Preparation, characterization and crystallization of an antibody Fab fragment that recognizes RNA. Crystal structures of native Fab and three Fab-mononucleotide complexes.
Fab fragments from Jel 103, an antibody which specifically binds to single-stranded poly(rl), were prepared by papain digestion, separated into eight isoforms and characterized by mass spectrometry. One of the purified isoforms yielded crystals suitable for structural studies by X-ray diffraction and its crystal structure was determined to 2.4 A resolution. Soaking the crystals in solutions containing either of the mononucleotides inosine-5'-diphosphate, guanosine-5'-diphosphate or deoxyinosine-5'-monophosphate resulted in binding of the nucleotide in a single binding site. However, adenosine-5'-diphosphate does not bind to this antibody. The recognition of the base is achieved through hydrogen bonds to the C6 carbonyl oxygen and the imino NH group of the purine in a pattern similar to that of the base-base interactions in a double-stranded nucleic acid. Additional binding energy is provided by stacking of the base and the Tyr32L side-chain and by interaction of the alpha-phosphate with the antibody in an anionic binding site. Most of the side-chains interacting with the nucleotide come from the light chain. Surprisingly, this antibody shares the VL sequence with another nucleic acid-binding antibody, BV04-1. The latter binds to a single stranded DNA with a high preference for thymine bases. The structures of the unliganded and complexed Jel 103 Fab are compared to those of BV-04-1 Fab and while they show similarity in recognition of the base of the immunodominant nucleotide, their 5' phosphates occupy different positions, suggesting different orientation of the nucleic acid bound to these two antibodies. Differences in the conformations of the L1 loops between the two Fabs have been noted.
Pokkuluri
P R
PR
Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec.
Bouthillier
F
F
Li
Y
Y
Kuderova
A
A
Lee
J
J
Cygler
M
M
D000595:Amino Acid Sequence; D000918:Antibody Specificity; D001483:Base Sequence; D001666:Binding Sites, Antibody; D003460:Crystallization; D018360:Crystallography, X-Ray; D006153:Guanosine Diphosphate / Q000276:immunology; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D007290:Inosine Diphosphate / Q000276:immunology; D007291:Inosine Monophosphate / Q000031:analogs & derivatives / Q000276:immunology; D007526:Isoelectric Point; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012313:RNA / Q000276:immunology*; D012266:Ribose / Q000276:immunology
D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; D007291:Inosine Monophosphate; D006153:Guanosine Diphosphate; C017193:deoxyinosine monophosphate; D012313:RNA; D012266:Ribose; D007290:Inosine Diphosphate