The Three-dimensional Structure of Recombinant Leech-derived Tryptase Inhibitor in Complex with Trypsin: IMPLICATIONS FOR THE STRUCTURE OF HUMAN MAST CELL TRYPTASE AND ITS INHIBITION

Milton T Stubbs,Robert Morenweiser,Jörg Stürzebecher,Margit Bauer,Wolfram Bode,Robert Huber,Gerd P Piechottka,Gabriele Matschiner,Christian P Sommerhoff,Hans Fritz,Ennes A Auerswald

doi:10.1074/jbc.272.32.19931

Abstract

The x-ray crystal structure of recombinant leech-derived tryptase inhibitor (rLDTI) has been solved to a resolution of 1.9 A in complex with porcine trypsin. The nonclassical Kazal-type inhibitor exhibits the same overall architecture as that observed in solution and in rhodniin. The complex reveals structural aspects of the mast cell proteinase tryptase. The conformation of the binding region of rLDTI suggests that tryptase has a restricted active site cleft. The basic amino terminus of rLDTI, apparently flexible from previous NMR measurements, approaches the 148-loop of trypsin. This loop has an acidic equivalent in tryptase, suggesting that the basic amino terminus could make favorable electrostatic interactions with the tryptase molecule. A series of rLDTI variants constructed to probe this hypothesis confirmed that the amino-terminal Lys-Lys sequence plays a role in inhibition of human lung tryptase but not of trypsin or chymotrypsin. The location of such an acidic surface patch is in accordance with the known low molecular weight inhibitors of tryptase.

Highlights

The x-ray crystal structure of recombinant leech-derived tryptase inhibitor has been solved to a resolution of 1.9 Å in complex with porcine trypsin
These studies indicate the location of putative heparin binding sites and suggest that the limited activity of human tryptase may be due to a restricted active site cleft
With the exception of the side chain of Tyr217, which swings out to accommodate the amino-terminal residues of leech-derived tryptase inhibitor (LDTI) (Fig. 1; see below), no significant conformational changes are seen in the trypsin component compared with other porcine trypsin structures [29, 47,48,49]