The crystal structure of calcium-free human m-calpain suggests an electrostatic switch mechanism for activation by calcium - PubMed (original) (raw)
. 2000 Jan 18;97(2):588-92.
doi: 10.1073/pnas.97.2.588.
C Fernandez-Catalan, M Braun, R Huber, H Masumoto, K Nakagawa, A Irie, H Sorimachi, G Bourenkow, H Bartunik, K Suzuki, W Bode
Affiliations
- PMID: 10639123
- PMCID: PMC15374
- DOI: 10.1073/pnas.97.2.588
The crystal structure of calcium-free human m-calpain suggests an electrostatic switch mechanism for activation by calcium
S Strobl et al. Proc Natl Acad Sci U S A. 2000.
Abstract
Calpains (calcium-dependent cytoplasmic cysteine proteinases) are implicated in processes such as cytoskeleton remodeling and signal transduction. The 2.3-A crystal structure of full-length heterodimeric [80-kDa (dI-dIV) + 30-kDa (dV+dVI)] human m-calpain crystallized in the absence of calcium reveals an oval disc-like shape, with the papain-like catalytic domain dII and the two calmodulin-like domains dIV+dVI occupying opposite poles, and the tumor necrosis factor alpha-like beta-sandwich domain dIII and the N-terminal segments dI+dV located between. Compared with papain, the two subdomains dIIa+dIIb of the catalytic unit are rotated against one another by 50 degrees, disrupting the active site and the substrate binding site, explaining the inactivity of calpains in the absence of calcium. Calcium binding to an extremely negatively charged loop of domain dIII (an electrostatic switch) could release the adjacent barrel-like subdomain dIIb to move toward the helical subdomain dIIa, allowing formation of a functional catalytic center. This switch loop could also mediate membrane binding, thereby explaining calpains' strongly reduced calcium requirements in vivo. The activity status at the catalytic center might be further modulated by calcium binding to the calmodulin domains via the N-terminal linkers.
Figures
Figure 1
Ribbon structure of human m-calpain in the absence of calcium, shown in reference orientation. The 80-kDa L-chain starts in the molecular center (green, dI), folds into the surface of the dIIa subdomain (gold, I→II linker), forms the papain-like left-side part of the catalytic domain dII (gold, dIIa) and the right-side barrel-like subdomain dIIb (red), descends through the open II→III loop (red), builds domain dIII (blue), runs down (magenta, III→IV), and forms the right-side calmodulin-like domain dIV (yellow). The 30-kDa S-chain becomes visible from Thr95S onwards (magenta, dV) before forming the left-side calmodulin domain dVI (orange). The catalytic residues Cys105L, His262L, and Asn286L together with Trp106L, Pro287L, and Trp288L (top) are shown with all non-hydrogen atoms. The figure was made with
setor
(34).
Figure 2
Sequence of the 80-kDa L- and the 30-kDa S-chain of human m-calpain [accession nos. P17655 and P04632 (6, 7)]. β-strands and α-helices are indicated by arrows and cylinders colored as in Fig. 1, and the topologically defined domain boundaries are given by meeting black arrows. Active-site residues and other notable residues (dIIa and dIIb) are marked by red and black triangles, acidic residues of the switch loop (dIII) and opposing alkaline residues (dIIb and II->III) by red and blue circles, and the residues of domain dVI known to be involved in normal calcium binding (8) by black circles, respectively. The figure was made with
alscript
(35).
Figure 3
Superposition of the m-calpain catalytic domain and papain. The papain-like part of the catalytic domain (gold, dIIa) and the barrel-like subdomain dIIb (red) are superimposed with papain (18) (blue) after optimal fit of the left-side papain half to the helical subdomain dIIa. The active site residues Cys105L, His262L, and Asn286L, and Pro287L, Trp288L, and Trp106L are shown in full structure. This “standard view” of papain-like cysteine proteinases (18) is obtained from Fig. 1 by a 90° rotation around a horizontal axis. The figure was made with
setor
(34).
Figure 4
Ribbon plot of domain dIII (blue) and contacting segments from subdomains dIIa (gold) and dIIb (red). The side chains of the right-side acidic loop and of the left-side basic loop, and some polar and hydrophobic residues forming the polar (center, left) and hydrophobic (center, right) interface between both catalytic subdomains and domain dIII are given. Reference orientation is as in Fig. 1. The figure was made with
setor
(34).
Figure 5
Ribbon plot of the calmodulin-like domains dIV (yellow) and dVI (orange) superimposed with the calcium-containing rat dVI-dVI homodimer (8, 9) (white and green). Shown are also the 2 × 3 calcium ions (pink spheres I to III) and the acidic residues found in the homodimer at medium calcium levels and involved in calcium binding, respectively (8). Reference orientation is as in Fig. 1. The figure was made with
setor
(34).
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