PDBsum entry 2fo5

Hydrolase/hydrolase inhibitor

PDB id: 2fo5

Contents

Protein chains

224 a.a. *

Ligands

ACE-LEU-LEU-AR7 ×4

SO4 ×9

Waters ×509

* Residue conservation analysis

PDB id:

2fo5

Name:

Hydrolase/hydrolase inhibitor

Title:

Crystal structure of recombinant barley cysteine endoprotease b isoform 2 (ep-b2) in complex with leupeptin

Structure:

Cysteine proteinase ep-b 2. Chain: a, b, c, d. Fragment: cystein proteinase ep-b2 domain, residues 133-356. Engineered: yes. Ace-leu-leu-argininal (leupeptin). Chain: e, f, g, h. Engineered: yes

Source:

Hordeum vulgare. Organism_taxid: 4513. Gene: epb2. Expressed in: escherichia coli bl21. Expression_system_taxid: 511693. Synthetic: yes. Actinomycetes streptomyces roseus ma 839-a1. Organism_taxid: 66430. Other_details: chemically synthesized

Biol. unit:

Dimer (from PQS)

Resolution:

2.20Å R-factor: 0.201 R-free: 0.233

Authors:

M.T.Bethune,P.Strop,A.T.Brunger,C.Khosla

Key ref:

M.T.Bethune et al. (2006). Heterologous expression, purification, refolding, and structural-functional characterization of EP-B2, a self-activating barley cysteine endoprotease. Chem Biol, 13, 637-647. PubMed id: 16793521 DOI: 10.1016/j.chembiol.2006.04.008

Date:

12-Jan-06 Release date: 18-Jul-06

Protein chains

P25250  (CYSP2_HORVU) -  Cysteine proteinase EP-B 2 from Hordeum vulgare

Seq: 373 a.a.

Struc: 224 a.a.

Enzyme reactions

• Enzyme class: E.C.3.4.22.- - ?????

DOI no: 10.1016/j.chembiol.2006.04.008

Chem Biol 13:637-647 (2006)

PubMed id: 16793521

Heterologous expression, purification, refolding, and structural-functional characterization of EP-B2, a self-activating barley cysteine endoprotease.

M.T.Bethune, P.Strop, Y.Tang, L.M.Sollid, C.Khosla.

ABSTRACT

We describe the heterologous expression in Escherichia coli of the proenzyme precursor to EP-B2, a cysteine endoprotease from germinating barley seeds. High yields (50 mg/l) of recombinant proEP-B2 were obtained from E. coli inclusion bodies in shake flask cultures following purification and refolding. The zymogen was rapidly autoactivated to its mature form under acidic conditions at a rate independent of proEP-B2 concentration, suggesting a cis mechanism of autoactivation. Mature EP-B2 was stable and active over a wide pH range and efficiently hydrolyzed a recombinant wheat gluten protein, alpha2-gliadin, at sequences with known immunotoxicity in celiac sprue patients. The X-ray crystal structure of mature EP-B2 bound to leupeptin was solved to 2.2 A resolution and provided atomic insights into the observed subsite specificity of the endoprotease. Our findings suggest that orally administered proEP-B2 may be especially well suited for treatment of celiac sprue.

Selected figure(s)

Figure 2.
Figure 2. Crystal Structure of Mature EP-B2 Complexed with Leupeptin

Figure 6.
Figure 6. Activity of EP-B2 against the Proteolytically Resistant 33-mer Peptide from α2-Gliadin

The above figures are reprinted by permission from Cell Press: Chem Biol (2006, 13, 637-647) copyright 2006.

Figures were selected by the author.