The importance of framework residues H6, H7 and H10 in antibody heavy chains: Experimental evidence for a new structural subclassification of antibody V-H domains

The N-terminal segment (FR-H1) of the heavy chain (V-H) of antibodies shows significant conformational variability correlating with the nature of the amino acids H6, H7 and H10 (Kabat H9). In this study, we have established a causal relationship between the local sequence and the structure of this f ramework region and linked this relationship to important biophysical prope rties such as affinity, folding yield and stability. We have generated six mutants of the scFv fragment aL2, covering some of the most abundant amino acid combinations in positions H6, H7 and H10 (according to a new consensus nomenclature, Kabat H9). For the aL2 wild-type (w.t.) with the sequence 6( Q)7(P)10(A) and for two of the mutants, the X-ray structures have been dete rmined. The structure of the triple mutant aL2-6(E)7(S)10(G) shows the FR-H 1 backbone conformations predicted for this amino acid combination, which i s distinctly different from the structure of the w.t, thus supporting our h ypothesis that these residues determine the conformation of this segment. T he mutant aL2-6(E)7(P)10(G) represents a residue combination not occurring in natural antibody sequences. It shows a completely different, unique stru cture in the first X -strand of V-H, not observed in natural Fv fragments a nd forms a novel type of diabody. Two V-H domains of the mutant associate b y swapping the first P-strand. Concentration-dependent changes in Trp fluor escence indicate that this dimerization also occurs in solution. The mutati ons in amino acids H6, H7 and H10 (Kabat H9) influence the dimerization beh avior of the scFv and its thermodynamic stability. All the observations rep orted here have practical implications for the cloning of Fv fragments with degenerate primers, as well as for the design of new antibodies by CDR gra fting or synthetic Libraries. (C) 2001 Academic Press.