Design, characterization, and structure of a biologically active single-chain mutant of human IFN-gamma.
A mutant form of human interferon-gamma (IFN-gamma SC1) that binds one IFN-gamma receptor alpha chain (IFN-gamma R alpha) has been designed and characterized. IFN-gamma SC1 was derived by linking the two peptide chains of the IFN-gamma dimer by a seven-residue linker and changing His111 in the first chain to an aspartic acid residue. Isothermal titration calorimetry shows that IFN-gamma SC1 forms a 1:1 complex with its high-affinity receptor (IFN-gamma R alpha) with an affinity of 27(+/- 9) nM. The crystal structure of IFN-gamma SC1 has been determined at 2.9 A resolution from crystals grown in 1.4 M citrate solutions at pH 7.6. Comparison of the wild-type receptor-binding domain and the Asp111-containing domain of IFN-gamma SC1 show that they are structurally equivalent but have very different electrostatic surface potentials. As a result, surface charge rather than structural changes is likely responsible for the inability of the His111-->Asp domain of to bind IFN-gamma R alpha. The AB loops of IFN-gamma SC1 adopt conformations similar to the ordered loops of IFN-gamma observed in the crystal structure of the IFN-gamma/IFN-gamma R alpha complex. Thus, IFN-gamma R alpha binding does not result in a large conformational change in the AB loop as previously suggested. The structure also reveals the final six C-terminal amino acid residues of IFN-gamma SC1 (residues 253-258) that have not been observed in any other reported IFN-gamma structures. Despite binding to only one IFN-gamma R alpha, IFN-gamma SC1 is biologically active in cell proliferation, MHC class I induction, and anti-viral assays. This suggests that one domain of IFN-gamma is sufficient to recruit IFN-gamma R alpha and IFN-gamma R beta into a complex competent for eliciting biological activity. The current data are consistent with the main role of the IFN-gamma dimer being to decrease the dissociation constant of IFN-gamma for its cellular receptors.
Landar
A
A
Center for Macromolecular Crystallography, University of Alabama, Birmingham 35294, USA.
Curry
B
B
Parker
M H
MH
DiGiacomo
R
R
Indelicato
S R
SR
Nagabhushan
T L
TL
Rizzi
G
G
Walter
M R
MR
D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D002151:Calorimetry; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D003460:Crystallization; D018360:Crystallography, X-Ray; D019281:Dimerization; D015395:Histocompatibility Antigens Class I / Q000378:metabolism; D006801:Humans; D007371:Interferon-gamma / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D011485:Protein Binding; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017471:Receptors, Interferon / Q000737:chemistry / Q000378:metabolism; D055672:Static Electricity; D013816:Thermodynamics; D015854:Up-Regulation / Q000187:drug effects; D000096743:Interferon gamma Receptor
D000998:Antiviral Agents; D015395:Histocompatibility Antigens Class I; D007371:Interferon-gamma; D017471:Receptors, Interferon; D000096743:Interferon gamma Receptor