BacMam system for high-level expression of recombinant soluble and membrane glycoproteins for structural studies

The ubiquitous EBV causes infectious mononucleosis and is associated with several types of cancers. The EBV genome encodes an early gene product, BARF1, which contributes to pathogenesis, potentially through growth-altering and immune-modulating activities, but the mechanisms for such activities are poorly understood. We have determined the crystal structure of BARF1 in complex with human macrophage-colony stimulating factor (M-CSF), a hematopoietic cytokine with pleiotropic functions in development and immune response. BARF1 and M-CSF form a high-affinity, stable, ring-like complex in both solution and the crystal, with a BARF1 hexameric ring surrounded by three M-CSF dimers in triangular array. The binding of BARF1 to M-CSF dramatically reduces but does not completely abolish M-CSF binding and signaling through its cognate receptor FMS. A three-pronged down-regulation mechanism is proposed to explain the biological effect of BARF1 on M-CSF:FMS signaling. These prongs entail control of the circulating and effective local M-CSF concentration, perturbation of the receptor-binding surface of M-CSF, and imposition of an unfavorable global orientation of the M-CSF dimer. Each prong may reduce M-CSF:FMS signaling to a limited extent but in combination may alter M-CSF:FMS signaling dramatically. The downregulating mechanism of BARF1 underlines a viral modulation strategy, and provides a basis for understanding EBV pathogenesis.

Section: Resultsmentioning

confidence: 99%

“…The BARF1 and M-CSF proteins were expressed from HEK293S N-acetylglucosaminyltransferase I-negative cells (33) using the BacMam method previously described (20). The N-linked glycans of these proteins were trimmed with endoglycosidase F1 (Sigma) before crystallization.…”

Section: Methodsmentioning

confidence: 99%

Multipronged attenuation of macrophage-colony stimulating factor signaling by Epstein–Barr virus BARF1

Shim¹,

Chang

Chen³

et al. 2012

“…To construct the single-chain FSH, we joined the FSHB coding sequence (N1-G122) and the common glycoprotein hormone alpha chain (CGA) coding sequence (A1-S92) with a GGGSGGNSGGGSGGGS linker, generally following the scheme described previously (8). This joined DNA fragment and the cDNA fragment encoding the full ectodomain of human FSHR ED (S16-R366) were attached to an N-terminal Gaussian luciferase signal peptide and a C-terminal 7-histidine tag, and were then subcloned into the baculovirus-mediated mammalian cell gene transduction (BacMam) vector pVLAD6 (30). The constructs and the BacVector-3000 baculovirus DNA (EMD Chemicals) were used to cotransfect Sf9 cells in six-well plates in the presence of the Insect GeneJuice reagent (EMD Chemicals).…”

Section: Methodsmentioning

confidence: 99%

Structure of follicle-stimulating hormone in complex with the entire ectodomain of its receptor

Jiang

Liu

Chen

et al. 2012

232

293

FSH, a glycoprotein hormone, and the FSH receptor (FSHR), a G protein-coupled receptor, play central roles in human reproduction. We report the crystal structure of FSH in complex with the entire extracellular domain of FSHR (FSHR ED ), including the enigmatic hinge region that is responsible for signal specificity. Surprisingly, the hinge region does not form a separate structural unit as widely anticipated but is part of the integral structure of FSHR ED . In addition to the known hormone-binding site, FSHR ED provides interaction sites with the hormone: a sulfotyrosine (sTyr) site in the hinge region consistent with previous studies and a potential exosite resulting from putative receptor trimerization. Our structure, in comparison to others, suggests FSHR interacts with its ligand in two steps: ligand recruitment followed by sTyr recognition. FSH first binds to the high-affinity hormone-binding subdomain of FSHR and reshapes the ligand conformation to form a sTyr-binding pocket. FSHR then inserts its sTyr (i.e., sulfated Tyr335) into the FSH nascent pocket, eventually leading to receptor activation.F SH is a gonadotropin that stimulates steroidogenesis and gametogenesis in the gonads. Secreted by the anterior pituitary gland, FSH regulates the menstrual cycle and ovarian follicular maturation in women and supports sperm production in men. FSH acts by binding to the FSH receptor (FSHR) on the granulosa cell surface in ovaries and the Sertoli cell surface in testes. The stimulated receptor leads to the dissociation of α-and βγ-subunits of G protein heterotrimer inside the cell. The α-subunit activates adenylyl cyclase, resulting in an increase of cAMP levels, and ultimately leads to the increased steroid production that is necessary for follicular growth and ovulation in women. The free βγ dimers recruit G protein-coupled receptor (GPCR) kinases to the receptor, which, in turn, lead to the recruitment of β-arrestin to the receptor (1). FSH is used clinically for controlled ovarian stimulation in women treated with assisted reproductive technologies and also for the treatment of anovulatory infertility in women and hypogonadotropic hypogonadism in men. The central role of FSH in human reproduction makes its receptor a unique pharmaceutical target in the field of fertility regulation (2-4).The glycoprotein hormone (GPH) family has four members: FSH; two other pituitary hormones, luteinizing hormone and thyroid-stimulating hormone (TSH); and one placental hormone, chorionic gonadotropin. The four members are homologous in sequence, structure, and function. Each member is a heterodimer composed of a common α-subunit and a hormone-specific β-subunit. The crystal structures of FSH and human CG (hCG) revealed that both α-and β-subunits adopt similar folds of cystineknot architecture (5-7). The assembled α-and β-heterodimers bind to their respective receptors with high affinity and hormone specificity, resulting in similar signaling pathways but distinct biological responses.

