INTERFERON-GAMMA VARIANTS WITH DELETIONS IN THE AB SURFACE LOOP - FLEXIBILITY IS A CRITICAL-POINT FOR RECEPTOR-BINDING

The receptor-binding AB loop of recombinant human interferon-gamma (IF N-gamma) has multiple contacts with the extracellular part of the IFN- gamma receptor a chain (LFN-gamma R alpha). We explored the possible l ength of truncated AB loops and their conformations by molecular model ling. Deletions of two amino acids at the tip of the loop were tolerat ed in the model without van der Waals collisions of the AB loop with h elix F. Based on these modelling results, two deletion mutants were co nstructed by overlap-extension PCR mutagenesis: des-(A23, D24)-IFN-gam ma and des-(N25, G26)-IFN-gamma. Both mutations were tolerated by the folding pattern of recombinant human IFN-gamma, as proved by CD spectr oscopy. The stability of both mutants against cosolvent-induced unfold ing was equal to that of wild-type IFN-gamma. In contrast to the bioph ysical similarities of wild-type and mutant IFN-gamma proteins, the bi ological activities of both mutants dropped significantly. Antiviral a ctivity and human leucocyte antigen (HLA)-DR induction of des-(N25, G2 6)-IFN-gamma was 10% that of wild-type activity. des-(A23, D24)-IFN-ga mma had only 1% remaining activity. Receptor-binding experiments confi rmed that both deletions had a negative influence on the affinity of r ecombinant human IFN-gamma to its cellular receptor. We conclude from this combined molecular modelling and mutagenesis experiments, that th e reduced flexibility of the truncated AB loop abrogates the possibili ty of the formation of a 3(10) helix in the receptor-bound state as ob served in the X-ray structure of the IFN-gamma R alpha-IFN-gamma compl ex.