1GKT

Neutron Laue diffraction structure of endothiapepsin complexed with transition state analogue inhibitor H261

Summary for 1GKT

• Entry DOI: 10.2210/pdb1gkt/pdb
• Related: 1E5O 1E80 1E81 1E82 1EED 1ENT 1EPL 1EPM 1EPN 1EPO 1EPP 1EPQ 1EPR 1ER8 2ER0 2ER6 2ER7 2ER9 3ER3 3ER5 4APE 4ER1 4ER2 4ER4 5ER1 5ER2
• Related PRD ID: PRD_000267
• Descriptor: ENDOTHIAPEPSIN, INHIBITOR, H261 (3 entities in total)
• Functional Keywords: hydrolase-hydrolase inhibitor complex, protease-inhibitor, aspartic proteinase, hydrolysis, hydrolase- hydrolase inhibitor complex, hydrolase/hydrolase inhibitor
• Biological source: CRYPHONECTRIA PARASITICA (CHESTNUT BLIGHT FUNGUS); More
• Total number of polymer chains: 2
• Total formula weight: 34899.17

Authors

Coates, L.,Erskine, P.T.,Wood, S.P.,Myles, D.A.A.,Cooper, J.B. (deposition date: 2001-08-20, release date: 2001-11-20, Last modification date: 2023-11-15)

Primary citation

Coates, L.,Erskine, P.T.,Wood, S.P.,Myles, D.A.A.,Cooper, J.B.
A Neutron Laue Diffraction Study of Endothiapepsin: Implications for the Aspartic Proteinase Mechanism
Biochemistry, 40:13149-, 2001

PubMed Abstract: Current proposals for the catalytic mechanism of aspartic proteinases are largely based on X-ray structures of bound oligopeptide inhibitors possessing nonhydrolyzable analogues of the scissile peptide bond. However, the positions of protons on the catalytic aspartates and the ligand in these complexes have not been determined with certainty. Thus, our objective was to locate crucial protons at the active site of an inhibitor complex since this will have major implications for a detailed understanding of the mechanism of action. We have demonstrated that high-resolution neutron diffraction data can be collected from crystals of the fungal aspartic proteinase endothiapepsin bound to a transition state analogue (H261). The neutron structure of the complex has been refined at a resolution of 2.1 A to an R-factor of 23.5% and an R(free) of 27.4%. This work represents the largest protein structure studied to date by neutron crystallography at high resolution. The neutron data demonstrate that 49% of the main chain nitrogens have exchanged their hydrogen atoms with D2O in the mother liquor. The majority of residues resisting exchange are buried within core beta-sheet regions of the molecule. The neutron maps confirm that the protein has a number of buried ionized carboxylate groups which are likely to give the molecule a net negative charge even at very low pH, thereby accounting for its low pI. The functional groups at the catalytic center have clearly undergone H-D exchange despite being buried by the inhibitor occupying the active site cleft. Most importantly, the data provide convincing evidence that Asp 215 is protonated and that Asp 32 is the negatively charged residue in the transition state complex. This has an important bearing on mechanistic proposals for this class of proteinase.

PubMed: 11683623
DOI: 10.1021/BI010626H

Experimental method

NEUTRON DIFFRACTION (2.1 Å)