Mu-calpain binds to lipid bilayers via the exposed hydrophobic surface of its Ca2+-activated conformation.
Mu- 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 mu-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 mu-calpain is due to the exposed hydrophobic surface of the active conformation and does not reduce the Ca2+ requirement for activation.
Fernández-Montalván
Amaury
A
Abteilung für Klinische Chemie und Klinische Biochemie, Chirurgische Klinik, Ludwig-Maximilians-Universität München, Nussbaumstr. 20, D-80336 München, Germany.
Assfalg-Machleidt
Irmgard
I
Pfeiler
Dietmar
D
Fritz
Hans
H
Jochum
Marianne
M
Machleidt
Werner
W
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D002154:Calpain / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008051:Lipid Bilayers / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D020349:Surface Plasmon Resonance
D008051:Lipid Bilayers; D010743:Phospholipids; D011994:Recombinant Proteins; D002154:Calpain; C449002:mu-calpain; D002118:Calcium