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Structure of an allosteric inhibitor of LFA-1 bound to the I-domain studied by crystallography, NMR, and calorimetry.

Crump, Matthew P, Ceska, Thomas A, Spyracopoulos, Leo, Henry, Alistair, Archibald, Sarah C, Alexander, Rikki, Taylor, Richard J, Findlow, Stuart C., O'Connell, James, Robinson, Martyn K and Shock, Anthony (2004) Structure of an allosteric inhibitor of LFA-1 bound to the I-domain studied by crystallography, NMR, and calorimetry. Biochemistry, 43, (9), pp. 2394-2404.

Record type: Article

Abstract

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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Published date: 9 March 2004

Identifiers

Local EPrints ID: 156683
URI: http://eprints.soton.ac.uk/id/eprint/156683
ISSN: 0006-2960
PURE UUID: 2d9c1850-68a5-4e22-8745-f9260ae0d3ca

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Date deposited: 01 Jun 2010 13:30
Last modified: 18 Jul 2017 12:42

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Contributors

Author: Matthew P Crump
Author: Thomas A Ceska
Author: Leo Spyracopoulos
Author: Alistair Henry
Author: Sarah C Archibald
Author: Rikki Alexander
Author: Richard J Taylor
Author: Stuart C. Findlow
Author: James O'Connell
Author: Martyn K Robinson
Author: Anthony Shock

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