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.
Davis
S J
SJ
Nuffield Department of Clinical Medicine, The University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DU, England. sdavis@molbiol.ox.ac.uk
Ikemizu
S
S
Collins
A V
AV
Fennelly
J A
JA
Harlos
K
K
Jones
E Y
EY
Stuart
D I
DI
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