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PDBsum entry 4e1z
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Transferase/transferase inhibitor
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PDB id
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4e1z
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References listed in PDB file
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Key reference
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Title
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Enabling structure-Based drug design of tyk2 through co-Crystallization with a stabilizing aminoindazole inhibitor.
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Authors
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M.A.Argiriadi,
E.R.Goedken,
D.Banach,
D.W.Borhani,
A.Burchat,
R.W.Dixon,
D.Marcotte,
G.Overmeyer,
V.Pivorunas,
R.Sadhukhan,
S.Sousa,
N.S.Moore,
M.Tomlinson,
J.Voss,
L.Wang,
N.Wishart,
K.Woller,
R.V.Talanian.
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Ref.
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Bmc Struct Biol, 2012,
12,
22.
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PubMed id
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Abstract
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ABSTRACT: BACKGROUND: Structure-based drug design (SBDD) can accelerate
inhibitor lead design and optimization, and efficient methods including protein
purification, characterization, crystallization, and high-resolution diffraction
are all needed for rapid, iterative structure determination. Janus kinases are
important targets that are amenable to structure-based drug design. Here we
present the first mouse Tyk2 crystal structures, which are complexed to
3-aminoindazole compounds. RESULTS: A comprehensive construct design effort
included N- and C-terminal variations, kinase-inactive mutations, and multiple
species orthologs. High-throughput cloning and expression methods were coupled
with an abbreviated purification protocol to optimize protein solubility and
stability. In total, 50 Tyk2 constructs were generated. Many displayed poor
expression, inadequate solubility, or incomplete affinity tag processing. One
kinase-inactive murine Tyk2 construct, complexed with an ATP-competitive
3-aminoindazole inhibitor, provided crystals that diffracted to 2.5--2.6 A
resolution. This structure revealed initial "hot-spot" regions for
SBDD, and provided a robust platform for ligand soaking experiments. Compared to
previously reported human Tyk2 inhibitor crystal structures (Chrencik et al.
(2010) J Mol Biol 400:413), our structures revealed a key difference in the
glycine-rich loop conformation that is induced by the inhibitor. Ligand binding
also conferred resistance to proteolytic degradation by thermolysin. As crystals
could not be obtained with the unliganded enzyme, this enhanced stability is
likely important for successful crystallization and inhibitor soaking methods.
CONCLUSIONS: Practical criteria for construct performance and prioritization,
the optimization of purification protocols to enhance protein yields and
stability, and use of high-throughput construct exploration enable structure
determination methods early in the drug discovery process. Additionally,
specific ligands stabilize Tyk2 protein and may thereby enable crystallization.
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