Structural basis for the constitutive activity and immunomodulatory properties of the Epstein-Barr virus-encoded G protein-coupled receptor BILF1.

Epstein-Barr virus (EBV) encodes a G protein-coupled receptor (GPCR) termed BILF1 that is essential for EBV-mediated immunosuppression and oncogenesis. BILF1 couples with inhibitory G protein (Gi), the major intracellular signaling effector for human chemokine receptors, and exhibits constitutive signaling activity; the ligand(s) for BILF1 are unknown. We studied the origins of BILF1's constitutive activity through structure determination of BILF1 bound to the inhibitory G protein (Gi) heterotrimer. The 3.2-Å resolution cryo-electron microscopy structure revealed an extracellular loop within BILF1 that blocked the typical chemokine binding site, suggesting ligand-autonomous receptor activation. Rather, amino acid substitutions within BILF1 transmembrane regions at hallmark ligand-activated class A GPCR "microswitches" stabilized a constitutively active BILF1 conformation for Gi coupling in a ligand-independent fashion. Thus, the constitutive activity of BILF1 promotes immunosuppression and virulence independent of ligand availability, with implications for the function of GPCRs encoded by related viruses and for therapeutic targeting of EBV.

An extracellular interactome of immunoglobulin and LRR proteins reveals receptor-ligand networks.

Extracellular domains of cell surface receptors and ligands mediate cell-cell communication, adhesion, and initiation of signaling events, but most existing protein-protein "interactome" data sets lack information for extracellular interactions. We probed interactions between receptor extracellular domains, focusing on a set of 202 proteins composed of the Drosophila melanogaster immunoglobulin superfamily (IgSF), fibronectin type III (FnIII), and leucine-rich repeat (LRR) families, which are known to be important in neuronal and developmental functions. Out of 20,503 candidate protein pairs tested, we observed 106 interactions, 83 of which were previously unknown. We "deorphanized" the 20 member subfamily of defective-in-proboscis-response IgSF proteins, showing that they selectively interact with an 11 member subfamily of previously uncharacterized IgSF proteins. Both subfamilies interact with a single common "orphan" LRR protein. We also observed interactions between Hedgehog and EGFR pathway components. Several of these interactions could be visualized in live-dissected embryos, demonstrating that this approach can identify physiologically relevant receptor-ligand pairs.

Structural insights into human MHC-II association with invariant chain.

The loading of processed peptides on to major histocompatibility complex II (MHC-II) molecules for recognition by T cells is vital to cell-mediated adaptive immunity. As part of this process, MHC-II associates with the invariant chain (Ii) during biosynthesis in the endoplasmic reticulum to prevent premature peptide loading and to serve as a scaffold for subsequent proteolytic processing into MHC-II-CLIP. Cryo-electron microscopy structures of full-length Human Leukocyte Antigen-DR (HLA-DR) and HLA-DQ complexes associated with Ii, resolved at 3.0 to 3.1 Å, elucidate the trimeric assembly of the HLA/Ii complex and define atomic-level interactions between HLA, Ii transmembrane domains, loop domains, and class II-associated invariant chain peptides (CLIP). Together with previous structures of MHC-II peptide loading intermediates DO and DM, our findings complete the structural path governing class II antigen presentation.

Structure of the Wnt-Frizzled-LRP6 initiation complex reveals the basis for coreceptor discrimination.

Wnt morphogens are critical for embryonic development and tissue regeneration. Canonical Wnts form ternary receptor complexes composed of tissue-specific Frizzled (Fzd) receptors together with the shared LRP5/6 coreceptors to initiate ß-catenin signaling. The cryo-EM structure of a ternary initiation complex of an affinity-matured XWnt8-Frizzled8-LRP6 complex elucidates the basis of coreceptor discrimination by canonical Wnts by means of their N termini and linker domains that engage the LRP6 E1E2 domain funnels. Chimeric Wnts bearing modular linker "grafts" were able to transfer LRP6 domain specificity between different Wnts and enable non-canonical Wnt5a to signal through the canonical pathway. Synthetic peptides comprising the linker domain serve as Wnt-specific antagonists. The structure of the ternary complex provides a topological blueprint for the orientation and proximity of Frizzled and LRP6 within the Wnt cell surface signalosome.

Molecular architecture of the αß T cell receptor-CD3 complex.