“…Additionally, 3C protease sites (LVELFQGP) were installed 9 aa after the predicted end of the transmembrane region for each CD3 chain. The constructs were cloned into the BacMam expression vector pVLAD6 and transfected to create baculovirus as previously described (27). Viruses were amplified to P2 before protein expression.…”

Section: Methodsmentioning

confidence: 99%

“…1G4 TCR has also recently been functionally reconstituted in HEK-293 cells (26). We created one baculovirus each for TCR and CD3 expression in mammalian cells (27), with individual TCR/CD3 chains cleaved into individual polypeptides through use of viral 2A peptides ( Fig. 2A) (28).…”

Section: Significancementioning

confidence: 99%

Molecular architecture of the αβ T cell receptor–CD3 complex

Birnbaum

Berry

Hsiao

et al. 2014

Self Cite

107

αβ T-cell receptor (TCR) activation plays a crucial role for T-cell function. However, the TCR itself does not possess signaling domains. Instead, the TCR is noncovalently coupled to a conserved multisubunit signaling apparatus, the CD3 complex, that comprises the CD3eγ, CD3eδ, and CD3ζζ dimers. How antigen ligation by the TCR triggers CD3 activation and what structural role the CD3 extracellular domains (ECDs) play in the assembled TCR-CD3 complex remain unclear. Here, we use two complementary structural approaches to gain insight into the overall organization of the TCR-CD3 complex. Small-angle X-ray scattering of the soluble TCR-CD3eδ complex reveals the CD3eδ ECDs to sit underneath the TCR α-chain. The observed arrangement is consistent with EM images of the entire TCR-CD3 integral membrane complex, in which the CD3eδ and CD3eγ subunits were situated underneath the TCR α-chain and TCR β-chain, respectively. Interestingly, the TCR-CD3 transmembrane complex bound to peptide-MHC is a dimer in which two TCRs project outward from a central core composed of the CD3 ECDs and the TCR and CD3 transmembrane domains. This arrangement suggests a potential ligand-dependent dimerization mechanism for TCR signaling. Collectively, our data advance our understanding of the molecular organization of the TCR-CD3 complex, and provides a conceptual framework for the TCR activation mechanism.T-cell receptor | electron microscopy | small-angle X-ray scattering T cells are key mediators of the adaptive immune response.Each αβ T cell contains a unique αβ T-cell receptor (TCR), which binds antigens (Ags) displayed by major histocompatibility complexes (MHCs) and MHC-like molecules (1). The TCR serves as a remarkably sensitive driver of cellular function: although TCR ligands typically bind quite weakly (1-200 μM), even a handful of TCR ligands are sufficient to fully activate a T cell (2, 3). The TCR does not possess intracellular signaling domains, uncoupling Ag recognition from T-cell signaling. The TCR is instead noncovalently associated with a multisubunit signaling apparatus, consisting of the CD3eγ and CD3eδ heterodimers and the CD3ζζ homodimer, which collectively form the TCR-CD3 complex (4, 5). The CD3γ/δ/e subunits each consist of a single extracellular Ig domain and a single immunoreceptor tyrosine-based activation motif (ITAM), whereas CD3ζ has a short extracellular domain (ECD) and three ITAMs (6-11). The TCR-CD3 complex exists in 1:1:1:1 stoichiometry for the αβTCR: CD3eγ:CD3eδ:CD3ζζ dimers (12). Phosphorylation of the intracellular CD3 ITAMs and recruitment of the adaptor Nck lead to T-cell activation, proliferation, and survival (13,14). Understanding the underlying principles of TCR-CD3 architecture and T-cell signaling is of therapeutic interest. For example, TCR-CD3 is the target of therapeutic antibodies such as the immunosuppressant OKT3 (15), and there is increasing interest in manipulating T cells in an Ag-dependent manner by using naturally occurring and engineered TCRs (16).Assembly of the TCR-CD3 complex is p...