PDBsum entry: 2nqi

Hydrolase

PDB-id: 2nqi

Main view

Contents

Description

Header details

Header records

References

PROCHECK

Protein chain

322 a.a. *

Ligands

NQI

Metal ions

_CA ×2

Waters ×153

* Residue conservation analysis

Tools

Clefts Calculation

Right view	Bottom view

PDB id:

2nqi

Name:

Hydrolase

Title:

Calpain 1 proteolytic core inactivated by wr13(r,r), an epoxysuccinyl-type inhibitor.

Structure:

Calpain-1 catalytic subunit. Chain: a. Fragment: calpain catalytic domain, residues 27-356. Synonym: calpain-1 large subunit, calcium-activated neutral proteinase 1, canp 1, calpain mu-type, mucanp, micromolar-calpain. Engineered: yes

Source:

Rattus norvegicus. Norway rat. Organism_taxid: 10116. Gene: capn1, cls1. Expressed in: escherichia coli bl21. Expression_system_taxid: 511693.

UniProt:

P97571 (CAN1_RAT)

Seq:

Struc:

Seq:

Struc:

Seq:

713 a.a.

Struc:

322 a.a.

Key:

PfamA domain

Secondary structure

Enzyme class:

E.C.3.4.22.52   [IntEnz]   [ExPASy]   [KEGG]   [BRENDA]

Cofactor:

Calcium

Resolution:

2.04Å

R-factor:

0.184

R-free:

0.247

Authors:

D.Cuerrier,P.L.Davies,R.L.Campbell,T.Moldoveanu

Key ref:

D.Cuerrier et al. (2007). Development of calpain-specific inactivators by screening of positional scanning epoxide libraries.. J Biol Chem, 282, 9600-9611. [PubMed id: 17218315] [DOI: 10.1074/jbc.M610372200]

Date:

31-Oct-06

Release date:

09-Jan-07

Related entries:

2nqg
crystal structure of the same protein fragment, inactivated
by wr18(s,s), another epoxysuccinyl inhibitor with opposite
stereochemistry.
1tlo
crystal structure of the same protein fragment, inactivated
by e-64, another epoxysuccinyl inhibitor.
1kxr
crystal structure of the same protein fragment, uncomplexed
1tl9
... plus others (see Header records)

Quick_links

Procheck

Clefts

Surface

Key reference

DOI no: 10.1074/jbc.M610372200

J Biol Chem 282:9600-9611 (2007)

PubMed id: 17218315

Development of calpain-specific inactivators by screening of positional scanning epoxide libraries.

D.Cuerrier, T.Moldoveanu, R.L.Campbell, J.Kelly, B.Yoruk, S.H.Verhelst, D.Greenbaum, M.Bogyo, P.L.Davies.

ABSTRACT

Calpains are calcium-dependent proteases that are required for numerous intracellular processes but also play an important role in the development of pathologies such as ischemic injury and neurodegeneration. Many current small molecule calpain inhibitors also inhibit other cysteine proteases, including cathepsins, and need improved selectivity. The specificity of inhibition of several calpains and papain was profiled using synthetic positional scanning libraries of epoxide-based compounds that target the active-site cysteine. These peptidomimetic libraries probe the P4, P3, and P2 positions, display (S,S)- or (R,R)-epoxide stereochemistries, and incorporate both natural and non-natural amino acids. To facilitate library screening, an SDS-PAGE assay that measures the extent of hydrolysis of an inactive recombinant m-calpain was developed. Individual epoxide inhibitors were synthesized guided by calpain-specific preferences observed from the profiles and tested for inhibition against calpain. The most potent compounds were assayed for specificity against cathepsins B, L, and K. Several compounds demonstrated high inhibition specificity for calpains over cathepsins. The best of these inhibitors, WRH(R,R), irreversibly inactivates m- and mu-calpain rapidly (k(2)/K(i) = 131,000 and 16,500 m(-1) s(-1), respectively) but behaves exclusively as a reversible and less potent inhibitor toward the cathepsins. X-ray crystallography of the proteolytic core of rat mu-calpain inactivated by the epoxide compounds WR gamma-cyano-alpha-aminobutyric acid (S,S) and WR allylglycine (R,R) reveals that the stereochemistry of the epoxide influences positioning and orientation of the P2 residue, facilitating alternate interactions within the S2 pocket. Moreover, the WR gamma-cyano-alpha-aminobutyric acid (S,S)-complexed structure defines a novel hydrogen-bonding site within the S2 pocket of calpains.

Selected figure(s)

Figure 7.
FIGURE 7. Crystal structures of µI–II inactivated by WR18(S,S) and WR13(R,R). A and B, fitted electron density of WR18(S,S) and WR13(R,R), respectively. The observed electron density for the ligands is shown by an F[o] – F[c] (omit) map calculated for the structures omitting the ligands and contoured at 2 . C and D, stereo images demonstrating the hydrogen bonds formed by WR18(S,S) and WR13(R,R), respectively. All polar bonds shorter than 3.2 Å are shown by dashes. Carbon atoms of the polypeptide are colored white, and water molecules are shown as red spheres. The flexible P3-Arg and P4-Trp moieties of the ligands were omitted for clarity. Arrows point to the hydrogen bond between WR18(S,S) and a water molecule within the S2 pocket in C and to the (R,R)-epoxide specific hydrogen bond between the P2 backbone amide of WR13(R,R) and Gly-271 in D.

Figure 8.
FIGURE 8. Structural overlap of three epoxide-derived ligands. The overlap of WR18(S,S), WR13(R,R) and E-64 was obtained by the structural alignment of the -carbons of the polypeptide chain. The color scheme is as in Fig. 7, with carbon atoms of E-64 in pink. The active site for the WR18(S,S) complex is shown as a surface. Arrows point to the position of the -carbons of the P2 residues. The flexible P3-Arg and P4-Trp moieties of WR13(R,R) and WR18(S,S) were omitted for clarity.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2007, 282, 9600-9611) copyright 2007.

Figures were selected by an automated process.