The unique C termini of orthopoxvirus gamma interferon binding proteins are essential for ligand binding.
The orthopoxviruses ectromelia virus (ECTV) and vaccinia virus (VACV) express secreted gamma interferon binding proteins (IFN-gammaBPs) with homology to the ligand binding domains of the host's IFN-gamma receptor (IFN-gammaR1). Homology between these proteins is limited to the extracellular portions of the IFN-gammaR1 and the first approximately 200 amino acids of the IFN-gammaBPs. The remaining 60 amino acids at the C termini of the IFN-gammaBPs contain a single cysteine residue shown to be important in covalent dimerization of the secreted proteins. The function of the remaining C-terminal domain (CTD) has remained elusive, yet this region is conserved within all orthopoxvirus IFN-gammaBPs. Using a series of C-terminal deletion constructs, we have determined that the CTD is essential for IFN-gamma binding despite having no predicted homology to the IFN-gammaR1. Truncation of the ECTV IFN-gammaBP by more than two amino acid residues results in a complete loss of binding activity for both murine IFN-gamma and human IFN-gamma (hIFN-gamma), as measured by surface plasmon resonance (SPR) and bioassay. Equivalent truncation of the VACV IFN-gammaBP resulted in comparable loss of hIFN-gamma binding activity by SPR. Full-length IFN-gammaBPs were observed to form higher-ordered structures larger than the previously reported dimers. Mutants that were unable to bind IFN-gamma with high affinity in SPR experiments failed to assemble into these higher-ordered structures and migrated as dimers. We conclude that the unique CTD of orthopoxvirus IFN-gammaBPs is important for the assembly of covalent homodimers as well as the assembly of higher-ordered structures essential for IFN-gamma binding.
Nuara
Anthony A
AA
Saint Louis University, Department of Molecular Microbiology and Immunology, 1402 South Grand Blvd., St. Louis, MO 63104, USA.
Bai
Hongdong
H
Chen
Nanhai
N
Buller
R Mark L
RM
Walter
Mark R
MR
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002522:Chlorocebus aethiops; D019281:Dimerization; D004481:Ectromelia virus / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007371:Interferon-gamma / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014616:Vaccinia virus / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D002352:Carrier Proteins; D008024:Ligands; D011994:Recombinant Proteins; D014764:Viral Proteins; D007371:Interferon-gamma