αß 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 CD3εγ, CD3εδ, 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-CD3εδ complex reveals the CD3εδ 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 CD3εδ and CD3εγ subunits were situated underneath the TCR α-chain and TCR ß-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.

Structural basis of Janus kinase trans-activation.

Janus kinases (JAKs) mediate signal transduction downstream of cytokine receptors. Cytokine-dependent dimerization is conveyed across the cell membrane to drive JAK dimerization, trans-phosphorylation, and activation. Activated JAKs in turn phosphorylate receptor intracellular domains (ICDs), resulting in the recruitment, phosphorylation, and activation of signal transducer and activator of transcription (STAT)-family transcription factors. The structural arrangement of a JAK1 dimer complex with IFNλR1 ICD was recently elucidated while bound by stabilizing nanobodies. While this revealed insights into the dimerization-dependent activation of JAKs and the role of oncogenic mutations in this process, the tyrosine kinase (TK) domains were separated by a distance not compatible with the trans-phosphorylation events between the TK domains. Here, we report the cryoelectron microscopy structure of a mouse JAK1 complex in a putative trans-activation state and expand these insights to other physiologically relevant JAK complexes, providing mechanistic insight into the crucial trans-activation step of JAK signaling and allosteric mechanisms of JAK inhibition.

Atypical structural snapshots of human cytomegalovirus GPCR interactions with host G proteins.

Human cytomegalovirus (HCMV) encodes G protein-coupled receptors (GPCRs) US28 and US27, which facilitate viral pathogenesis through engagement of host G proteins. Here we report cryo-electron microscopy structures of US28 and US27 forming nonproductive and productive complexes with Gi and Gq, respectively, exhibiting unusual features with functional implications. The "orphan" GPCR US27 lacks a ligand-binding pocket and has captured a guanosine diphosphate-bound inactive Gi through a tenuous interaction. The docking modes of CX3CL1-US28 and US27 to Gi favor localization to endosome-like curved membranes, where US28 and US27 can function as nonproductive Gi sinks to attenuate host chemokine-dependent Gi signaling. The CX3CL1-US28-Gq/11 complex likely represents a trapped intermediate during productive signaling, providing a view of a transition state in GPCR-G protein coupling for signaling. Our collective results shed new insight into unique G protein-mediated HCMV GPCR structural mechanisms, compared to mammalian GPCR counterparts, for subversion of host immunity.

Structure of human Frizzled5 by fiducial-assisted cryo-EM supports a heterodimeric mechanism of canonical Wnt signaling.

Frizzleds (Fzd) are the primary receptors for Wnt morphogens, which are essential regulators of stem cell biology, yet the structural basis of Wnt signaling through Fzd remains poorly understood. Here we report the structure of an unliganded human Fzd5 determined by single-particle cryo-EM at 3.7 Å resolution, with the aid of an antibody chaperone acting as a fiducial marker. We also analyzed the topology of low-resolution XWnt8/Fzd5 complex particles, which revealed extreme flexibility between the Wnt/Fzd-CRD and the Fzd-TM regions. Analysis of Wnt/ß-catenin signaling in response to Wnt3a versus a 'surrogate agonist' that cross-links Fzd to LRP6, revealed identical structure-activity relationships. Thus, canonical Wnt/ß-catenin signaling appears to be principally reliant on ligand-induced Fzd/LRP6 heterodimerization, versus the allosteric mechanisms seen in structurally analogous class A G protein-coupled receptors, and Smoothened. These findings deepen our mechanistic understanding of Wnt signal transduction, and have implications for harnessing Wnt agonism in regenerative medicine.

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

Baculovirus mediated gene transduction of mammalian cells (BacMam) is an emerging technique for rapid recombinant protein expression in mammalian cells. We constructed two baculovirus transfer vectors that incorporate several mammalian transcriptional regulatory elements necessary for high-level protein expression in mammalian cells. Using these vectors, we show that the BacMam system in combination with the 293 GnTI(-) cell line can be used for production of milligram quantities of soluble glycoproteins. Moreover, for crystallization trials, the purified glycoproteins are sensitive to EndoH treatment resulting in a loss of the bulk of the attached N-linked glycosylation. In addition, we also show that a combination of the BacMam system and 293 GnTI(-) cell line can be used for producing milligram quantities of a GPCR-protein ligand complex suitable for crystallization trials.

