1rd4
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An allosteric inhibitor of LFA-1 bound to its I-domain
OverviewOverview
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.
About this StructureAbout this Structure
1RD4 is a Single protein structure of sequence from Homo sapiens with as ligand. Full crystallographic information is available from OCA.
ReferenceReference
Structure of an allosteric inhibitor of LFA-1 bound to the I-domain studied by crystallography, NMR, and calorimetry., Crump MP, Ceska TA, Spyracopoulos L, Henry A, Archibald SC, Alexander R, Taylor RJ, Findlow SC, O'Connell J, Robinson MK, Shock A, Biochemistry. 2004 Mar 9;43(9):2394-404. PMID:14992576
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