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 (IFN-gamma) has multiple contacts with the extracellular part of the IFN-gamma receptor a chain (IFN-gammaRalpha). We explored the possible length of truncated AB loops and their conformations by molecular modelling. Deletions of two amino acids at the tip of the loop were tolerated in the model without van der Waals collisions of the AB loop with helix F. Based on these modelling results, two deletion mutants were constructed by overlap-extension PCR mutagenesis: des-(A23, D24)-IFN-gamma and des-(N25, G26)-IFN-gamma. Both mutations were tolerated by the folding pattern of recombinant human IFN-gamma, as proved by CD spectroscopy. The stability of both mutants against cosolvent-induced unfolding was equal to that of wild-type IFN-gamma. In contrast to the biophysical similarities of wild-type and mutant IFN-gamma proteins, the biological activities of both mutants dropped significantly. Antiviral activity and human leucocyte antigen (HLA)-DR induction of des-(N25, G26)-IFN-gamma was 10% that of wild-type activity. des-(A23, D24)-IFN-gamma had only 1% remaining activity. Receptor-binding experiments confirmed that both deletions had a negative influence on the affinity of recombinant human IFN-gamma to its cellular receptor. We conclude from this combined molecular modelling and mutagenesis experiments, that the reduced flexibility of the truncated AB loop abrogates the possibility of the formation of a 3(10) helix in the receptor-bound state as observed in the X-ray structure of the IFN-gammaRalpha-IFN-gamma complex.
Fraunhofer Institut für Grenzflächen und Bioverfahrenstechnik (IGB), Abteilung Gentechnologie, Hannover, Germany. guenther@ita.fhg.de
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G
Waschütza
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Dengler
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Villmann
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Böttinger
B
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Otto
D000998:Antiviral Agents / Q000378:metabolism; D001665:Binding Sites / Q000502:physiology; D001667:Binding, Competitive; D002460:Cell Line; D002942:Circular Dichroism; D003001:Cloning, Molecular; D005434:Flow Cytometry; D019791:Guanidine / Q000494:pharmacology; D006684:HLA-DR Antigens / Q000276:immunology; D006801:Humans; D007371:Interferon-gamma / Q000737:chemistry*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D011487:Protein Conformation; D017510:Protein Folding; D017471:Receptors, Interferon / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D016415:Sequence Alignment; D017384:Sequence Deletion / Q000235:genetics
D000998:Antiviral Agents; D006684:HLA-DR Antigens; D017471:Receptors, Interferon; D011994:Recombinant Proteins; D007371:Interferon-gamma; D019791:Guanidine