PDBsum entry 1rd4

Immune system

PDB id: 1rd4

Contents

Protein chain

184 a.a. *

Ligands

L08 ×4

* Residue conservation analysis

PDB id:

1rd4

Name:

Immune system

Title:

An allosteric inhibitor of lfa-1 bound to its i-domain

Structure:

Integrin alpha-l. Chain: a, b, c, d. Fragment: i domain, residues 125-311. Synonym: leukocyte adhesion glycoprotein lfa-1 alpha chain, leukocyte function associated molecule 1 alpha chain, cd11a. Engineered: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: itgal, cd11a. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

2.40Å R-factor: 0.236 R-free: 0.290

Authors:

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

Key ref:

M.P.Crump et al. (2004). Structure of an allosteric inhibitor of LFA-1 bound to the I-domain studied by crystallography, NMR, and calorimetry. Biochemistry, 43, 2394-2404. PubMed id: 14992576 DOI: 10.1021/bi035422a

Date:

05-Nov-03 Release date: 30-Mar-04

Protein chains

P20701  (ITAL_HUMAN) -  Integrin alpha-L from Homo sapiens

Seq: 1170 a.a.

Struc: 184 a.a.

DOI no: 10.1021/bi035422a

Biochemistry 43:2394-2404 (2004)

PubMed id: 14992576

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

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.

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.