Conformational multiplicity of the antibody combining site of a monoclonalantibody specific for a (6-4) photoproduct

The antigen binding site of monoclonal antibody 64M5, which possesses a hig h degree of affinity for DNA containing pyrimidine (6-4) pyrimidone photopr oducts, were investigated by use of stable-isotope-assisted NMR spectroscop y. A variety of 64M5 Fab fragments specifically labeled with C-13 and N-15 backbone amide groups were prepared. Extensive assignments of amide resonan ces originating from the variable region of 64M5 were made by using 2D-HN(C O) measurements along with recombination of the heavy and light chains of 6 4M5. 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 re sponsible for the binding to (6-4) photodimer, exhibit conformational multi plicities in the absence of antigens, and take different conformations betw een the d(T[6-4]T) and d(GTAT[6-4]TATG)-bound forms. On the basis of spectr al comparison with another Fab fragment with a similarity in the amino acid sequence of the V-L domain of 64M5, we suggest that the conformational mul tiplicities 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 struct ure, which leads to a preferable effect on the antigen binding, and by a sp ecific combination of L1 and L3 canonical structures. (C) 1999 Academic Pre ss.