title: Crystal structure and interaction studies of the human FBxo3 ApaG domain.
abstract: Transcriptional activation of proinflammatory cytokines, mediated by tumor necrosis factor receptor-associated factors (TRAFs), is in part triggered by the degradation of the F-box protein, FBxl2, via an E3 ligase that contains another F-box protein, FBxo3. The ApaG domain of FBxo3 is required for the interaction with and degradation of FBxl2 [Mallampalli RK et al., (2013) J Immunol 191, 5247-5255]. Here, we report the X-ray structure of the human FBxo3 ApaG domain, residues 278-407, at 2.0 Å resolution. Like bacterial ApaG proteins, this domain is characterized by a classic Immunoglobin/Fibronectin III-type fold, comprising a seven-stranded β-sheet core, surrounded by four extended loops. Although cation binding had been proposed for bacterial ApaG proteins, no interactions with Mg(2+) or Co(2+) were detected for the human ApaG domain. In addition, dinucleotide polyphosphates, which have been reported to be second messengers in the inflammation response and targets of the bacterial apaG-containing operon, are not bound by the human ApaG domain. In the context of the full-length protein, loop 1, comprising residues 294-303, is critical for the interaction with FBxl2. However, titration of the individual ApaG domain with a 15-mer FBxl2 peptide that was phosphorylated on the crucial T404, as well as the inability of the ApaG domain to interact with full-length FBxl2, assessed by coimmunoprecipitation, indicate that the ApaG domain alone is necessary, but not sufficient for binding and degradation of FBxl2.
  
  
  
  PDB ID (5HDW).
authors.affiliation: Department of Structural Biology, Pittsburgh Center for HIV Protein Interactions, University of Pittsburgh School of Medicine, PA, USA.
authors.forename: Troy C
authors.initials: TC
authors.lastname: Krzysiak
authors.affiliation: Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, University of Pittsburgh School of Medicine, PA, USA.|Acute Lung Injury Center of Excellence, University of Pittsburgh School of Medicine, PA, USA.
authors.forename: Bill B
authors.initials: BB
authors.lastname: Chen
authors.affiliation: Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, University of Pittsburgh School of Medicine, PA, USA.|Acute Lung Injury Center of Excellence, University of Pittsburgh School of Medicine, PA, USA.|Department of Environmental and Occupational Health, School of Public Health, University of Pittsburgh, PA, USA.
authors.forename: Travis
authors.initials: T
authors.lastname: Lear
authors.affiliation: Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, University of Pittsburgh School of Medicine, PA, USA.|Acute Lung Injury Center of Excellence, University of Pittsburgh School of Medicine, PA, USA.|Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, PA, USA.|Medical Specialty Service Line, Veterans Affairs Pittsburgh Healthcare System, PA, USA.
authors.forename: Rama K
authors.initials: RK
authors.lastname: Mallampalli
authors.affiliation: Department of Structural Biology, Pittsburgh Center for HIV Protein Interactions, University of Pittsburgh School of Medicine, PA, USA.
authors.forename: Angela M
authors.initials: AM
authors.lastname: Gronenborn
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D001665:Binding Sites; D018360:Crystallography, X-Ray; D044783:F-Box Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D000072417:Protein Domains; D054730:Protein Interaction Domains and Motifs / Q000235:genetics*; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000235:genetics; D054875:Ubiquitination / Q000235:genetics
chemical_list: D044783:F-Box Proteins; D044767:Ubiquitin-Protein Ligases; C122028:FBXL2 protein, human; C533903:FBXO3 protein, human
keywords: ApaG; F-box; FBxl2; FBxo3; inflammation