2F9N

Crystal Structure of the Recombinant Human Alpha I Tryptase Mutant K192Q/D216G in Complex with Leupeptin

Summary for 2F9N

• Entry DOI: 10.2210/pdb2f9n/pdb
• Related: 2F9O 2F9P
• Related PRD ID: PRD_000216
• Descriptor: alpha I tryptase, Leupeptin, alpha-L-fucopyranose-(1-3)-[2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)][alpha-L-fucopyranose-(1-6)]2-acetamido-2-deoxy-beta-D-glucopyranose, ... (6 entities in total)
• Functional Keywords: serine proteinase, leupeptin, trypsin-like, difucosylation, hydrolase-hydrolase inhibitor complex, hydrolase/hydrolase inhibitor
• Biological source: Homo sapiens (human); More
• Total number of polymer chains: 8
• Total formula weight: 114593.14

Authors

Rohr, K.B.,Selwood, T.,Marquardt, U.,Huber, R.,Schechter, N.M.,Bode, W.,Than, M.E. (deposition date: 2005-12-06, release date: 2006-01-31, Last modification date: 2024-10-30)

Primary citation

Rohr, K.B.,Selwood, T.,Marquardt, U.,Huber, R.,Schechter, N.M.,Bode, W.,Than, M.E.
X-ray Structures of Free and Leupeptin-complexed Human alpha I-Tryptase Mutants: Indication for an alpha to beta-Tryptase Transition
J.Mol.Biol., 357:195-209, 2005

PubMed Abstract: Tryptases alpha and beta are trypsin-like serine proteinases expressed in large amounts by mast cells. Beta-tryptase is a tetramer that has enzymatic activity, but requires heparin binding to maintain functional and structural stability, whereas alpha-tryptase has little, if any, enzymatic activity but is a stable tetramer in the absence of heparin. As shown previously, these differences can be mainly attributed to the different conformations of the 214-220 segment. Interestingly, the replacement of Asp216 by Gly, which is present in beta-tryptase, results in enzymatically active but less stable alpha-tryptase mutants. We have solved the crystal structures of both the single (D216G) and the double (K192Q/D216G) mutant forms of recombinant human alphaI-tryptase in complex with the peptide inhibitor leupeptin, as well as the structure of the non-inhibited single mutant. The inhibited mutants exhibited an open functional substrate binding site, while in the absence of an inhibitor, the open (beta-tryptase-like) and the closed (alpha-tryptase-like) conformations were present simultaneously. This shows that both forms are in a two-state equilibrium, which is influenced by the residues in the vicinity of the active site and by inhibitor/substrate binding. Novel insights regarding the observed stability differences as well as a potential proteolytic activity of wild-type alpha-tryptase, which may possess a cryptic active site, are discussed.

PubMed: 16414069
DOI: 10.1016/j.jmb.2005.12.037

Experimental method

X-RAY DIFFRACTION (1.6 Å)