5WI6

Human beta-1 tryptase mutant Ile99Cys

Summary for 5WI6

• Entry DOI: 10.2210/pdb5wi6/pdb
• Related PRD ID: PRD_000288
• Descriptor: Tryptase alpha/beta-1, SULFATE ION, L-alpha-glutamyl-N-{(1S)-4-{[amino(iminio)methyl]amino}-1-[(1S)-2-chloro-1-hydroxyethyl]butyl}glycinamide, ... (4 entities in total)
• Functional Keywords: disulfide mutation, hydrolase, hydrolase-hydrolase inhibitor complex, hydrolase/hydrolase inhibitor
• Biological source: Homo sapiens (Human)
• Total number of polymer chains: 4
• Total formula weight: 112121.23

Authors

Eigenbrot, C.,Maun, H.R. (deposition date: 2017-07-18, release date: 2018-04-25, Last modification date: 2024-10-16)

Primary citation

Maun, H.R.,Liu, P.S.,Franke, Y.,Eigenbrot, C.,Forrest, W.F.,Schwartz, L.B.,Lazarus, R.A.
Dual functionality of beta-tryptase protomers as both proteases and cofactors in the active tetramer.
J. Biol. Chem., 293:9614-9628, 2018

PubMed Abstract: Human β-tryptase, a tetrameric trypsin-like serine protease, is an important mediator of the allergic inflammatory responses in asthma. During acute hypersensitivity reactions, mast cells degranulate, releasing active tetramer as a complex with proteoglycans. Extensive efforts have focused on developing therapeutic β-tryptase inhibitors, but its unique activation mechanism is less well-explored. Tryptase is active only after proteolytic removal of the pro-domain followed by tetramer formation via two distinct symmetry-related interfaces. We show that the cleaved I16G mutant cannot tetramerize, likely due to impaired insertion of its N terminus into its "activation pocket," indicating allosteric linkage at multiple sites on each protomer. We engineered cysteines into each of the two distinct interfaces (Y75C for small or I99C for large) to assess the activity of each tetramer and disulfide-locked dimer. Using size-exclusion chromatography and enzymatic assays, we demonstrate that the two large tetramer interfaces regulate enzymatic activity, elucidating the importance of this protein-protein interaction for allosteric regulation. Notably, the I99C large interface dimer is active, even in the absence of heparin. We show that a monomeric β-tryptase mutant (I99C*/Y75A/Y37bA, where C* is cysteinylated Cys-99) cannot form a dimer or tetramer, yet it is active but only in the presence of heparin. Thus heparin both stabilizes the tetramer and allosterically conditions the active site. We hypothesize that each β-tryptase protomer in the tetramer has two distinct roles, acting both as a protease and as a cofactor for its neighboring protomer, to allosterically regulate enzymatic activity, providing a rationale for direct correlation of tetramer stability with proteolytic activity.

PubMed: 29661938
DOI: 10.1074/jbc.M117.812016

Experimental method

X-RAY DIFFRACTION (2.72 Å)