Three-dimensional structures of single-chain Fv-neuraminidase complexes.
The structure of the complex between a recombinant single-chain Fv construct of antibody NC10 with a five-residue peptide linker between VH and VL (termed scFv(5)), and its antigen, tetrameric neuraminidase from influenza virus (NA), has been determined and refined at 2.5 A resolution. The antibody-antigen binding interface is very similar to that of a similar NC10 scFv-NA complex in which the scFv has a 15-residue peptide linker (scFv(15)), and the NC10 Fab-NA complex. However, scFv(5) and scFv(15) have different stoichiometries in solution. While scFv(15) is predominantly monomeric in solution, scFv(5) forms dimers exclusively, because the five-residue linker is not long enough to permit VH and VL domains from the same polypeptide associating and forming an antigen-binding site. Upon forming a complex with NA, scFv(15) forms a approximately 300 kDa complex corresponding to one NA tetramer binding four scFv(15) monomers, while scFv(5) forms a approximately 590 kDa complex, corresponding to two NA tetramers crosslinked by four bivalent scFv(5) dimers. However, the dimeric scFv(5) in the scFv(5)-NA crystals does not crosslink NA tetramers, and modelling studies indicate that it is not possible to pack four dimeric and simultaneously bivalent scFvs between the NA tetramers with only a five-residue linker between VH and VL. The inability arises from the exacting requirement to orient the two antigen-binding surfaces to bind the tetrameric NA antigen while avoiding steric clashes with NC10 scFv(5) dimers bound to other sites on the NA tetramer. The utility of bivalent or bifunctional scFvs with short linkers may therefore be restricted by the steric constraints imposed by binding multivalent antigens.
Malby
R L
RL
Biomolecular Research Institute, 343 Royal Parade, Parkville, 3052, Australia.
McCoy
A J
AJ
Kortt
A A
AA
Hudson
P J
PJ
Colman
P M
PM
D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D009439:Neuraminidase / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; C055966:immunoglobulin Fv; D009439:Neuraminidase