The influence of the buried glutamine or glutamate residue in position 6 on the structure of immunoglobulin variable domains.
Immunoglobulin V(H) domain frameworks can be grouped into four distinct types, depending on the main-chain conformation of framework 1. Based on the analysis of over 200 X-ray structures representing more than 100 non-redundant V(H) domain sequences, we have come to the conclusion that the marked structural variability of the V(H) framework 1 region is caused by three residues: the buried side-chain of H6, which can be either a glutamate or a glutamine residue, the residue in position H7, which may be proline only if H6 is glutamine, and by H9 (H10 according to a new consensus nomenclature), which has to be either glycine or proline if H6 is a glutamate residue. In natural antibodies, these three residues are encoded in combinations that are compatible with each other and with the rest of the structure and therefore will yield functional molecules. However, the degenerate primer mixtures commonly used for PCR cloning of antibody fragments can and frequently do introduce out-of-context mutations to combinations that can lead to severe reduction of stability, production yield and antigen affinity.
Honegger
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Biochemisches Institut der Universität Zürich, Winterthurerstrasse 190, Zürich, CH-8057, Switzerland.
Plückthun
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D000595:Amino Acid Sequence; D000818:Animals; D000941:Antigens / Q000276:immunology; D001666:Binding Sites, Antibody; D016384:Consensus Sequence; D018360:Crystallography, X-Ray; D019992:Databases as Topic; D019281:Dimerization; D018095:Germ-Line Mutation; D018698:Glutamic Acid / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism*; D006801:Humans; D006860:Hydrogen Bonding; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D012984:Software; D013329:Structure-Activity Relationship
D000941:Antigens; D007135:Immunoglobulin Variable Region; D005973:Glutamine; D018698:Glutamic Acid