Catalytic Site Atlas
LITERATURE entry for 1kfx
|Species||Homo sapiens (Human)|
E.C. Number (IntEnz)
|CSA Homologues of 1kfx||There are 22 Homologs
CSA Entries With UniProtID
CSA Entries With EC Number
|MACiE Entry ||1kfx|
|Introduction||Calpain 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 atr 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.
The catalytic triad is not assembled prior to calcium binding. The calcium ion induces conformational changes to bring together the domain I Cis residue and the domain II His and Asp residues. In the inactive form, the residues are too far apart to form the triad since the Cis-His ion pair cannot be formed over such a distance.
Calpain is also capable of autoproteolysis.
|Mechansim||The 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 sulfhydryl 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
Catalytic Sites for 1kfx
| Annotated By Reference To The Literature - Site 2 (Perform Site Search)|
|Residue||Chain||Number||UniProtKB Number||Functional Part||Function||Target||Description|
|Asn||L||286||286||macie:sideChain||Asn 286 is adjacent to the catalytic His 262 residue, and stabilises the Cys/His ion pair by forming a hydrogen bond withits side chain amide oxygen to the N-e2 hydrogen of the His 262 residue. This also keeps the imidazole ring in favourable orientation.|
|His||L||262||262||macie:sideChain||The basic His 262 side chain deprotonates the Cys 105 thiol to activate it towards nucleophilic attack of the substrate peptide bond. The His 262 imidazole ring is approximately coplanar to the thiolate ion, and forms a transition state stabilising ion pair. The His 262 side chain amide oxygen forms a hydrogen bond to Asn 286. His 262 also has a weak, stabilising interaction with the Trp 288 aromatic side chain.|
|Gln||L||99||99||macie:sideChain||The Gln 99 and Cys 105 residues form an oxyanion hole, which stabilises the transition state. The Gln 99 residue also stabilises the ion pair. |
|Cys||L||105||105||macie:sideChain||Deprotonation of the cysteine thiol by the His 262 basic side chain activates the cysteine S to carry out nucleophilic attack on the carbonyl carbon of the peptide bond in the substrate. The thiolate ion is stabilised by the formation of an active site ion pair with the His 262 imidazole ring. The main chain NH of Cys 105 forms part of the oxyanion hole, which stabilises the transition state.|
|Cys||L||105||105||macie:mainChainAmide||Deprotonation of the cysteine thiol by the His 262 basic side chain activates the cysteine S to carry out nucleophilic attack on the carbonyl carbon of the peptide bond in the substrate. The thiolate ion is stabilised by the formation of an active site ion pair with the His 262 imidazole ring. The main chain NH of Cys 105 forms part of the oxyanion hole, which stabilises the transition state.|
|Trp||L||288||288||macie:sideChain||The aromatic side chain of the conserved Trp 288 of D-II has a weak interaction with His, and helps to maintain His orientation.|
|Notes:||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.
Crystal structure of calcium bound domain VI of calpain at 1.9 A resolution and its role in enzyme assembly, regulation, and inhibitor binding.
Nat Struct Biol 1997 4 539-547
Calpain silencing by a reversible intrinsic mechanism.
Nat Struct Biol 2003 10 371-378
A structural model for the inhibition of calpain by calpastatin: crystal structures of the native domain VI of calpain and its complexes with calpastatin peptide and a small molecule inhibitor.
J Mol Biol 2003 328 131-146
Crystal structure of calpain reveals the structural basis for Ca(2+)-dependent protease activity and a novel mode of enzyme activation.
EMBO J 1999 18 6880-6889
Calcium-dependent proteolysis and isopeptide bond formation:Calpains and transglutaminases
Pure and Appl. Chem 1992 64 1093-1097
Catalytic-site characteristics of the porcine calpain II 80 kDa/18 kDa heterodimer revealed by selective reaction of its essential thiol group with two-hydronic-state time-dependent inhibitors: evidence for a catalytic site Cys/His interactive system and an ionizing modulatory group.
Biochem J 1993 290 ( Pt 1) 75-83