PDBsum entry 1df0

Hydrolase

PDB id: 1df0

Contents

Protein chains

624 a.a. *

176 a.a. *

Waters ×358

* Residue conservation analysis

PDB id:

1df0

Name:

Hydrolase

Title:

Crystal structure of m-calpain

Structure:

M-calpain. Chain: a. Fragment: large (catalytic) subunit. Synonym: calcium-activated neutral proteinase, calpain 2. Engineered: yes. Mutation: yes. Calpain. Chain: b. Fragment: domain vi (calcium-binding domain), small (regulatory)

Source:

Rattus norvegicus. Norway rat. Organism_taxid: 10116. Expressed in: escherichia coli. Expression_system_taxid: 562. Expression_system_taxid: 562

Biol. unit:

Dimer (from PQS)

Resolution:

2.60Å R-factor: 0.223 R-free: 0.293

Authors:

C.M.Hosfield,J.S.Elce,P.L.Davies,Z.Jia

Key ref:

C.M.Hosfield et al. (1999). Crystal structure of calpain reveals the structural basis for Ca(2+)-dependent protease activity and a novel mode of enzyme activation. EMBO J, 18, 6880-6889. PubMed id: 10601010 DOI: 10.1093/emboj/18.24.6880

Date:

16-Nov-99 Release date: 21-Jun-00

Protein chain

Q07009  (CAN2_RAT) -  Calpain-2 catalytic subunit from Rattus norvegicus

Seq: 700 a.a.

Struc: 624 a.a.*

Protein chain

Q64537  (CPNS1_RAT) -  Calpain small subunit 1 from Rattus norvegicus

Seq: 270 a.a.

Struc: 176 a.a.

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class 1: Chain A: E.C.3.4.22.53 - calpain-2.
• Cofactor: Ca(2+)
• Enzyme class 2: Chain B: E.C.?

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

DOI no: 10.1093/emboj/18.24.6880

EMBO J 18:6880-6889 (1999)

PubMed id: 10601010

Crystal structure of calpain reveals the structural basis for Ca(2+)-dependent protease activity and a novel mode of enzyme activation.

C.M.Hosfield, J.S.Elce, P.L.Davies, Z.Jia.

ABSTRACT

The combination of thiol protease activity and calmodulin-like EF-hands is a feature unique to the calpains. The regulatory mechanisms governing calpain activity are complex, and the nature of the Ca(2+)-induced switch between inactive and active forms has remained elusive in the absence of structural information. We describe here the 2.6 A crystal structure of m-calpain in the Ca(2+)-free form, which illustrates the structural basis for the inactivity of calpain in the absence of Ca(2+). It also reveals an unusual thiol protease fold, which is associated with Ca(2+)-binding domains through heterodimerization and a C(2)-like beta-sandwich domain. Strikingly, the structure shows that the catalytic triad is not assembled, indicating that Ca(2+)-binding must induce conformational changes that re-orient the protease domains to form a functional active site. The alpha-helical N-terminal anchor of the catalytic subunit does not occupy the active site but inhibits its assembly and regulates Ca(2+)-sensitivity through association with the regulatory subunit. This Ca(2+)-dependent activation mechanism is clearly distinct from those of classical proteases.

Selected figure(s)

Figure 3.
Figure 3 Calpain has a unique N-terminal anchor. (A) The helical anchor (residues 2 -16 are shown) makes contacts only with D-VI (colors as in Figure 1). (B) View down the helical axis highlights interactions between the residues in the anchor (magenta type) and D-VI (black type), represented as an electrostatic GRASP surface (Nicholls et al., 1991) (red, acidic; blue, basic). (C) Side view of (B) illustrates the depth of the hydrophobic pocket in D-VI, which interacts with hydrophobic residues Ala2, Gly3, Ile4, Ala5, Leu8 and Ala9 of the anchor. This anchor inhibits active site assembly by associating with the regulatory subunit, thus restricting flexibility of protease D-I. The anchor also acts as a co-chaperone in concert with D-VI, ensuring proper folding of the catalytic subunit. (B) and (C) were created with the program GRASP (Nicholls et al., 1991).

Figure 4.
Figure 4 Domain III shares similar characteristics with a C[2] domain. A typical C[2] domain exists as an anti-parallel -sandwich with several acidic residues at one end that form a binding cradle for Ca^2+. The first C[2] domain from synaptotagmin (cyan, PDB accession code 1RSY) (Sutton et al., 1995) and D-III (green) have approximately the same overall dimensions, though slightly differing topologies. Numerous acidic residues (red) result in a highly negative potential, which is partially stabilized by adjacent basic residues (blue).

The above figures are reprinted from an Open Access publication published by Macmillan Publishers Ltd: EMBO J (1999, 18, 6880-6889) copyright 1999.

Figures were selected by an automated process.