Five atomic resolution structures of endothiapepsin inhibitor complexes: Implications for the aspartic proteinase mechanism

L. Coates, P. T. Erskine, M. P. Crump, S. P. Wood, J. B. Cooper

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Abstract

Endothiapepsin is derived from the fungus Endothia parasitica and is a member of the aspartic proteinase class of enzymes. This class of enzyme is comprised of two structurally similar lobes, each lobe contributing an aspartic acid residue to form a catalytic dyad that acts to cleave the substrate peptide bond. The three-dimensional structures of endothiapepsin bound to five transition state analogue inhibitors (H189, H256, CP-80,794, PD-129,541 and PD-130,328) have been solved at atomic resolution allowing full anisotropic modelling of each complex. The active sites of the five structures have been studied with a view to studying the catalytic mechanism of the aspartic proteinases by locating the active site protons by carboxyl bond length differences and electron density analysis. In the CP-80,794 structure there is excellent electron density for the hydrogen on the inhibitory statine hydroxyl group which forms a hydrogen bond with the inner oxygen of Asp32. The location of this proton has implications for the catalytic mechanism of the aspartic proteinases as it is consistent with the proposed mechanism in which Asp32 is the negatively charged aspartate. A number of short hydrogen bonds (∼ 2.6 Å) with ESD values of around 0.01 Å that may have a role in catalysis have been identified within the active site of each structure; the lengths of these bonds have been confirmed using NMR techniques. The possibility and implications of low barrier hydrogen bonds in the active site are considered.

Original languageEnglish
Pages (from-to)1405-1415
Number of pages11
JournalJournal of Molecular Biology
Volume318
Issue number5
DOIs
StatePublished - 2002
Externally publishedYes

Funding

The ESRF (Grenoble, France) and EMBL (Desy, Hamburg, Germany) are thanked for the provision of X-ray beam time and support during data collection. We also thank Professor M Akhtar FRS for many helpful discussions on catalytic mechanisms and Gordon Beaven for assistance in the protein purification. We gratefully acknowledge the BBSRC (UK) for project grant support and the EPSRC for funding a research studentship (to L.C.) and the Wellcome Trust for an equipment grant for a 600 MHz spectrometer and associated computing equipment.

FundersFunder number
Wellcome Trust
Engineering and Physical Sciences Research Council
Biotechnology and Biological Sciences Research Council

    Keywords

    • Anisotropic refinement
    • Aspartic proteinase mechanism
    • Atomic resolution
    • Tetrahedral intermediate
    • Transition-state analogues

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