The Crystal Structure of Human a1-Tryptase Reveals a Blocked Substrate-binding Region Ulf Marquardt 1 , Frank Zettl 2 , Robert Huber 1 , Wolfram Bode 1 * and Christian P. Sommerhoff 2 1 Max-Planck-Institut fu ¨r Biochemie, Abteilung Strukturforschung Am Klopferspitz 18a, D-82152 Martinsried bei Mu ¨ nchen Germany 2 Abteilung fu ¨r Klinische Chemie und Klinische Biochemie, Klinikumsstandort Innenstadt der Ludwig- Maximilians-Universita ¨t Nußbaumstraße 20, D-80336 Mu ¨nchen, Germany Human mast cell tryptases represent a subfamily of trypsin-like serine proteinases implicated in asthma. Unlike b-tryptases, a-tryptases apparently are proteolytically inactive. We have solved the 2.2 A ˚ crystal structure of mature human a1-tryptase. It reveals a frame-like tetrameric architecture that, surprisingly, does not require heparin-binding for stability. In marked contrast to b2-tryptase, the Ser214-Gly219 segment, which normally provides the template for substrate binding, is kinked in a-tryptase, thereby blocking its non-primed subsites. This so far unobserved subsite distortion is incompatible with productive substrate binding and processing. a-Tryptase apparently is trapped in this off-con- formation by repulsions and attractions of the Asp216 side-chain. How- ever, proteolytic activity could be generated by an induced-fit mechanism. q 2002 Elsevier Science Ltd. All rights reserved Keywords: tryptase; asthma; mast cells; allergy; X-ray crystallography *Corresponding author Introduction The dense granules of human mast cells, a cell type involved in acquired and innate immunity and the initiation of inflammatory and allergic responses, contain large amounts of tryptases. Human mast cell tryptases (EC 3.4.21.59) comprise a family of trypsin-like serine proteinases that are encoded by at least four non-allelic genes. 1–3 Several isoenzymes, called a1-, a2-, b1a-, b1b-, b2-, b3-, mMCP-7-like-1, mMCP-7-like-2 and TMT/ g-tryptases have been identified and characterized by their sequence in man. 1–7 Activation of the b-tryptase zymogens is believed to occur in a mixed autocatalytic/dipeptidyl peptidase I catalyzed manner on the way from the trans Golgi to the secretory granules, from where the active tetramer bound to and stabilized by granular pro- teoglycans (upon mast cell stimulation) is released into the surrounding medium together with hista- mine and other effectors. 8,9 The heparin-stabilized active b-tryptase tetramer has been shown in vitro to cleave e.g. fibrinogen, fibronectin, and kinino- gen, and to activate pro-urokinase, pro-MMP3 and PAR-2 receptors. 10 – 13 The inactivation of some bronchodilating peptides has been proposed as a mechanism, by which b-tryptases might enhance bronchoconstriction in asthmatic patients. 14 The restricted specificity of the b-tryptases, their pre- ferred cleavage after Lys and Arg residues, 15,16 their resistance to endogenous proteinaceous inhibitors and their heparin requirement for tetramer stabilization 17 have been well explained by the crystal structure of b2-tryptase. 18 Despite , 90% amino acid identity with the human b2 isoenzyme, human a-tryptases have been considered as non-functional proteinases that are not stored in secretory granules, but are rather secreted constitutively. 19 They are expressed in human lung and skin mast cells in smaller amounts than b-tryptases, but seem to predomi- nate in basophils. 20 a-Tryptases have been found in the blood of most normal individuals and at elevated concentrations in sera of systemic masto- cytosis patients. 21,22 Their non-functionality had been assigned to an Arg ! Gln substitution at position 2 3 in the a-tryptase propeptide, which was believed to hinder autocatalytic trimming and subsequent activation of the resulting zymogen. 9 More recently, Huang et al. demonstrated, by expression of a pseudo-zymogen in insect cells, that human a-tryptase can be activated and assembled into a tetramer. These authors further- more showed that this “mature” a-tryptase tetramer can be labeled with [ 3 H]DFP but exhibits 0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved E-mail address of the corresponding author: bode@biochem.mpg.de Abbreviations used: LDTI, leech-derived tryptase inhibitor; MCP, mast cell protease;MMP, matrix metalloproteinase; NCS, non-crystallographic symmetry; PAR, protease-activated receptor. doi:10.1016/S0022-2836(02)00625-3 available online at http://www.idealibrary.com on B w J. Mol. Biol. (2002) 321, 491–502