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PDBsum entry 4x1r
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Hydrolase inhibitor/hydrolase
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PDB id
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4x1r
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References listed in PDB file
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Key reference
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Title
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A cyclic peptidic serine protease inhibitor: increasing affinity by increasing peptide flexibility.
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Authors
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B.Zhao,
P.Xu,
L.Jiang,
B.Paaske,
T.Kromann-Hansen,
J.K.Jensen,
H.P.Sørensen,
Z.Liu,
J.T.Nielsen,
A.Christensen,
M.Hosseini,
K.K.Sørensen,
N.C.Nielsen,
K.J.Jensen,
M.Huang,
P.A.Andreasen.
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Ref.
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Plos One, 2014,
9,
e115872.
[DOI no: ]
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PubMed id
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Abstract
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Peptides are attracting increasing interest as protease inhibitors. Here, we
demonstrate a new inhibitory mechanism and a new type of exosite interactions
for a phage-displayed peptide library-derived competitive inhibitor, mupain-1
(CPAYSRYLDC), of the serine protease murine urokinase-type plasminogen activator
(uPA). We used X-ray crystal structure analysis, site-directed mutagenesis,
liquid state NMR, surface plasmon resonance analysis, and isothermal titration
calorimetry and wild type and engineered variants of murine and human uPA. We
demonstrate that Arg6 inserts into the S1 specificity pocket, its carbonyl group
aligning improperly relative to Ser195 and the oxyanion hole, explaining why the
peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with
novel unnatural Arg analogues with aliphatic or aromatic ring structures led to
an increased affinity, depending on changes in both P1 - S1 and exosite
interactions. Site-directed mutagenesis showed that exosite interactions, while
still supporting high affinity binding, differed substantially between different
uPA variants. Surprisingly, high affinity binding was facilitated by
Ala-substitution of Asp9 of the peptide, in spite of a less favorable binding
entropy and loss of a polar interaction. We conclude that increased flexibility
of the peptide allows more favorable exosite interactions, which, in combination
with the use of novel Arg analogues as P1 residues, can be used to manipulate
the affinity and specificity of this peptidic inhibitor, a concept different
from conventional attempts at improving inhibitor affinity by reducing the
entropic burden.
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