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PDBsum entry 1rd4
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Immune system
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PDB id
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1rd4
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Structure of an allosteric inhibitor of lfa-1 bound to the i-Domain studied by crystallography, Nmr, And calorimetry.
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Authors
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M.P.Crump,
T.A.Ceska,
L.Spyracopoulos,
A.Henry,
S.C.Archibald,
R.Alexander,
R.J.Taylor,
S.C.Findlow,
J.O'Connell,
M.K.Robinson,
A.Shock.
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Ref.
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Biochemistry, 2004,
43,
2394-2404.
[DOI no: ]
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PubMed id
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Abstract
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LFA-1 (lymphocyte function-associated antigen-1) plays a role in intercellular
adhesion and lymphocyte trafficking and activation and is an attractive
anti-inflammatory drug target. The alpha-subunit of LFA-1, in common with
several other integrins, has an N-terminally inserted domain (I-domain) of
approximately 200 amino acids that plays a central role in regulating ligand
binding to LFA-1. An additional region, termed the I-domain allosteric site
(IDAS), has been identified exclusively within the LFA-1 I-domain and shown to
regulate the function of this protein. The IDAS is occupied by small molecule
LFA-1 inhibitors when cocrystallized or analyzed by (15)N-(1)H HSQC
(heteronuclear single-quantum coherence) NMR (nuclear magnetic resonance)
titration experiments. We report here a novel arylthio inhibitor that binds the
I-domain with a K(d) of 18.3 nM as determined by isothermal titration
calorimetry (ITC). This value is in close agreement with the IC(50) (10.9 nM)
derived from a biochemical competition assay (DELFIA) that measures the level of
inhibition of binding of whole LFA-1 to its ligand, ICAM-1. Having established
the strong affinity of the arylthio inhibitor for the isolated I-domain, we have
used a range of techniques to further characterize the binding, including ITC,
NMR, and X-ray crystallography. We have first developed an effective ITC binding
assay for use with low-solubility inhibitors that avoids the need for
ELISA-based assays. In addition, we utilized a fast NMR-based assay for the
generation of I-domain-inhibitor models. This is based around the collection of
HCCH-TOCSY spectra of LFA-1 in the bound form and the identification of a subset
of side chain methyl groups that give chemical shift changes upon binding of
LFA-1 inhibitors. This subset was used in two-dimensional (13)C-(15)N and
(15)N-filtered and -edited two-dimensional NMR experiments to identify a minimal
set of intraligand and ligand-protein NOEs, respectively (nuclear Overhauser
enhancements). Models from the NMR data were assessed by comparison to an X-ray
crystallographic structure of the complex, confirming that the method correctly
predicted the essential features of the bound ligand.
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