Single-chain Fv multimers of the anti-neuraminidase antibody NC10: the residue at position 15 in the V(L) domain of the scFv-0 (V(L)-V(H)) molecule is primarily responsible for formation of a tetramer-trimer equilibrium.
Single-chain variable fragment of the murine monoclonal antibody NC10 specific to influenza virus N9 neuraminidase, joined directly in the V(L) to V(H) orientation (scFv-0), forms an equilibrium mixture of tetramer and trimer with the tetramer as the preferred multimeric species. In contrast, the V(H)-V(L) isomer was previously shown to exist exclusively as a trimer. Computer-generated trimeric and tetrameric scFv models, based on the refined crystal structure for NC10 Fv domain, were constructed and used to evaluate factors influencing the transition between V(L)-V(H) trimer and tetramer. These model structures indicated that steric restrictions between loops spanning amino acid residues L55-L59 and L13-L17 from the two adjacent V(L) domains within the V(L)-V(H) trimer were responsible for four scFv-0 molecules assembling to form a tetramer. In particular, leucine at position L15 and glutamate at position L57 appeared to interfere significantly with each other. To minimize this steric interference, the site-directed mutagenesis technique was used to construct several NC10 scFv-0 clones with mutations at these positions. Size-exclusion chromatographic analyses revealed that several of these mutations resulted in the production of NC10 scFv-0 proteins with significantly altered tetramer-trimer equilibrium ratios. In particular, introduction of a polar residue, such as asparagine or threonine, at position L15 generated a highly stable NC10 scFv-0 trimer.
Dolezal
Olan
O
CSIRO Health Sciences and Nutrition, 343 Royal Parade, Parkville 3052, Victoria, Australia. olan.dolezal@csiro.au
De Gori
Ross
R
Walter
Mark
M
Doughty
Larissa
L
Hattarki
Meghan
M
Hudson
Peter J
PJ
Kortt
Alexander A
AA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D007930:Leucine / Q000737:chemistry / Q000235:genetics; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009439:Neuraminidase / Q000276:immunology*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000906:Antibodies; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D018698:Glutamic Acid; D009439:Neuraminidase; D007930:Leucine