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Search The CSA
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Catalytic Site Atlas

CSA LITERATURE entry for 1kfu

E.C. namecalpain-2
SpeciesHomo sapiens (Human)
E.C. Number (IntEnz) 3.4.22.53
CSA Homologues of 1kfuThere are 22 Homologs
CSA Entries With UniProtID P17655
CSA Entries With EC Number 3.4.22.53
PDBe Entry 1kfu
PDBSum Entry 1kfu
MACiE Entry 1kfu

Literature Report

IntroductionCalpain is an intracellular, cytostolic, calcium dependent cysteine protease containing a conserved papain-like catalytic fold. Thus, in the presence of clacium, calpain catalyses the hydrolysis of peptide bonds in proteins. Two major, heterodimeric isoforms exist, micro- and m-calpain, which differ in their concentration requirements of calcium ions for activation. Calpains are the only known mammalian enzymes that combine protease activity with dependence on calcium ion binding to EF-hands in one molecule.
Calcium regulates calpain-1 activity by affecting the orientation of the two catalytic lobes (domains I and II). calcium binding occurs at multiple sites, including EF-hands and loops near the active site itself. At low calcium levels, domains I and II are locked by the regulatory domains of the proteins in an open, inhibited conformation, where the catalytic residues are too far apart to form the triad. (Cys of domain I, His and Asp of domain II).
Calpain exists in animals, but not in plants, yeast or bacteria. Through proteolysis of key cellular components, calpain plays a significant physiological role and is involved in a variety of cellular functions, including nerve growth, muscle homeostasis, signal transduction and apoptosis. Calpains have been known as biomodulators since their physiological activity involves cleavage of substrates at inter-domain boundaries, hence modulating the function of these substartes rather than digesting them.
Excessive activation and calcium dependent proteolysis by calpain has been implicated in neurodegenerative and demyelinating diseases including cerebral ischemia, ischemic myocardial infarction, spinal cord injury, muscular dystrophy and cataract. Calpain inhibition should help to control symptoms, hence calpains are ideal targets for pharmacological intervention.
MechansimThe calpain active site contains a Cis-His-Asp catalytic triad. Upon calcium binding, conformational changes brings the Cys105 (D-I) close to His 262 and Asn 286 (D-II).
The overall mechanism involves a nucleophilic cysteine thiol in a catalytic triad.
1. Deprotonation of the Cys 105 thiol group by His 262 with a basic side chain. The thiolate ion is stabilised through the formation of an ion pair with the neighbouring, approximately coplanar imidazolium group of His 262. The ion pair is stabilised by Gln 99 and Asn 286. The Gln 99 and Cys 105 residues form an oxyanion hole, which stabilises the transition state. Asn 286 is adjacent to the catalytic His 262, and its side chain amide oxygen is bonded to the N(e2)H of His 262. This effect of this is to both stabilise the ion pair and also keep the imidazole ring of the His residue in favourable orientation. The aromatic side chain of the conserved Trp 288 of D-II has a weak interaction with His, and helps to maintain His orientation.
2. Nucleophilic attack of the anionic cysteine S (thiolate ion) on the peptide carbonyl carbon. In this step, a fragment of the substrate is released with an amine terminus, the histidine residue in the protease is restored to its deprotonated form, and a thioester intermediate linking the new carboxy-terminus of the substrate to the cysteine thiol is formed.
3. The thioester bond is subsequently hydrolysed to generate a carboxylic acid moiety on the remaining substrate fragment, whilst regenerating the free enzyme
Note: Calcium ion binding results in a conformational change in the alpha-helical N-terminal anchor of the catalytic subunit. Binding causes conformation in domain IV, which allows the transducer to release constraints on other domains, thus increasing flexibility to D-II. Thus, calcium ion binding to D-IV causes release of the anchor, yielding a more flexible D-I, which can move towards D-II, bringing the residues of the catalytic triad close enough for activation of the enzyme.
Reaction

Catalytic Sites for 1kfu

Annotated By Reference To The Literature - Site 1 (Perform Site Search)
ResidueChainNumberUniProtKB NumberFunctional PartFunctionTargetDescription
CysL105105macie:sideChainWhen deprotonated by His262, Cys105 functions in nucleophilic attack on the peptide carbonyl carbon. Collapse of the tetrahedral intermediate leads to release of a substrate with an amine terminus. Cys105 is regenerated when nucleophilic attack of water on the carbonyl carbon, cleaves the enzyme-substrate bond to form a carboxylic acid. Backbone amide stabilises oxyanion hole.
CysL105105macie:mainChainAmideWhen deprotonated by His262, Cys105 functions in nucleophilic attack on the peptide carbonyl carbon. Collapse of the tetrahedral intermediate leads to release of a substrate with an amine terminus. Cys105 is regenerated when nucleophilic attack of water on the carbonyl carbon, cleaves the enzyme-substrate bond to form a carboxylic acid. Backbone amide stabilises oxyanion hole.
HisL262262macie:sideChainDeprotonates thiol side chain in order for it to function as a nucleophile, aided in this functions by Gln99 and Asn286. Subsequently acts as a general acid to protonate leaving groups.
AsnL286286macie:sideChainElectrostatically stabilises His262.
GlnL9999macie:sideChainActs to stabilise both His262 residue, and forms an oxyanion hole to stabilise the increase in negative charge during nucleophilic attack in conjunction with Cys105.
TrpL288288macie:sideChainH-bonds to His262 to amintain its orientation.

Literature References

Notes:Note this pdb is at 2.5Angstroms resolution, a previosu file 1kfx identical protein but at 3.2Angstroms was present. E.C class has changed from 3.4.22.17 to 3.4.22.53 too.
Strobl S
The crystal structure of calcium-free human m-calpain suggests an electrostatic switch mechanism for activation by calcium.
Proc Natl Acad Sci U S A 2000 97 588-592
PubMed: 10639123
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