Crystallization and functional analysis of a soluble deglycosylated form of the human costimulatory molecule B7-1.
The interactions of B7-1 with CD28 and CTLA-4 modulate the course of human immune responses, making B7-1 an important target for developing structure-based therapeutics. B7-1 is, however, one of the most heavily glycosylated proteins found at the leukocyte cell surface, complicating the structural analysis of this molecule. Methods for the production, crystallization and selenomethionine labelling of a soluble deglycosylated form of this molecule are described. The protein readily forms both tetragonal plate and bipyramidal crystals belonging to space groups I4(1)22, with unit-cell parameters a = b = 56.9, c = 298.7 A, and P4(1)22 (or P4(3)22), with unit-cell parameters a = b = 89.0, c = 261.9 A, respectively. The I4(1)22 and primitive crystal forms diffract to 2.7 and 3.5 A, respectively. Surface plasmon resonance-based assays indicate that the ligand-binding properties of sB7-1 are unaffected by deglycosylation. Since none of the methods relied on any special structural properties of sB7-1, it is proposed that this novel combination of procedures could in principle be adapted to the systematic analysis of many other glycoproteins of structural or functional interest.
Nuffield Department of Clinical Medicine, The University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DU, England. sdavis@molbiol.ox.ac.uk
S J
SJ
Davis
S
S
Ikemizu
A V
AV
Collins
J A
JA
Fennelly
K
K
Harlos
E Y
EY
Jones
D I
DI
Stuart
D000069594:Abatacept; D015703:Antigens, CD; D000943:Antigens, Differentiation / Q000378:metabolism; D018122:B7-1 Antigen / Q000737:chemistry* / Q000378:metabolism*; D018106:CD28 Antigens / Q000378:metabolism; D060908:CTLA-4 Antigen; D003460:Crystallization; D006031:Glycosylation; D006801:Humans; D018796:Immunoconjugates; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012645:Selenomethionine / Q000378:metabolism; D012995:Solubility; D020349:Surface Plasmon Resonance; D014961:X-Ray Diffraction
D015703:Antigens, CD; D000943:Antigens, Differentiation; D018122:B7-1 Antigen; D018106:CD28 Antigens; D060908:CTLA-4 Antigen; C556706:CTLA4 protein, human; D018796:Immunoconjugates; D011993:Recombinant Fusion Proteins; D000069594:Abatacept; D012645:Selenomethionine