Structure of an allosteric inhibitor of LFA-1 bound to the I-domain studied by crystallography, NMR, and calorimetry.
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.
Crump
Matthew P
MP
School of Biological Sciences, University of Southampton, Bassett Crescent East, UK. matt.crump@bristol.ac.uk
Ceska
Thomas A
TA
Spyracopoulos
Leo
L
Henry
Alistair
A
Archibald
Sarah C
SC
Alexander
Rikki
R
Taylor
Richard J
RJ
Findlow
Stuart C
SC
O'Connell
James
J
Robinson
Martyn K
MK
Shock
Anthony
A
D000495:Allosteric Site; D000577:Amides / Q000737:chemistry; D001667:Binding, Competitive; D002151:Calorimetry; D002934:Cinnamates / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D015195:Drug Design; D006801:Humans; D008024:Ligands; D016169:Lymphocyte Function-Associated Antigen-1 / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000737:chemistry; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry
D000577:Amides; D002934:Cinnamates; D008024:Ligands; D016169:Lymphocyte Function-Associated Antigen-1; D021122:Protein Subunits; D011994:Recombinant Proteins