Interaction of Kazal-type inhibitor domains with serine proteinases: biochemical and structural studies |
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Authors: | Schlott Bernhard Wöhnert Jens Icke Christian Hartmann Manfred Ramachandran Ramadurai Gührs Karl-Heinz Glusa Erika Flemming Joachim Görlach Matthias Grosse Frank Ohlenschläger Oliver |
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Affiliation: | Institut für Molekulare Biotechnologie e.V., Postfach 100813, D-07708 Jena, Germany. |
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Abstract: | The interaction of domains of the Kazal-type inhibitor protein dipetalin with the serine proteinases thrombin and trypsin is studied. The functional studies of the recombinantly expressed domains (Dip-I+II, Dip-I and Dip-II) allow the dissection of the thrombin inhibitory properties and the identification of Dip-I as a key contributor to thrombin/dipetalin complex stability and its inhibitory potency. Furthermore, Dip-I, but not Dip-II, forms a complex with trypsin resulting in an inhibition of the trypsin activity directed towards protein substrates. The high resolution NMR structure of the Dip-I domain is determined using multi-dimensional heteronuclear NMR spectroscopy. Dip-I exhibits the canonical Kazal-type fold with a central alpha-helix and a short two-stranded antiparallel beta-sheet. Molecular regions essential for inhibitor complex formation with thrombin and trypsin are identified. A comparison with molecular complexes of other Kazal-type thrombin and trypsin inhibitors by molecular modeling shows that the N-terminal segment of Dip-I fulfills the structural prerequisites for inhibitory interactions with either proteinase and explains the capacity of this single Kazal-type domain to interact with different proteinases. |
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Keywords: | dipetalin thrombin trypsin serine proteinase inhibitor heteronuclear NMR |
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