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FIG. 8. Models of the interaction of the human tryptase tetramer with proteinaceous inhibitors. The tryptase tetramers are shown as green ribbons. An inhibitor molecule (blue) is modeled into the active site of monomer A by superposition of the proteinase moiety of known proteinase-inhibitor complexes to a tryptase monomer. For LDTI and BPTI the target proteinase was trypsin (17, 49), for MPI chymotrypsin (47). The active sites of the other tryptase monomers are occupied by APPA molecules (orange). Parts of the inhibitors clashing with the structure of tryptase (i.e., a distance smaller than 1.5 Å between the Ca-atoms of the respective molecules) are highlighted in red. (a) In addition to one molecule of the “atypical” Kazal-type inhibitor LDTI bound to the tryptase monomer A a second molecule (shown in pink and yellow) can bind to the active site of either monomer B or C. (b) Bovine pancreatic trypsin inhibitor (aprotinin). (c) Human mucous proteinase inhibitor bound to tryptase with its inhibitorily active second domain.

atic trypsin inhibitor indicate strong collisions of their distal pole segments with the neighboring monomers D and B, in particular with the 147-loops, explaining the observed inactivity of these inhibitors toward tryptase (Fig. 8b). The central portion of the two-domain mucous proteinase inhibitor (MPI=SLPI=HUSI-I) would clash with the A–D interface region of the tryptase tetramer if bound to the active site of monomer A (Fig. 8c) via its inhibitorily active second domain (47). Similarly, elafin (=SKALP), an inhibitor corresponding to the MPI second domain (48), should not be able to inhibit tryptase. The much larger plasma proteinase inhibitors are clearly far too bulky to fit into the narrow pore of the tryptase tetramer and gain access to one of the active centers.

CONCLUSION

In summary, the structure of the ßII-tryptase tetramer has been identified based on the four crystallographically independent quasiidentical monomers and the analysis of their arrangement within the crystal packing. With its frame-like architecture and its active centers facing a narrow central pore, the resulting tryptase tetramer structure explains most of the distinct properties of the biologically active tryptase tetramer in solution. The unusual substrate specificity, with a preference for peptidergic substrates, and the resistance to proteinaceous inhibitors other than LDTI are both caused by the limited accessibility of the active sites within the narrow central pore. The tetramer can be stabilized by heparin glycosaminoglycan chains larger than ˜20 sugar residues, a length required to bridge the weaker of the two distinct monomer-monomer interfaces. The loss of enzymatic activity on dissociation of the tetramer is caused by stabilization by internal molecular groups of a zymogen-like rather than the active state. Finally, the knowledge of the structure of the active center of the monomer as well as of the distances between neighboring active sites allows the rational design of multifunctional inhibitors. Such inhibitors that bind to more than one active center will ideally have potentiated affinity, conferring selectivity for the tryptase tetramer. Such inhibitors will be valuable as pharmacological tools to probe the pathophysiological function(s) of tryptases in vivo and may have therapeutic potential against asthma and other mast-cell related disorders.

We are grateful to R.Huber and H.Fritz for their generous support. We thank D.Grosse and R.Mentele for their excellent help in crystallization and amino acid sequence analysis. This work was supported by Sonderforschungsbereich 469 of the University of Munich, the Deutsche Forschungsgemeinschaft (STU 161, BO 1279), the Fonds der Chemischen Industrie, and programs BIO4-CT98–0418 and TMR ERBFXCT 98–0193 of the European Union.

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