PREPARATION, CHARACTERIZATION AND CRYSTALLIZATION OF AN ANTIBODY FAB FRAGMENT THAT RECOGNIZES RNA - CRYSTAL-STRUCTURES OF NATIVE FAB AND 3 FAB-MONONUCLEOTIDE COMPLEXES

Fab fragments from Jel 103, an antibody which specifically binds to si ngle-stranded poly(rl), were prepared by papain digestion, separated i nto 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 Angs trom resolution. Soaking the crystals in solutions containing either o f the mononucleotides inosine-5'-diphosphate, guanosine-5'-diphosphate or deoxyinosine-5'-monophosphate resulted in binding of the nucleotid e in a single binding site. However, adenosine-5'-diphosphate does not bind to this antibody. The recognition of the base is achieved throug h hydrogen bonds to the C6 carbonyl oxygen and the imino NH group of t he purine in a pattern similar to that of the base-base interactions i n a double-stranded nucleic acid. Additional binding energy is provide d by stacking of the base and the Tyr32L side-chain and by interaction of the a-phosphate with the antibody in an anionic binding site. Most of the side-chains interacting with the nucleotide come from the ligh t chain. Surprisingly, this antibody shares the V-L sequence with anot her nucleic acid-binding antibody, BV04-1. The latter binds to a singl e stranded DNA with a high preference for thymine bases. The structure s of the unliganded and complexed Jel 103 Fab are compared to those of BV-04-1 Feb and while they show similarity in recognition of the base of the immunodominant nucleotide, their 5' phosphates occupy differen t positions, suggesting different orientation of the nucleic acid boun d to these two antibodies. Differences in the conformations of the L1 loops between the two Fabs have been noted.