μ-Calpain binds to lipid bilayers via the exposed hydrophobic surface of its Ca2+-activated conformation
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Amaury Fernández-Montalván
Abstract
μ- and m-calpain are cysteine proteases requiring micro- and millimolar Ca2+ concentrations for their activation in vitro. Among other mechanisms, interaction of calpains with membrane phospholipids has been proposed to facilitate their activation by nanomolar [Ca2+] in living cells. Here the interaction of non-autolysing, C115A active-site mutated heterodimeric human μ-calpain with phospholipid bilayers was studied in vitro using protein-to-lipid fluorescence resonance energy transfer and surface plasmon resonance. Binding to liposomes was Ca2+-dependent, but not selective for specific phospholipid head groups. [Ca2+]0.5 for association with lipid bilayers was not lower than that required for the exposure of hydrophobic surface (detected by TNS fluorescence) or for enzyme activity in the absence of lipids. Deletion of domain V reduced the lipid affinity of the isolated small subunit (600-fold) and of the heterodimer (10- to 15-fold), thus confirming the proposed role of domain V for membrane binding. Unexpectedly, mutations in the acidic loop of the ‘C2-like’ domain III, a putative Ca2+ and phospholipid-binding site, did not affect lipid affinity. Taken together, these results support the hypothesis that in vitro membrane binding of μ-calpain is due to the exposed hydrophobic surface of the active conformation and does not reduce the Ca2+ requirement for activation.
References
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- Protein aggregation in crowded environments
- Nitrite, a naturally occurring precursor of nitric oxide that acts like a ‘prodrug’
- Functional studies of the small subunit of EcoHK31I DNA methyltransferase
- Functional analysis of amino acid residues at the dimerisation interface of KpnI DNA methyltransferase
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- Pathogenicity of catalytic antibodies: catalytic activity of Bence Jones proteins from myeloma patients with renal impairment can elicit cytotoxic effects
- Transgenic expression of gallerimycin, a novel antifungal insect defensin from the greater wax moth Galleria mellonella, confers resistance to pathogenic fungi in tobacco
- Catalytic pathways of Euphorbia characias peroxidase reacting with hydrogen peroxide
- Biochemical and pharmacological characterization of the human bradykinin subtype 2 receptor produced in mammalian cells using the Semliki Forest virus system
- A spectroscopic analysis of the interaction between the human regulatory proteins RACK1 and Ki-1/57
- Subcellular localisation of human inositol 1,4,5-trisphosphate 3-kinase C: species-specific use of alternative export sites for nucleo-cytoplasmic shuttling indicates divergent roles of the catalytic and N-terminal domains
- The gating effect of calmodulin and calcium on the connexin50 hemichannel
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