PDBsum entry 3df0

Hydrolase

PDB id: 3df0

Contents

Protein chains

676 a.a. *

174 a.a. *

56 a.a. *

Metals

_CA ×10

* Residue conservation analysis

PDB id:

3df0

Name:

Hydrolase

Title:

Calcium-dependent complex between m-calpain and calpastatin

Structure:

Calpain-2 catalytic subunit. Chain: a. Synonym: calpain-2 large subunit, calcium-activated neutral proteinase 2, canp 2, calpain m-type, m-calpain, millimolar-calpain. Engineered: yes. Mutation: yes. Calpain small subunit 1. Chain: b. Fragment: unp residues 87-270.

Source:

Rattus norvegicus. Rat. Organism_taxid: 10116. Gene: capn2. Expressed in: escherichia coli. Gene: capns1, capn4, css1. Gene: cast.

Resolution:

2.95Å R-factor: 0.232 R-free: 0.299

Authors:

T.Moldoveanu,K.Gehring,D.R.Green

Key ref:

T.Moldoveanu et al. (2008). Concerted multi-pronged attack by calpastatin to occlude the catalytic cleft of heterodimeric calpains. Nature, 456, 404-408. PubMed id: 19020622 DOI: 10.1038/nature07353

Date:

11-Jun-08 Release date: 11-Nov-08

Protein chain

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

Seq: 700 a.a.

Struc: 676 a.a.*

Protein chain

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

Seq: 270 a.a.

Struc: 174 a.a.

Protein chain

P27321  (ICAL_RAT) -  Calpastatin from Rattus norvegicus

Seq: 713 a.a.

Struc: 56 a.a.

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

Enzyme reactions

• Enzyme class 2: Chain A: E.C.3.4.22.53 - calpain-2.
• Cofactor: Ca(2+)
• Enzyme class 3: Chains B, C: 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: 10.1038/nature07353

Nature 456:404-408 (2008)

PubMed id: 19020622

Concerted multi-pronged attack by calpastatin to occlude the catalytic cleft of heterodimeric calpains.

T.Moldoveanu, K.Gehring, D.R.Green.

ABSTRACT

The Ca(2+)-dependent cysteine proteases, calpains, regulate cell migration, cell death, insulin secretion, synaptic function and muscle homeostasis. Their endogenous inhibitor, calpastatin, consists of four inhibitory repeats, each of which neutralizes an activated calpain with exquisite specificity and potency. Despite the physiological importance of this interaction, the structural basis of calpain inhibition by calpastatin is unknown. Here we report the 3.0 A structure of Ca(2+)-bound m-calpain in complex with the first calpastatin repeat, both from rat, revealing the mechanism of exclusive specificity. The structure highlights the complexity of calpain activation by Ca(2+), illustrating key residues in a peripheral domain that serve to stabilize the protease core on Ca(2+) binding. Fully activated calpain binds ten Ca(2+) atoms, resulting in several conformational changes allowing recognition by calpastatin. Calpain inhibition is mediated by the intimate contact with three critical regions of calpastatin. Two regions target the penta-EF-hand domains of calpain and the third occupies the substrate-binding cleft, projecting a loop around the active site thiol to evade proteolysis.

Selected figure(s)

Figure 1.
Figure 1: Complex between Ca^2+-bound m-calpain and calpastatin. a, Overall structure shows regions A, B and C of calpastatin bound to DIV, DI–III and DVI of calpain, respectively. The intervening sequences of calpastatin are devoid of electron density (red dots). The central part of the inhibitory region B forms the occluding loop at the active site. The active site in the protease core DI–II is stabilized by DIII. Calpain heterodimerization is largely defined at the DIV–DVI interface. Alternate conformations at the interface between the DI–III core and the DIV–DVI heterodimer (black dots) are possible^30. b, ^15N,^1H-HSQC (heteronuclear single quantum correlation) spectrum of the complex between ^13C,^15N-labelled calpastatin (residues 128–226, Supplementary Fig. 2b) and unlabelled calpain identified flexible/disordered residues of calpastatin. Connected sample strips from the HNCA NMR experiment are inset.

Figure 4.
Figure 4: Calpain–calpastatin proteolytic system. A schematic diagram illustrating the Ca^2+-induced activation of calpain and its inhibition by calpastatin. DIII has a fundamental role in relaying the Ca^2+-induced structural changes (red dotted arrows) from the peripheral domains to the catalytically competent yet labile protease core. Concerted binding of the intrinsically unstructured protein (IUP) calpastatin to peripheral domains and the active site of calpain results in low-nanomolar inhibition.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nature (2008, 456, 404-408) copyright 2008.

Figures were selected by an automated process.