local (2-fold; see below) rotation axis along the crystallographic 41 screw axis. The largely complementary interaction surfaces between the monomers of the tetramer are typical for intersubunit contacts, whereas neighboring tetramers interact with one another via much more usual crystal contacts. Thus, within a tetramer, monomer A (Fig. 2) interacts with monomers B and D via interfaces of sizes 540 Å2 and 1,075 Å2, respectively (solvent inaccessible surface probed by using a sphere of 1.4-Å radius; Collaborative Computational Project No. 4 suite). In contrast, the four monomers of one given tetramer interact with monomers from neighboring tetramers via interfaces of less than 280 Å2 (in the x-y plane) and 265 Å2 (along the z-axis), respectively. The contacts between tetramers include a number of hydrogen bonds and six unique salt bridges and thus are qualitatively similar to those usually observed in typical crystal contacts.
These packing considerations suggest that the tetramer emphasized in Fig. 1 represents the enzymatically active tetramer of human ß-tryptase. This tetramer selection is supported by the finding that the six loops that deviate most from the structures of other trypsin-like proteinases are aII involved in forming monomer-monomer contacts within a tetramer. More important, this unique tetramer perfectly explains the distinguishing properties of tryptase in solution, e.g., the resistance to proteinaceous inhibitors other than LDTI, the unusual substrate specificity, and the stabilization by the binding of heparin-like glycosaminoglycans (see below).
Overall Tetramer Structure. In the tryptase tetramer, monomers (arbitrarily assigned A, B, C, and D in Fig. 2) are positioned at the corners of a flat rectangular frame leaving a continuous central pore. The tetramer displays almost perfect 222 symmetry that, however, is not exact because of the crystallographically asymmetric environment and an imperfect internal packing (see below). The horizontal and the vertical 2-fold axes, which cross each other in the center of the tetramer, relate monomers A to B and C to D, or A to D and B to C, respectively. The third 2-fold symmetry axis relating monomers A to C and B to D is arranged virtually perpendicular to the other 2-fold axes and runs almost through their point of intersection in the central pore.
The active centers of the four monomers are directed toward the central pore (Fig. 2). This pore exhibits a rectangular cross section and is twisted by ˜30° about the tetramer axis. It possesses two narrow openings of dimension 40 Å×15 Å, and widens in its central part to a cross section of 50 Å×25 Å, just large enough for elongated peptides of the diameter of an a-helix to thread though the exits and to interact with the active sites. Both pore entrances are partially obscured by the 147-loops (see below), which project from each of the monomers but on alternative entrance sides, so that only two diagonally arranged active centers can be viewed directly (Fig. 2). With 33 basic (including 12 His residues) and 24 acidic residues per monomer, human tryptase exhibits an average percentage of charged residues comparable to related serine proteinases, but is only slightly positively charged at neutral pH. These charges are not evenly distributed along the molecular surface, however. Rather, negatively charged residues cluster preferentially on the inner pore-facing surface, conferring the pore with a quite negative electrostatic potential, and along the peripheral A–D (and B–C) edges. In contrast, the A–B (and C–D) peripheries and one front side of the monomer surface are positively charged and probably are involved in heparin binding (see below and Fig. 6).
Monomer Structure. The tryptase monomer exhibits the typical ß-strand-dominated fold seen in other trypsin-like serine proteinases. The core is made by two six-stranded