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PDBsum entry 2f9o
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References listed in PDB file
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Key reference
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Title
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X-Ray structures of free and leupeptin-Complexed human alphai-Tryptase mutants: indication for an alpha-->Beta-Tryptase transition.
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Authors
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K.B.Rohr,
T.Selwood,
U.Marquardt,
R.Huber,
N.M.Schechter,
W.Bode,
M.E.Than.
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Ref.
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J Mol Biol, 2006,
357,
195-209.
[DOI no: ]
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PubMed id
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Abstract
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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.
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Figure 2.
Figure 2. Stereo ribbon representation of the rHTaQGI
tetramer surrounded by a semitransparent surface. The four
monomers A to D are represented in gray, orange, yellow and
blue. Sugar chains, visible only in monomers B and D, and the
four leupeptin molecules binding to the active sites are
represented as ball-and-stick models with nitrogen blue, oxygen
red and carbonyl atoms black.
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Figure 7.
Figure 7. The uncomplexed tryptase single mutant rHTaG. (a)
Stick representation of the active site of rHTaG, showing the
open conformation (  vert,
similar 75% occupancy, blue) and the closed conformation ( vert,
similar 25% occupancy, light blue) as thick sticks.
Additionally, the 214-220 segments of aI-tryptase (orange) and
bII-tryptase (green) are superimposed as thin stick models to
show the high level of similarity of both conformations with the
two conformations seen in free active rHTaG. (b) B-factors of
the inhibited single mutant rHTaGI (continuous line), of the
inhibited double mutant rHTaQGI (dotted line) and of the
uncomplexed single mutant rHTaG (broken line) are plotted
against residue number (chymotrypsinogen numbering). A striking
increase of the B-factors can be seen for the 214-220 segment of
rHTaG. (c) Electron density of an averaged kicked omit map
(orange mesh) shown together with the open (blue) and the closed
(light blue) conformation of the 214-220 segment. The map was
calculated for residues 214-220 in the closed conformation and
contoured at 1.8s. The active site residues are shown in green.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2006,
357,
195-209)
copyright 2006.
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