship program of the SNBNCBS.

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