Communication
Unexpected Active-Site Flexibility in the Structure of Human Neutrophil Elastase in Complex with a New Dihydropyrimidone Inhibitor

https://doi.org/10.1016/j.jmb.2011.04.047Get rights and content

Abstract

Human neutrophil elastase (HNE), a trypsin-type serine protease, is of pivotal importance in the onset and progression of chronic obstructive pulmonary disease (COPD). COPD encompasses a group of slowly progressive respiratory disorders and is a major medical problem and the fifth leading cause of death worldwide. HNE is a major target for the development of compounds that inhibit the progression of long-term lung function decline in COPD patients.

Here, we present the three-dimensional structure of a potent dihydropyrimidone inhibitor (DHPI) non-covalently bound to HNE at a resolution of 2.0 Å. The inhibitor binds to the active site in a unique orientation addressing S1 and S2 subsites of the protease. To facilitate further analysis of this binding mode, we determined the structure of the uncomplexed enzyme at a resolution of 1.86 Å. Detailed comparisons of the HNE:DHPI complex with the uncomplexed HNE structure and published structures of other elastase:inhibitor complexes revealed that binding of DHPI leads to large conformational changes in residues located in the S2 subsite. The rearrangement of residues Asp95–Leu99B creates a deep, well-defined cavity, which is filled by the P2 moiety of the inhibitor molecule to almost perfect shape complementarity. The shape of the S2 subsite in complex with DHPI clearly differs from all other observed HNE structures. The observed structural flexibility of the S2 subsite is a key feature for the understanding of the binding mode of DHPIs in general and the development of new HNE selective inhibitors.

Section snippets

Inhibitor binding site and HNE:DHPI interactions

HNE adopts the typical fold of trypsin-like serine proteases.31 The determined overall three-dimensional structures of HNE in the DHPI complex (Fig. 1a) and free HNE are very similar and closely resemble those of previously published HNE:inhibitor complexes24, 25, 26, 27, 28, 29, 30 with RMSD between 0.14 Å and 0.47 Å for Cα atoms. We observed N-linked sugar moieties at both potential glycosylation sites. At Asn109, clear electron density was visible for one β-N-acetyl-d-glucosamine (NAG). At

Comparison of the HNE:DHPI complex and free HNE

The binding of DHPI leads to major structural changes in the active-site region. The most significant difference in the active-site topology between free HNE and the HNE:DHPI complex is found within the S2 binding pocket. The main-chain loop containing the consecutive residues Tyr94, Asp95, Pro98, Val99, Asn99A, and Leu99B is situated in proximity to the S2 subsite, and its side chains contribute to the paneling of the S2 subsite. The loop itself adopts a β-sheet-like topology with several

Comparison of the HNE:DHPI complex with other published HNE-complexes

Seven different crystal structures of HNE complexes have been described in the literature. Bode et al. reported a non-covalent complex of HNE with the third domain of the ovomucoid inhibitor (OMTKY3), a 56-amino-acid protease inhibitor.29 More recently, the structure of HNE in complex with the inhibitory domain of secretory leukocyte protease inhibitor (SLPI) has been reported.30 In addition, five complexes with suicide small-molecule inhibitors forming covalent bonds with Ser195 or His57 have

PDB accession codes

The final refined atomic coordinates and structure factors of the HNE:DHPI complex and the uncomplexed enzyme have been deposited in the Research Collaboratory for Structural Bioinformatics PDB with PDB ID: 3Q77 and PDB ID: 3Q76, respectively.

Acknowledgements

We thank Sabine Lohmer for the excellent characterization of HNE protein samples and initial screening for crystals. We are grateful to the staff of European Molecular Biology Laboratory outstation in Hamburg for advice during data collection with synchrotron radiation.

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    Present addresses: G. Hansen, Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany; P. Reinemer, InterMed Discovery GmbH, Otto-Hahn-Str. 15, D-44227 Dortmund, Germany; D. Schomburg, Department of Bioinformatics and Biochemistry, Technical University Braunschweig, Langer Kamp 19B, D-38106 Braunschweig, Germany.

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