Structure and calcium-binding studies of calmodulin-like domain of human non-muscle α-actinin-1

Abstract

The activity of several cytosolic proteins critically depends on the concentration of calcium ions. One important intracellular calcium-sensing protein is α-actinin-1, the major actin crosslinking protein in focal adhesions and stress fibers. The actin crosslinking activity of α-actinin-1 has been proposed to be negatively regulated by calcium, but the underlying molecular mechanisms are poorly understood. To address this, we determined the first high-resolution NMR structure of its functional calmodulin-like domain (CaMD) in calcium-bound and calcium-free form. These structures reveal that in the absence of calcium, CaMD displays a conformationally flexible ensemble that undergoes a structural change upon calcium binding, leading to limited rotation of the N- and C-terminal lobes around the connecting linker and consequent stabilization of the calcium-loaded structure. Mutagenesis experiments, coupled with mass-spectrometry and isothermal calorimetry data designed to validate the calcium binding stoichiometry and binding site, showed that human non-muscle α-actinin-1 binds a single calcium ion within the N-terminal lobe. Finally, based on our structural data and analogy with other α-actinins, we provide a structural model of regulation of the actin crosslinking activity of α-actinin-1 where calcium induced structural stabilisation causes fastening of the juxtaposed actin binding domain, leading to impaired capacity to crosslink actin.

Figure 1. Domain structure of α-actinin-1.

Domain composition of α-actinin antiparallel dimer, as inspired by the structure of human α-actinin-2 (ref. 11). ABD is shown in red, neck peptide in yellow, SR1−SR4 in green, EF1-2 in magenta and EF3-4 in violet.

Figure 2. Sequence alignment of α-actinin-1 EF1-4 and calcium-binding domains of selected representative human proteins.

(a) Both putative and experimentally confirmed calcium-binding motifs are shown as grey rectangles. Residues critical for Ca²⁺ coordination (positions X, Y, Z, -Y, -X, -Z) in EF hands with known calcium-binding activity are shown in green and bold. ACTN1 - α-actinin-1, ACTN2 - α-actinin-2, SPTN1 - α chain of non-erythrocytic spectrin, SPTA1 - α chain of erythrocytic spectrin, CALM - calmodulin, CALL3 - calmodulin-like protein 3, CANB1 - subunit B of type I calcineurin. Alignments were prepared using ClustalW. Residue numbering follows α-actinin-1. α-helices of the apo form are denoted as pink (EF1-2) or violet (EF3-4) rectangles, β-strand secondary structure elements as orange rectangles, and the linker between the two lobes as a curved line. (b) Calcium coordination by the canonical EF hand (EF1 of calmodulin, PDB ID 1CLL) illustrating the pentagonal bipyramidal coordination of the Ca²⁺ ion (broken lines). NH groups of coordinating amino acids are indicated in dark blue, oxygen atoms in red, the Ca²⁺ ion in green-cyan and the coordinating water molecule in red. (c) Sequence preference of the EF hand loop. Ca²⁺ ligands are indicated by their positions in the coordination sphere (X, Y, Z, -X, -Y and -Z). Coordination occurs via side chains (sc) or through the backbone (bb) of amino acids shown in red. The asterisk highlights the ligand typically provided by a water molecule that is hydrogen-bonded to the side chain of the amino acid at position 9, the label sc2 indicates bidentate ligand. The most common amino acids at each position, with their corresponding percentages of occurrence, and those that occur with a frequency greater than 5% in known EF-loops, are shown. Residues mutated in our study are circled.

Figure 3. Calcium titration of CaMD as monitored by ITC.

(a) Wild-type CaMD, (b) CaMD_D759A mutant, (c) CaMD_E770A mutant, (d) CaMD_D800A mutant. The upper panels show measured raw heat changes as a function of time, while the lower panels show integrated heat changes after subtracting the heat of dilution at different Ca²⁺/protein molar ratios. Calculated thermodynamic parameters are shown in Table 1.

Figure 4. Ensemble of 20 lowest energy structures of the apo and holo forms of CaMD of α-actinin-1.

