A second binding site revealed by C‐terminal truncation of calpain small subunit, a penta‐EF‐hand protein

Abstract

The subunits in calpain and in the related penta-EF-hand (PEF) proteins are bound through contacts between the unpaired EF-hand 5 from each subunit. To study subunit binding further, a tetra-EF-hand 18 kDa N- and C-terminally truncated form of the calpain small subunit was prepared (18k). This protein does not combine with the calpain large subunit to form active calpain, but forms homodimers in solution, as shown by ultracentrifugation. The X-ray structure of the 18k protein in the presence of cadmium was solved to a resolution of 2.0 A. The structure of the monomer is almost identical to the known structure of the calpain small subunit, but the 18k protein forms an oligomer in the crystal by the use of two binding sites. One of these sites is an artefact arising from the C-terminal truncation, but the other is a naturally occurring site that is fully exposed to water in intact purified calpain. The characteristics of this site suggest that it may be important in binding other protein modulators involved in the regulation of calpain and of PEF proteins.

Crystal Structure of the Endophilin-A1 BAR Domain

“…It is noteworthy that three of the Cd 2C co-ordinations are not involved in crystal contacts. As the Cd 2C co-ordinations are typically found in the binding of Ca 2C , consistent with similar ionic radii of Cd 2C and Ca 2C (0.97 Å and 0.99 Å , respectively) and previous data showing protein Ca 2C -binding sites occupied by Cd 2C , [39][40][41] in vivo, endo-BAR may bind Ca 2C instead of Cd 2C . Ca 2C binding is also consistent with a Ca 2C -dependent interaction of endophilin with either voltage-gated Ca 2C channels or dynamin.…”

Caspase-dependent and -independent pathways for cadmium-induced apoptosis in cultured kidney proximal tubule cells

“…Calpains are activated by Ca 2ϩ and also thought to mediate apoptosis (13). Cd 2ϩ has been previously reported to release Ca 2ϩ from intracellular pools (23,24), and because Cd 2ϩ and Ca 2ϩ have similar ionic radii, Cd 2ϩ may also directly activate calpains (21). Indeed, calpain activity was significantly enhanced at 6 h of Cd 2ϩ treatment (Fig.…”

Interaction between catalytically inactive calpain and calpastatin

“…In recent years, information has accumulated on the 3D structure of l-calpain and m-calpain [1][2][3][4][5][6][7][8][9][10], as well as their isolated catalytic cores [11][12][13][14][15][16]. Much less is known about the process by which calpain is activated [3,4,6,17,18].…”