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Structure of a novel leech carboxypeptidase inhibitor determined free in solution and in complex with human carboxypeptidase A2

Nature Structural Biology volume 7, pages 322–328 (2000)Cite this article

Abstract

Leech carboxypeptidase inhibitor (LCI) is a novel protein inhibitor present in the medicinal leech Hirudo medicinalis. The structures of LCI free and bound to carboxypeptidase A2 (CPA2)have been determined by NMR and X-ray crystallography, respectively. The LCI structure defines a new protein motif that comprises a five-stranded antiparallel β-sheet and one short α-helix. This structure is preserved in the complex with human CPA2 in the X-ray structure, where the contact regions between the inhibitor and the protease are defined. The C-terminal tail of LCI becomes rigid upon binding the protease as shown in the NMR relaxation studies, and it interacts with the carboxypeptidase in a substrate-like manner. The homology between the C-terminal tails of LCI and the potato carboxypeptidase inhibitor represents a striking example of convergent evolution dictated by the target protease. These new structures are of biotechnological interest since they could elucidate the control mechanism of metallo-carboxypeptidases and could be used as lead compounds for the search of fibrinolytic drugs.

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Figure 1: Amino acid sequence of LCI and NMR data used for identifying the secondary structure elements.
Figure 2: Three-dimensional structures of LCI.
Figure 3: Steady-state heteronuclear 1H-15N NOEs for the backbone amides of LCI.
Figure 4: Crystal structure of LCI–CPA2 and comparison between LCI and PCI structures.
Figure 5: Stereo view of the final 1.65 Å 2Fo − Fc electron density map (blue) contoured at 1σ around the ‘primary’ interaction site of LCI (residues suffixed with an i) with the active center of CPA2.
Figure 6: Surface representation of CPA2 in complex with LCI (yellow worm).

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Acknowledgements

D. Reverter is a recipient of a fellowship from the European Community Biotechnology Marie Curie program. Thanks to John Richardson for helpful discussions. This work was supported by the Deutsche Forschungsgemeinschaft, the Ministerio de Educación y Cultura (Spain), by the Centre de Referència en Biotecnologia (Generalitat de Catalunya) and by the E.C. Biotechnology Programme.

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Authors and Affiliations

  1. Department de Bioquímica i Biologia Molecular, Unitat de Ciències and Institut de Biologia Fonamental, Universitat Autonoma de Barcelona, Bellaterra, 08193, Barcelona, Spain

    David Reverter, Josep Vendrell & Francesc X. Avilés

  2. Max Planck Institute for Biochemistry, Martinsried, D-82152, Germany

    David Reverter, Carlos Fernández-Catalán, Roland Baumgartner, Ruth Pfänder, Robert Huber, Wolfram Bode & Tad A. Holak

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Correspondence to Tad A. Holak or Francesc X. Avilés.

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Reverter, D., Fernández-Catalán, C., Baumgartner, R. et al. Structure of a novel leech carboxypeptidase inhibitor determined free in solution and in complex with human carboxypeptidase A2. Nat Struct Mol Biol 7, 322–328 (2000). https://doi.org/10.1038/74092

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