Conformational multiplicity of the antibody combining site of a monoclonal antibody specific for a (6-4) photoproduct.
The antigen binding site of monoclonal antibody 64M5, which possesses a high degree of affinity for DNA containing pyrimidine (6-4) pyrimidone photoproducts, were investigated by use of stable-isotope-assisted NMR spectroscopy. A variety of 64M5 Fab fragments specifically labeled with 13C and 15N at backbone amide groups were prepared. Extensive assignments of amide resonances originating from the variable region of 64M5 were made by using 2D-HN(CO) measurements along with recombination of the heavy and light chains of 64M5. On the basis of chemical shift changes of the amide resonances caused upon addition of d(T[6-4]T) and d(GTAT[6-4]TATG), the binding sites of 64M5 Fab for the (6-4) photodimer and for the oligodeoxynucleotides flanking it were identified. It was revealed that the L1 and L3 segments, which are responsible for the binding to (6-4) photodimer, exhibit conformational multiplicities in the absence of antigens, and take different conformations between the d(T[6-4]T) and d(GTAT[6-4]TATG)-bound forms. On the basis of spectral comparison with another Fab fragment with a similarity in the amino acid sequence of the VL domain of 64M5, we suggest that the conformational multiplicities observed in the present study is caused by a substitution of an amino acid residue at the position of a key residue in L3 canonical structure, which leads to a preferable effect on the antigen binding, and by a specific combination of L1 and L3 canonical structures.
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, 113-0033, Japan.
T
T
Torizawa
K
K
Kato
J
J
Kato
H
H
Kobayashi
Y
Y
Komatsu
H
H
Morioka
O
O
Nikaido
E
E
Ohtsuka
I
I
Shimada
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D000918:Antibody Specificity; D000941:Antigens / Q000276:immunology; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011740:Pyrimidine Dimers / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000941:Antigens; D007140:Immunoglobulin Fab Fragments; D011740:Pyrimidine Dimers; C058406:pyrimidine-pyrimidone dimer