USE OF 2D NMR, PROTEIN ENGINEERING, AND MOLECULAR MODELING TO STUDY THE HAPTEN-BINDING SITE OF AN ANTIBODY FV FRAGMENT AGAINST 2-PHENYLOXAZOLONE

被引:42
作者
MCMANUS, S [1 ]
RIECHMANN, L [1 ]
机构
[1] MRC, MOLEC BIOL LAB, HILLS RD, CAMBRIDGE CB2 2QH, ENGLAND
关键词
D O I
10.1021/bi00238a007
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Two-dimensional (2D) H-1 NMR spectroscopy was used to study the hapten-binding site of a recombinant antibody F(v) fragment expressed in Escherichia coli. Point mutations of residues in the CDR loops of the F(v) fragment were designed in order to investigate their influence on hapten binding and to make site-specific assignments of aromatic NMR proton signals. Two tyrosines giving NOEs to the ligand 2-phenyloxazolone were identified, residue 33 in CDR1 of the heavy chain and residue 32 in CDR1 of the light chain. The benzyl portion of 2-phenyloxazolone is located between these two residues. The binding site is close to the surface of the F(v) fragment. Comparison with a different anti-2-phenyloxazolone antibody, the crystal structure of which has recently been solved, shows that the general location of the hapten-binding site in both antibodies is similar. However, in the crystallographically solved antibody, the hapten is bound farther from the surface in a pocket created by a short CDR3 loop of the heavy chain. In the binding site identified in the F(v) fragment studied in this report, this space is probably filled by the extra seven residues of the CDR3.
引用
收藏
页码:5851 / 5857
页数:7
相关论文
共 28 条
  • [11] DOMAIN ASSOCIATION IN IMMUNOGLOBULIN MOLECULES - THE PACKING OF VARIABLE DOMAINS
    CHOTHIA, C
    NOVOTNY, J
    BRUCCOLERI, R
    KARPLUS, M
    [J]. JOURNAL OF MOLECULAR BIOLOGY, 1985, 186 (03) : 651 - 663
  • [12] STRUCTURE OF AN ANTIBODY COMBINING SITE BY MAGNETIC-RESONANCE
    DWEK, RA
    WAINHOBSON, S
    DOWER, S
    GETTINS, P
    SUTTON, B
    PERKINS, SJ
    GIVOL, D
    [J]. NATURE, 1977, 266 (5597) : 31 - 37
  • [13] 3-DIMENSIONAL STRUCTURE OF A FLUORESCEIN FAB COMPLEX CRYSTALLIZED IN 2-METHYL-2,4-PENTANEDIOL
    HERRON, JN
    HE, XM
    MASON, ML
    VOSS, EW
    EDMUNDSON, AB
    [J]. PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS, 1989, 5 (04) : 271 - 280
  • [14] INVESTIGATION OF EXCHANGE PROCESSES BY 2-DIMENSIONAL NMR-SPECTROSCOPY
    JEENER, J
    MEIER, BH
    BACHMANN, P
    ERNST, RR
    [J]. JOURNAL OF CHEMICAL PHYSICS, 1979, 71 (11) : 4546 - 4553
  • [15] MESSENGER-RNA SEQUENCES DEFINE AN UNUSUALLY RESTRICTED IGG RESPONSE TO 2-PHENYLOXAZOLONE AND ITS EARLY DIVERSIFICATION
    KAARTINEN, M
    GRIFFITHS, GM
    MARKHAM, AF
    MILSTEIN, C
    [J]. NATURE, 1983, 304 (5924) : 320 - 324
  • [16] Kabat E. A., 1987, SEQUENCES PROTEINS I, DOI 10.1016/0003-2697(84)90805-4
  • [17] 3-DIMENSIONAL TRIPLE-RESONANCE NMR-SPECTROSCOPY OF ISOTOPICALLY ENRICHED PROTEINS
    KAY, LE
    IKURA, M
    TSCHUDIN, R
    BAX, A
    [J]. JOURNAL OF MAGNETIC RESONANCE, 1990, 89 (03) : 496 - 514
  • [18] PROBING ANTIBODY DIVERSITY BY 2D NMR - COMPARISON OF AMINO-ACID SEQUENCES, PREDICTED STRUCTURES, AND OBSERVED ANTIBODY ANTIGEN INTERACTIONS IN COMPLEXES OF 2 ANTIPEPTIDE ANTIBODIES
    LEVY, R
    ASSULIN, O
    SCHERF, T
    LEVITT, M
    ANGLISTER, J
    [J]. BIOCHEMISTRY, 1989, 28 (18) : 7168 - 7175
  • [19] SEQUENCE-SPECIFIC [H-1]NMR RESONANCE ASSIGNMENTS AND SECONDARY STRUCTURE IDENTIFICATION FOR 1-ZINC AND 2-ZINC FINGER CONSTRUCTS FROM SWI5 - A HYDROPHOBIC CORE INVOLVING 4 INVARIANT RESIDUES
    NEUHAUS, D
    NAKASEKO, Y
    NAGAI, K
    KLUG, A
    [J]. FEBS LETTERS, 1990, 262 (02) : 179 - 184
  • [20] NEUHAUS D, 1989, NUCL OVERHAUSER EFFE, pCH5