Quantitation of the calcium and membrane binding properties of the C2 domains of dysferlin.
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.
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon.
Nazish
N
Abdullah
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon.
Murugesh
M
Padmanarayana
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon.
Naomi J
NJ
Marty
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon. Electronic address: colin.johnson@oregonstate.edu.
Colin P
CP
Johnson
D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D000073939:Dysferlin; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013324:Strontium / Q000378:metabolism
C114839:DYSF protein, human; D000073939:Dysferlin; D008565:Membrane Proteins; D009124:Muscle Proteins; D008274:Magnesium; D002118:Calcium; D013324:Strontium