The Dysferlin C2A Domain Binds PI(4,5)P2 and Penetrates Membranes

• Published in: Journal of molecular biology Vol. 435; no. 17; p. 168193
• Main Authors: Kwok, Ethiene, Otto, Shauna C., Khuu, Patricia, Carpenter, Andrew P., Codding, Sara J., Reardon, Patrick N., Vanegas, Juan, Kumar, Tanushri M., Kuykendall, Chapman J., Mehl, Ryan A., Baio, Joe, Johnson, Colin P.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier Ltd 01.09.2023
• Subjects: C2 domain; C2 Domains; calcium; Calcium - metabolism; Calcium-Binding Proteins - chemistry; CEST; dysferlin; Dysferlin - chemistry; Dysferlin - metabolism; genetic code expansion; Membrane Proteins - chemistry; Membrane Proteins - metabolism; Phosphatidylinositol 4,5-Diphosphate - chemistry; Protein Binding; calcium; PS; PC; dysferlin; PE; MD; Dysf; C2 domain; genetic code expansion; CEST; PI(4,5)P2
• ISSN: 0022-2836; 1089-8638; 1089-8638
• DOI: 10.1016/j.jmb.2023.168193

[Display omitted] •Dysferlin is a multi-C2 domain protein that regulates sarcolemma repair.•Dysferlin function is dependent on C2A through an unknown mechanism.•We find that C2A binds PI(4,5)P2 through interaction with Tyr23, K32, K33, and R77.•C2A domain inserts the domain’s Ca2+ binding loops into PI(4,5)P2 membranes.•Membrane binding and insertion of C2A likely facilitates dysf activity during membrane repair. Dysferlin is a large membrane protein found most prominently in striated muscle. Loss of dysferlin activity is associated with reduced exocytosis, abnormal intracellular Ca2+ and the muscle diseases limb-girdle muscular dystrophy and Miyoshi myopathy. The cytosolic region of dysferlin consists of seven C2 domains with mutations in the C2A domain at the N-terminus resulting in pathology. Despite the importance of Ca2+ and membrane binding activities of the C2A domain for dysferlin function, the mechanism of the domain remains poorly characterized. In this study we find that the C2A domain preferentially binds membranes containing PI(4,5)P2 through an interaction mediated by residues Y23, K32, K33, and R77 on the concave face of the domain. We also found that subsequent to membrane binding, the C2A domain inserts residues on the Ca2+ binding loops into the membrane. Analysis of solution NMR measurements indicate that the domain inhabits two distinct structural states, with Ca2+ shifting the population between states towards a more rigid structure with greater affinity for PI(4,5)P2. Based on our results, we propose a mechanism where Ca2+ converts C2A from a structurally dynamic, low PI(4,5)P2 affinity state to a high affinity state that targets dysferlin to PI(4,5)P2 enriched membranes through interaction with Tyr23, K32, K33, and R77. Binding also involves changes in lipid packing and insertion by the third Ca2+ binding loop of the C2 domain into the membrane, which would contribute to dysferlin function in exocytosis and Ca2+ regulation.