1RD4

An allosteric inhibitor of LFA-1 bound to its I-domain

Summary for 1RD4

• Entry DOI: 10.2210/pdb1rd4/pdb
• Descriptor: Integrin alpha-L, 1-ACETYL-4-(4-{4-[(2-ETHOXYPHENYL)THIO]-3-NITROPHENYL}PYRIDIN-2-YL)PIPERAZINE (2 entities in total)
• Functional Keywords: immune system
• Biological source: Homo sapiens (human)
• Cellular location: Cell membrane ; Single-pass type I membrane protein : P20701
• Total number of polymer chains: 4
• Total formula weight: 88593.84

Authors

Crump, M.P.,Ceska, T.A.,Spyracopoulos, L.,Henry, A.,Archibald, S.C.,Alexander, R.,Taylor, R.J.,Findlow, S.C.,O'Connell, J.,Robinson, M.K.,Shock, A. (deposition date: 2003-11-05, release date: 2004-03-30, Last modification date: 2023-08-23)

Primary citation

Crump, M.P.,Ceska, T.A.,Spyracopoulos, L.,Henry, A.,Archibald, S.C.,Alexander, R.,Taylor, R.J.,Findlow, S.C.,O'Connell, J.,Robinson, M.K.,Shock, A.
Structure of an allosteric inhibitor of LFA-1 bound to the I-domain studied by crystallography, NMR, and calorimetry
Biochemistry, 43:2394-2404, 2004

PubMed 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.

PubMed: 14992576
DOI: 10.1021/bi035422a

Experimental method

X-RAY DIFFRACTION (2.4 Å)