Sequence, specificity and crystallization of an oestrone-3-glucuronide antibody (3910).
We describe the specificity profile and V region sequences of a high-affinity monoclonal antibody (mAb), 3910, directed against oestrone-3-glucuronide (E3G). Inhibition studies show that the D-ring is critical for steroid specificity, while the glucuronic acid attached to the A ring is required for high binding affinity, suggesting that both 'ends' of the E3G ligand are recognized. The VH domain is encoded by a gene from the VH7183 family, while VL appears to be encoded by the Vk5.1 gene (kappa II subgroup) with a deletion of six residues from complementarity-determining region-1 (CDR1). The VH CDR3 is 10 amino acid residues in length, of which D/N contributes five residues. Comparison of VH CDR of 3910 with those of mAb against progesterone (DB3) and digoxin (26-10, 40-50), for which crystal structures have been determined, suggests that aromatic side chains are important for E3G binding and that tyrosine residues H50, H97 and H100 may interact with the ligand. The Fab fragment of 3910 has been crystallized in its native and steroid (E3G and oestriol-3-glucuronide) complexed forms. An X-ray diffraction data set to 3 A resolution has been collected for the native Fab.
He
M
M
Laboratory of Molecular Recognition, Babraham Institute, Cambridge, UK.
Gani
M
M
Livnah
O
O
Stura
E A
EA
Beale
D
D
Coley
J
J
Wilson
I A
IA
Taussig
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D000918:Antibody Specificity; D001483:Base Sequence; D003429:Cross Reactions; D003460:Crystallization; D004970:Estrone / Q000031:analogs & derivatives* / Q000737:chemistry / Q000276:immunology; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D007525:Isoelectric Focusing; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D014961:X-Ray Diffraction
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; C015856:estrone-3-glucuronide; D004970:Estrone