The structure of the human betaII-tryptase tetramer: fo(u)r better or worse

Abstract

Tryptases, the predominant serine proteinases of human mast cells, have recently been implicated as mediators in the pathogenesis of allergic and inflammatory conditions, most notably asthma. Their distinguishing features, their activity as a heparin-stabilized tetramer and resistance to most proteinaceous inhibitors, are perfectly explained by the 3-A crystal structure of human betaII-tryptase in complex with 4-amidinophenylpyruvic acid. The tetramer consists of four quasiequivalent monomers arranged in a flat frame-like structure. The active centers are directed toward a central pore whose narrow openings of approximately 40 A x 15 A govern the interaction with macromolecular substrates and inhibitors. The tryptase monomer exhibits the overall fold of trypsin-like serine proteinases but differs considerably in the conformation of six surface loops arranged around the active site. These loops border and shape the active site cleft to a large extent and form all contacts with neighboring monomers via two distinct interfaces. The smaller of these interfaces, which is exclusively hydrophobic, can be stabilized by the binding of heparin chains to elongated patches of positively charged residues on adjacent monomers or, alternatively, by high salt concentrations in vitro. On tetramer dissociation, the monomers are likely to undergo transformation into a zymogen-like conformation that is favored and stabilized by intramonomer interactions. The structure thus provides an improved understanding of the unique properties of the biologically active tryptase tetramer in solution and will be an incentive for the rational design of mono- and multifunctional tryptase inhibitors.

Figure 1

Packing of the human βII tryptase crystal. (a) View along the z-axis showing one layer of tryptase molecules in the x–y plane. The tryptase monomers are grouped into tetrameric aggregates that form extended sheets. Each of these tryptase tetramers is clearly delimited from its neighbors in both directions. A “reference” tetramer is shown in red for simplicity. (b) View across the z-axis. In the z direction, layers of tetramers are stacked on each other along the 4₁ screw axis. The local 2-fold symmetry axis is tilted from the z direction by ≈7°, causing increased crystal-stabilizing contacts between layers stacked in the z-direction. One unit cell (82.9 × 82.9 × 172.9Å), occupied by four tryptase tetramers, is indicated by a white bordered box.

Figure 2

Overall structure of the tryptase tetramer. The four monomers A, B, C, and D (clockwise) are shown as blue, red, green, and yellow ribbons, each surrounded by a semitransparent surface. The inhibitor molecules APPA are given as orange CPK models, each binding into one of the four S1 specificity pockets.

Figure 3

The tryptase monomer in standard orientation, i.e., as seen approximately from the middle of the central pore of the tetramer toward the active site of monomer A (represented by Ser-195, His-57, and Asp-102). (a) Ribbon representation of a tryptase monomer. The amidino group of the APPA molecule interacts with Asp-189 in the S1 pocket. Ser-195 O-γ is bound covalently to the APPA carbonyl group forming a hemiketal. The six unique surface loops of tryptase that surround the active site and are engaged in intermonomer contacts are shown in special colors, namely (anticlockwise) the 147-loop (light blue), the 70- to 80-loop (yellow), the 37-loop (orange), the 60-loop (magenta), the 97-loop (green), and the 173-flap (red). All other tryptase segments are given in dark blue. The side chains of the catalytic triad residues as well as Asp-143, Asp-145, and Asp-147 in the acidic 147-loop are shown as a ball-and-stick model. (b) Overlay of the structures of the tryptase monomer and bovine trypsin, both given as ropes. The color-coding of tryptase is as in a, whereas trypsin is shown in gray. The most relevant deviations from the trypsin backbone appear in the colored loop regions of tryptase.

Figure 4

Loop arrangements in the tetramer. The six special loops engaged in monomer–monomer interactions are shown in the color coding introduced in Fig. 3. (a) The D–A dimer as seen from outside of the tetramer along the local 2-fold axis. (b) The monomer viewed in standard orientation. (c) Front view of the tetramer. (d) The A–B dimer seen from outside of the tetramer along the local 2-fold axis.

Figure 5

Stick representation of the contact interfaces between monomers. (a) The AB-interface seen from inside the tetramer along the local 2-fold axis, shown together with the final 2F_o−F_c electron density map for both Tyr-75 side chains contoured at 1 σ level. The monomers and loops are given in the color coding introduced in Figs. 3 and 4. (b) The AD-interface (half side) observed approximately perpendicular to the local 2-fold axis, shown together with all intermonomer hydrogen bonds and salt bridges (green dots). Segments of monomers A and D are given in blue and yellow, respectively.

Figure 6

Model of the binding of a 20-mer heparin-like glycosaminoglycan chain along the A–B edge of the tryptase-tetramer. The solid-surface representation of tryptase indicates positive (blue) and negative (red) electrostatic potential contoured from −4 kT/e to 4 kT/e. The heparin chain (green/yellow/red stick model) is long enough to bind to clusters of positively charged residues on both sides of the monomer–monomer interface, thereby bridging and stabilizing the interface which is exclusively hydrophobic in nature (see Fig. 5a).

Figure 7

View from the LDTI inhibitor (represented only by its reactive site loop P7 to P3′) toward the active-site cleft. The P1 Lys residue is buried.

Figure 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 Cα-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.