Viral GPCR US28 can signal in response to chemokine agonists of nearly unlimited structural degeneracy.

Human cytomegalovirus has hijacked and evolved a human G-protein-coupled receptor into US28, which functions as a promiscuous chemokine 'sink' to facilitate evasion of host immune responses. To probe the molecular basis of US28's unique ligand cross-reactivity, we deep-sequenced CX3CL1 chemokine libraries selected on 'molecular casts' of the US28 active-state and find that US28 can engage thousands of distinct chemokine sequences, many of which elicit diverse signaling outcomes. The structure of a G-protein-biased CX3CL1-variant in complex with US28 revealed an entirely unique chemokine amino terminal peptide conformation and remodeled constellation of receptor-ligand interactions. Receptor signaling, however, is remarkably robust to mutational disruption of these interactions. Thus, US28 accommodates and functionally discriminates amongst highly degenerate chemokine sequences by sensing the steric bulk of the ligands, which distort both receptor extracellular loops and the walls of the ligand binding pocket to varying degrees, rather than requiring sequence-specific bonding chemistries for recognition and signaling.

Structural biology. Structural basis for chemokine recognition and activation of a viral G protein-coupled receptor.

Chemokines are small proteins that function as immune modulators through activation of chemokine G protein-coupled receptors (GPCRs). Several viruses also encode chemokines and chemokine receptors to subvert the host immune response. How protein ligands activate GPCRs remains unknown. We report the crystal structure at 2.9 angstrom resolution of the human cytomegalovirus GPCR US28 in complex with the chemokine domain of human CX3CL1 (fractalkine). The globular body of CX3CL1 is perched on top of the US28 extracellular vestibule, whereas its amino terminus projects into the central core of US28. The transmembrane helices of US28 adopt an active-state-like conformation. Atomic-level simulations suggest that the agonist-independent activity of US28 may be due to an amino acid network evolved in the viral GPCR to destabilize the receptor's inactive state.

Structural basis of Wnt recognition by Frizzled.

Wnts are lipid-modified morphogens that play critical roles in development principally through engagement of Frizzled receptors. The 3.25 angstrom structure of Xenopus Wnt8 (XWnt8) in complex with mouse Frizzled-8 (Fz8) cysteine-rich domain (CRD) reveals an unusual two-domain Wnt structure, not obviously related to known protein folds, resembling a "hand" with "thumb" and "index" fingers extended to grasp the Fz8-CRD at two distinct binding sites. One site is dominated by a palmitoleic acid lipid group projecting from serine 187 at the tip of Wnt's thumb into a deep groove in the Fz8-CRD. In the second binding site, the conserved tip of Wnt's "index finger" forms hydrophobic amino acid contacts with a depression on the opposite side of the Fz8-CRD. The conservation of amino acids in both interfaces appears to facilitate ligand-receptor cross-reactivity, which has important implications for understanding Wnt's functional pleiotropy and for developing Wnt-based drugs for cancer and regenerative medicine.

In vitro reconstitution and preparative purification of complexes between the chemokine receptor CXCR4 and its ligands SDF-1alpha, gp120-CD4 and AMD3100.

CXCR4 belongs to the family of G protein-coupled receptors and mediates the various developmental and regulatory effects of the chemokine SDF-1alpha. In addition, CXCR4 acts as a co-receptor along with CD4 for the HIV-1 viral glycoprotein gp120. Recently, there has also been a small molecule described that antagonizes both SDF-1 and gp120 binding to CXCR4. The structural and mechanistic basis for this dual recognition ability of CXCR4 is unknown largely due to the technical challenges of biochemically producing the components of the various complexes. We expressed the human CXCR4 receptor using a modified baculovirus expression vector that facilitates a single step antibody affinity purification of CXCR4 to >80% purity from Hi5 cells. The recombinant receptor undergoes N-linked glycosylation, tyrosine sulfation and is recognized by the 12G5 conformation specific antibody against human CXCR4. We are able to purify CXCR4 alone as well as complexed with its endogenous ligand SDF-1, its viral ligand gp120, and a small molecule antagonist AMD3100 by ion-exchange chromatography. We anticipate that the expression and purification scheme described in this paper will facilitate structure-function studies aimed at elucidating the molecular basis for CXCR4 recognition of its endogenous chemokine and viral ligands.