(a) Structure of the apo form; superposition of a region comprising residues 743-820 of EF1-2 (left) and 825-892 of EF3-4 (right). EF1-2 is shown in light pink, EF3-4 in violet, and β-strands in orange. (b) Structure of the holo form. Color-coding is the same as in (a) except for EF1-2, which is shown in magenta. Ca²⁺ ion is depicted as a green-cyan sphere. The three dotted circles mark three EF hand motifs which, do not bind calcium ions (EF2-4).

Figure 5. Stabilizing interactions between the linker and the N- and C-terminal lobes in the holo form of CaMD of α-actinin-1.

EF1-2 is shown in magenta, EF3-4 in violet, linker between them in dark green, and Ca²⁺ as green-cyan sphere. Residues involved in linker stabilization are shown as light green sticks. Hydrogen bonds and salt bridges are depicted as black and red broken lines, respectively.

Figure 6. Comparison of EF1-2 of apo and holo forms of CaMD of α-actinin-1.

(a) Structure of EF1-2 of apo (left) and holo (right) forms of CaMD of α-actinin-1. Calcium ion is shown as a green-cyan sphere. Residues involved in hydrophobic, cation-π and π-π interactions are shown as blue sticks. (b) Superposition of EF1-2 of apo and holo forms of CaMD. The most significant conformational changes upon calcium binding are observed at the linker loop between α1 and α2 helices of EF1 (encircled). (c) Superposition of separate EF1 and EF2 hands of apo and holo forms of CaMD demonstrate that upon calcium binding significant conformational changes occur within CaMD. Structures of the two forms of EF1 and EF2 were superimposed in the region corresponding to α1 and α3 helix, respectively. (d) Schematic display of the vector geometry mapping method (VGM) used to characterize EF motifs in terms of relative interhelical angles. The entering helix (α1, α3) of the EF hand is superimposed on a reference EF hand on the z-axis, and the corresponding position of the exiting helix (α2, α4) is evaluated using the angles θ and ϕ.

Figure 7. Interaction of CaMD of α-actinin-1 with Ca²⁺ ions.

(a) Overlay of ¹⁵N-HSQC spectra of the CaMD of α-actinin-1 during titration with Ca²⁺ ions. Only residues with significant chemical shift changes are labelled. For clarity, only nine out of fifteen titration data points between 0 and 20 eq. of Ca²⁺ ions are presented. (b) Chemical shift perturbations of the CaMD of α-actinin-1 in the presence of 20 eq. Ca²⁺ ions. Δδ(H,N) is defined by equation 1. Residues above the threshold value Δδ(H,N) of 0.3 ppm are coloured magenta. α-helical and β-strand secondary structure elements of the holo form are presented as rectangles at the top of the panel. (c) CSP of amides mapped on the structure of Ca²⁺-bound CaMD of α-actinin-1. Residues above the threshold value Δδ(H,N) of 0.3 ppm are coloured magenta. The Ca²⁺-coordinating loop of CaMD of α-actinin-1 with coordinating side chain groups of D759, D761, S763, E770, CO group of the T765 main chain and a water molecule hydrogen bonded to G767 are shown enlarged. Ca²⁺ ions are shown as green-cyan spheres.

Figure 8. Proposed model of structural changes accompanying Ca²⁺ binding to EF1-2 of α-actinin-1 and subsequent altered affinity for actin filaments.

(a) Model of the α-actinin-1 dimer depicting ordering of CaMD upon Ca²⁺-binding. For CaMD, NMR ensemble structures superposed in the EF1-2 region were used. Apo and holo forms of EF1-2 are shown in light pink and magenta surface representation, and EF3-4 as violet ribbons. The central feature of the model is increased rigidity in the linker region between the EF1-2 and EF3-4 lobes upon Ca²⁺ binding that could result in stabilization of the interaction between EF3-4 and the neck region. (b) Only α-actinin-1 with EF1-2 in the apo form is able to bundle actin filaments. Color coding is the same as in Fig. 1. Models were prepared using NMR structures of CaMD of α-actinin-1 reported in the present study, and structures of human α-actinin-2 (PDB ID 4D1E) and F-actin (PDB ID 3LUE).