CRYSTAL STRUCTURE OF THE CD2-BINDING DOMAIN OF CD58(LFA-3) AT 1.8&ARING RESOLUTION
Shinji Ikemizu1,
Lisa M. Sparks3, P. Anton van der Merwe4,
Karl Harlos1,2, David I. Stuart 1,2,
E. Yvonne Jones 1,2, and Simon J. Davis3.
1Laboratory of Molecular
Biophysics, The Rex Richards Building, South Parks Road, Oxford,
OX1 3QU, UK
2Oxford Centre for
Molecular Sciences, The Rex Richards Building, South Parks Road,
Oxford, OX1 3QU, UK
3Molecular Sciences
Division, Nuffield Department of Clinical Medicine, John
Radcliffe Hospital, Oxford, OX3 9DU
4MRC Cellular
Immunology Unit, Sir William Dunn School of Pathology, South
Parks Road, Oxford OX1 3RE, UK
Keywords: CD58, LFA-3, CD2, Cell
adhesion molecule, crystal structure
The binding of the cell surface molecule, CD58 (formerly lymphocyte function associated antigen-3, LFA-3), to its ligand, CD2, significantly increases the sensitivity of antigen recognition by T-cells, was the first heterophilic cell adhesion interaction to be discovered, and has become an important paradigm for analysing the structural basis of cell-cell recognition. The crystal structure of a CD2-binding chimeric form of CD58(cCD58), solved to 1.8 A resolution, reveals that the ligand binding domain of CD58 has the expected IgSF V-set topology and shares several of the hitherto unique structural features of CD2, underlining their close evolutionary relationship.
The chimeric protein was expressed in Lec3.2.8.1 cells(1) in the presence of 0.5mM N-butyldeoxynojirimycin and affinity-purified using mAb Bric-5 and deglycosylated with endo H(1).
Crystals grew under a large variety of conditions with the largest appearing in the presence of 1.1-1.5M ammonium sulphate, 50 mM Tris pH 8. Diffraction data were collected in-house at 20°C and on BM14 (l=0.8983 and 0.9463 A) of the European Synchrotron Radiation Facility (ESRF) at 100K using 18cm and 34.5cm MAR-Research detectors respectively. Data were processed and scaled using programs DENZO and SCALEPACK (2). The structure was determined by molecular replacement using the program AMoRe (3), the coordinates of rat CD2 (4) and the in-house X-ray data set (all observed data for 15 to 3.0 A resolution). An unambiguous solution was found for a single molecule which, after rigid body refinement, gave an Rcryst of 37.4%. Polyalanine was then substituted for the domain 1 sequence. Further refinement was carried out in program X-PLOR (5) with manual rebuilding in program O (6). Two domain rigid body refinement resulted in a relative reorientation of the domains. After simulated annealing, positional and overall B factor refinement, 2Fo-Fc and Fo-Fc electron density maps showed bias-free density and allowed the correct sequence to be substituted into domain 1. Subsequent refinement was carried out against the high resolution X-ray data set, with bulk solvent correction, using all observed data but with 5% of the data set aside for Rfree cross validation. The final Rcryst and Rfree are 0.202 and 0.243, respectively. The refined atomic model comprises residues 1 to 171 of the chimera, 3 N-linked GlcNAc carbohydrates and 197 ordered water molecules.
We will discuss about the CD2 binding domain of cCD58 comparison with another V-set domains [rat(4) and human(8) CD2 domain 1, CD4(7) domain 1, etc.] and heterophilic dimer models of cCD58 and CD2 based on crystal packing of CD2, CD8a(9) and P0(10)