Backbone assignment, secondary structure and protein A binding of an isolated, human antibody VH domain.
Antibody heavy chain variable domains (VH) lacking their light chain (VL) partner are prime candidates for the design of minimum-size immunoreagents. To obtain structural information about isolated VH domains, a human VH was labelled with 15N or doubly labelled with both 15N and 13C and was studied by heteronuclear nuclear magnetic resonance spectroscopy. Most (90%) of the 1H and 15N main-chain signals were assigned through two-dimensional TOCSY and NOESY experiments on the unlabelled VH and three-dimensional heteronuclear multiple quantum correlation TOCSY and NOESY experiments on the 15N-labelled VH. Four short stretches of the polypeptide chain could only be assigned on the basis of three-dimensional HNCA and HN(CO)CA experiments on the 13C-/15N-labelled protein. Long-range interstrand backbone NOEs suggest the presence of two adjacent beta-sheets formed by altogether nine antiparallel beta-strands. 3JNHC alpha H coupling constants and the location of slowly exchanging backbone amides support this interpretation. The secondary structure of the isolated VH is identical to that of heavy chain variable domains in intact antibodies, where VH domains are packed against a VL domain. The backbone assignments of the VH made it possible to locate its Protein A binding site. Chemical shift movements after complexing with the IgG binding fragment of Protein A indicate binding through one of the two beta-sheets of the VH. This beta-sheet is solvent exposed in intact antibodies. The Protein A binding site obviously differs from that on the Fc portion of immunoglobulins and is unique to members of the human VHIII gene subgroup.
Riechmann
L
L
MRC Laboratory of Molecular Biology, Cambridge, UK.
Davies
J
J
D000595:Amino Acid Sequence; D001665:Binding Sites; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013205:Staphylococcal Protein A / Q000378:metabolism*
D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D010446:Peptide Fragments; D013205:Staphylococcal Protein A