title: 1.8 A structure of murine GITR ligand dimer expressed in Drosophila melanogaster S2 cells.
abstract: Glucocorticoid-induced TNF receptor ligand (GITRL), a prominent member of the TNF superfamily, activates its receptor on both effector and regulatory T cells to generate critical costimulatory signals that have been implicated in a wide range of T-cell immune functions. The crystal structures of murine and human orthologs of GITRL recombinantly expressed in Escherichia coli have previously been determined. In contrast to all classical TNF structures, including the human GITRL structure, murine GITRL demonstrated a unique ;strand-exchanged' dimeric organization. Such a novel assembly behavior indicated a dramatic impact on receptor activation as well as on the signaling mechanism associated with the murine GITRL costimulatory system. In this present work, the 1.8 A resolution crystal structure of murine GITRL expressed in Drosophila melanogaster S2 cells is reported. The eukaryotic protein-expression system allows transport of the recombinant protein into the extracellular culture medium, thus maximizing the possibility of obtaining correctly folded material devoid of any folding/assembly artifacts that are often suspected with E. coli-expressed proteins. The S2 cell-expressed murine GITRL adopts an identical ;strand-exchanged' dimeric structure to that observed for the E. coli-expressed protein, thus conclusively demonstrating the novel quaternary structure assembly behavior of murine GITRL.
authors.affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
authors.forename: Kausik
authors.initials: K
authors.lastname: Chattopadhyay
authors.forename: Udupi A
authors.initials: UA
authors.lastname: Ramagopal
authors.forename: Stanley G
authors.initials: SG
authors.lastname: Nathenson
authors.forename: Steven C
authors.initials: SC
authors.lastname: Almo
mesh_terms: D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D019281:Dimerization; D004331:Drosophila melanogaster / Q000166:cytology; D004926:Escherichia coli; D006031:Glycosylation; D051379:Mice; D008958:Models, Molecular; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011487:Protein Conformation; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification; D013045:Species Specificity; D048069:Tumor Necrosis Factors / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D011993:Recombinant Fusion Proteins; C478966:Tnfsf18 protein, mouse; D048069:Tumor Necrosis Factors; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase