Interferon‐γ variants with deletions in the AB surface loop

Abstract

The receptor-binding AB loop of recombinant human interferon-γ (IFN-γ) has multiple contacts with the extracellular part of the IFN-γ receptor A chain (IFN-γRA). 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-γ and des-(N25, G26)-IFN-γ. Both mutations were tolerated by the folding pattern of recombinant human IFN-γ, as proved by CD spectroscopy. The stability of both mutants against cosolvent-induced unfolding was equal to that of wild-type IFN-γ. In contrast to the biophysical similarities of wild-type and mutant IFN-γ proteins, the biological activities of both mutants dropped significantly. Antiviral activity and human leucocyte antigen (HLA)-DR induction of des-(N25, G26)-IFN-γ was 10% that of wild-type activity. des-(A23, D24)-IFN-γ had only 1% remaining activity. Receptor-binding experiments confirmed that both deletions had a negative influence on the affinity of recombinant human IFN-γ 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-γRAϪIFN-γ complex.Keywords : interferon-γ; protein loops; computer modelling. 17 to 27. With an end-to-end distance of 15 Å (distance between the C A atoms of Ala17 and Thr27), it belongs to the class of Ω loops [7].The goal of our study was to examine the tolerance of the receptor-binding loop of recombinant human IFN-γ for deletions. An alignment of the AB-loop sequences from different species displays a one amino acid gap in mouse IFN-γ at Ala23 in the human sequence ( Table 1). This indicates a length variability in this region. Furthermore, the amino acid sequence of the AB loop determines the high species specificity of the cytokine [8]. MATERIALS AND METHODSModelling deletions in human IFN-γ. For computer modelling and documentation, the software packages BRAGI [9] and SYBYL (Tripos Associates Inc.) were used on a HP series 9000 model 735/99 workstation and on a Silicon Graphics Indigo 2 workstation, respectively. The starting model of recombinant human IFN-γ is based on the C A coordinates of the corresponding crystal structure (PDB code 1hig [2]) and has been described in an earlier report [6]. Deletions were constructed using a database search procedure [10]: a non-redundant fragment database, derived by hierarchical clustering (HCAPD database) is searched for suitable loops according to geometrical criteria derived from defined anchor groups in the template protein. The conformations of the highest ranked loops were inspected manually, and one loop fragment was selected. This selection was used to sim-