Effects on interaction kinetics of mutations at the VH-VL interface of Fabs depend on the structural context.
The influence of framework residues belonging to VH and VL modules of antibody molecules on antigen binding remains poorly understood. To investigate the functional role of such residues, we have performed semi-conservative amino acid replacements at the VH-VL interface. This work was carried out with (i) variants of the same antibody and (ii) with antibodies of different specificities (Fab fragments 145P and 1F1h), in order to check if functional effects are additive and/or similar for the two antibodies. Interaction kinetics of Fab mutants with peptide and protein antigens were measured using a BIACORE instrument. The substitutions introduced at the VH-VL interface had no significant effects on k(a) but showed small, significant effects on k(d). Mutations in the VH module affected k(d) not only for the two different antibodies but also for variants of the same antibody. These effects varied both in direction and in magnitude. In the VL module, the double mutation F(L37)L-Q(L38)L, alone or in combination with other mutations, consistently decreased k(d) about two-fold in Fab 145P. Other mutations in the VL module had no effect on k(d) in 145P, but always decreased k(d) in 1F1h. Moreover, in both systems, small-magnitude non-additive effects on k(d) were observed, but affinity variations seemed to be limited by a threshold. When comparing functional effects in antibodies of different specificity, no general rules could be established. In addition, no clear relationship could be pointed out between the nature of the amino acid change and the observed functional effect. Our results show that binding kinetics are affected by alteration of framework residues remote from the binding site, although these effects are unpredictable for most of the studied changes.
Khalifa
M B
MB
Institut de Biologie Structurale Jean-Pierre Ebel, CEA/CNRS, Grenoble, France.
Weidenhaupt
M
M
Choulier
L
L
Chatellier
J
J
Rauffer-Bruyère
N
N
Altschuh
D
D
Vernet
T
T
D000595:Amino Acid Sequence; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009029:Mosaic Viruses / Q000276:immunology; D016296:Mutagenesis; D009154:Mutation; D027383:Papillomaviridae / Q000276:immunology; D014764:Viral Proteins / Q000276:immunology
D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D014764:Viral Proteins