Calcium affinity of human α-actinin 1

Abstract

Due to alternative splicing, the human ACTN1 gene codes for three different transcripts of α-actinin; one isoform that is expressed only in the brain and two with a more general expression pattern. The sequence difference is located to the C-terminal domains and the EF-hand motifs. Therefore, any functional or structural distinction should involve this part of the protein. To investigate this further, the calcium affinities of these three isoforms of α-actinin 1 have been determined by isothermal calorimetry.

Keywords: EF-hand; calcium binding; α-actinin.

Figure 1. Exon organisation of the C-terminal domain.

Alternative splicing of exons 19a and b, give rise to three different transcripts of human α-actinin 1. The non-muscle isoform lacks exon 19b whereas smooth muscle α-actinin 1 lacks exon 19a. The brain isoform contains both exon 19a and 19b.

Figure 2. Far-UV CD spectra.

The far-UV CD spectra of the C-terminal domain of human brain (gold), non-muscle (black) and smooth muscle (red) α-actinin 1 were collected in 50 mM Tris-HCl, pH 7.6 and 200 mM KCl. The mean residue molar ellipticity was determined from three accumulated scans between 200 and 260 nm at 4 °C.

Figure 3. Representative isothermal calorimetry thermograms for calcium binding to α-actinin 1 C-terminal domains.

Heat traces were obtained by injecting calcium chloride to brain (A), non-muscle (B) or smooth muscle (C) isoform, using the indicated concentrations of protein and calcium. After integration, a single-site-binding isotherm was fitted to the data to obtain the enthalpy of binding ΔH, entropy ΔS and the dissociation constant K_d. Best fitting dissociation constants for the calcium binding to the brain or non-muscle isoforms were 44.8 and 54.7 µM, respectively, for the data displayed in the figure. The first point in ITC titration was not included in the fitting. Data shown are representative of at least 10 independent experiments. In all figures, red circles represent experimental data and the full drawn line the best fit.

Figure 4. Plausible calcium binding loops in the C-terminal domain of α-actinin 1.

The sequences of the C-terminal domain of brain, non-muscle and smooth muscle a-actinin 1 were aligned with the consensus sequence manually. In the calcium binding loop residues at the positions labelled X, Y, Z, −Y, −X and −Z coordinate the calcium ion, usually by side chain oxygenes. The residue at −Y coordinates the calcium ion through the back bone carbonyl oxygene. E, glutamate; G, glycine; I, isoleucine, leucine or valine; n, hydrophobic residue; ∗, any residue.