Quantitation of the calcium and membrane binding properties of the C2 domains of dysferlin

Abstract

Dysferlin is a large membrane protein involved in calcium-triggered resealing of the sarcolemma after injury. Although it is generally accepted that dysferlin is Ca(2+) sensitive, the Ca(2+) binding properties of dysferlin have not been characterized. In this study, we report an analysis of the Ca(2+) and membrane binding properties of all seven C2 domains of dysferlin as well as a multi-C2 domain construct. Isothermal titration calorimetry measurements indicate that all seven dysferlin C2 domains interact with Ca(2+) with a wide range of binding affinities. The C2A and C2C domains were determined to be the most sensitive, with Kd values in the tens of micromolar, whereas the C2D domain was least sensitive, with a near millimolar Kd value. Mutagenesis of C2A demonstrates the requirement for negatively charged residues in the loop regions for divalent ion binding. Furthermore, dysferlin displayed significantly lower binding affinity for the divalent cations magnesium and strontium. Measurement of a multidomain construct indicates that the solution binding affinity does not change when C2 domains are linked. Finally, sedimentation assays suggest all seven C2 domains bind lipid membranes, and that Ca(2+) enhances but is not required for interaction. This report reveals for the first time, to our knowledge, that all dysferlin domains bind Ca(2+) albeit with varying affinity and stoichiometry.

Figure 1

Schematic diagram of full-length human dysferlin. Dysferlin is composed of seven tandem C2 domains (ovals labeled A through G) and a single-pass transmembrane domain (labeled TMD). Non-C2 domain folds including Fer domains (small black rectangles) and the DYSF domain (large black rectangle) are also indicated. Recombinant proteins composed of isolated C2 domains were generated according to the amino acid designations listed.

Figure 2

Ca²⁺ binding by different C2 domains of dysferlin. The different domains are labeled, respectively. Controls include the MBP and otoferlin C2A. For each domain the top panel shows the raw heat rate and the lower panel shows the integrated heats and fit. The concentrations of proteins and Ca²⁺ have been indicated in the Materials and Methods. All titrations were carried out at 37°C. The models used for fitting the data are mentioned in Table 1.

Figure 3

The endogenous linker region between C2B and C2C does not affect Ca²⁺ binding. A construct containing the C2B and C2C domains of dysferlin was titrated with Ca²⁺. Analysis of the thermogram indicates that the affinities of the C2 domains are unaltered by the linker (Table 1).

Figure 4

The C2A domain of dysferlin binds weakly to Mg²⁺ and Sr²⁺. (A) Representative thermogram of C2A titrated with Mg²⁺. 640 μM dysferlin C2A was titrated with 10 mM MgCl₂. (B) Representative thermogram of C2A titrated with Sr²⁺. 330 μM C2A was titrated with 5 mM SrCl₂. (C) Representative thermogram showing that Ca²⁺ can compete with Mg²⁺ for binding to C2A. Protein was prebound to 750 μM Mg²⁺ and then titrated with 5 mM CaCl₂. (D). Representative thermogram of C2A prebound with 750 μM Ca²⁺ titrated with 5 mM MgCl₂. No binding was observed.

Figure 5

Conserved aspartates in C2A mediate Ca²⁺ binding (A) Sequence and (B) Homology model of the dysferlin C2A showing the location of the three aspartate residues chosen for mutation. (C) Model of loops 1 and 3 of dysferlin C2A indicating aspartate residues implicated in Ca²⁺ binding. Residues in gray represent homologous residues in synaptotagmin I C2A. (D) ITC data for D16A, D21A, and D71A. (E) ITC data for R79D and F80A mutants.

Figure 6

Association of dysferlin C2 domains with liposomes. (A) Interaction of C2 domains with liposomes composed of 65% POPS and 35% POPC under increasing Ca²⁺ concentrations. Sedimentation assays with 100% POPC liposomes indicate that the domains do not associate with neutral lipids. (B) Quantitation of the results of liposome assay from panel A. (C) Sedimentation assays conducted with mutant forms of C2A. (D) C2 domain-lipid interaction with different salt, lipid composition, and divalent cations. C2 domains have been labeled as A through G. Error bars represent standard deviation.