gp130 is the founding member of the Tall family of shared cytokine receptors including the Leukemia Inhibitory Factor Receptor (LIFR), which serve as signaling receptors for at least ten different four-helix bundle cytokines. We have a long standing interest in recognition and signaling issues regarding gp130 and LIFR (Wang-ARI-2009.pdf). We have solved the structures of several extracellular gp130 complexes for the cytokines Interleukin-6, the Kaposi Sarcoma Herpesvirus homolog of IL-6 (viral IL-6), and Leukemia Inhibitory Factor (LIF), which elucidated a new paradigm for the assembly and architectures of gp130 family signaling complexes. Together with biophysical studies, we have begun to explain how these shared receptors can interact with a diverse set of ligands in specific fashions. Understanding how extracellular cytokine recognition is communicated to intracellular signaling modules such as JAK and STAT remains an important future challenge in this system, as well as other cytokine receptors under study in the lab.
Structural elucidation of a codon of MHC restriction by the T cell receptor
Implications of pre-B cell receptor structure on antibody repertoire selection
During an immune response, antigen receptors on T cells have an extraordinary capability to recognize both our own self MHC, as well as foreign peptides and MHC molecules which they did not encounter during T cell development. This phenomenon, termed alloreactivity, has remained enigmatic. We solved the crystal structure of the 2C T cell receptor complexed to a foreign MHC H-2Ld-QL9, which we compared to a structure of this same TCR bound to a self MHC H-2Kb-dEV8. Surprisingly, we found that the TCR uses an entirely different recognition strategy for the self versus foreign MHC, and that TCR cross-reactivity is not dependent on molecular mimicry. However, importantly, we do find that allorecognition, in this case, is directed against conserved structural determinants of the MHC, suggesting that TCR reactivity with even foreign MHC is a result of co-evolution of TCR germline genes with MHC. Further studies in this system are ongoing in collaboration with the laboratory of Prof. David Kranz.
How a T cell receptor distinguishes self versus foreign Major Histocompatibility Complex proteins
Structural biology of gp130-cytokine receptor signaling
Neurotrophins, such as the Nerve Growth Factor (NGF), are necessary factors for the development and the maintenance of nervous system in vertebrates. Two known types of receptors for neurotrophins are Trk receptor tyrosine kinases and p75 neurotrophin receptors. We (He et al., 2004) solved the structure of the NGF-p75 complex, and revealed this complex to be an asymmetrical 2:1 assembly. Subsequently, in Wehrman et al. (2007)we determined the structure of the TrkA-NGF complex, and showed that a 1:2:1 complex of TrkA-NGF-p75 is sterically feasible, but probably not advantageous with regards to increased-affinity NGF binding. We examined the possibility of heterodimerization of p75 and TrkA in mammalian cells using a whole cell, enzyme complementation-based assay, and found no evidence for the presence of a Trk-p75 complex. Therefore, we propose that TrkA and p75 bind neurotrophins independently, and they communicate with each other though downstream signaling processes.
Nerve Growth Factor interactions with p75 and Trk receptors
Measles virus infectivity
Interleukin-2 and the common gamma chain (γc) cytokine receptor family
Pleiotropic signaling through the Interleukin-4 and Interleukin-13 receptors
Structural basis of γδ TCR recognition of antigen
Structural biology of Glial-cell line derived neurotrophic factor receptors
The cytokines IL-4 and Il-13 play critical roles in allergy and asthma by binding to different combinations of shared signaling receptors. In LaPorte et al., we explored both structural and signaling aspects of this system. We determined the structures of the three cytokine-receptor complexes that comprise the IL-4/IL-13 system, and elucidated the molecular basis of receptor and ligand sharing. These studies also revealed unexpectedly different assembly properties of the signaling complexes on cell surfaces, and more recently that the unusual structure of the IL-13 receptor enables tuning of cellular responsiveness (Ito et al-2009.pdf). Collectively, these studies show that extracellular receptor-ligand interactions are not simply on/off switches for signal transduction, but may instigate divergent signaling responses, which could be selectively targeted and manipulated for therapeutic purposes.
Measles is a highly pathogenic virus that infects roughly 20 million people per year. While measles has been effectively contained in modernized countries through vaccination, it remains a significant health risk in underdeveloped countries. We determined the crystal structure of the measles virus hemagglutinin, the surface glycoprotein responsible for measles virus binding to its human host cell receptors. We found that, although the protein lacks the neuraminidase activity often used by viruses for cell attachment and entry, the structure resembles a dead neuraminidase fold, presenting spatially distinct receptor binding sites for its receptors CD46 and SLAM.
T cell receptor genes have co-evolved with MHC genes, and as a result the TCR gene products appear to have a bias for recognition of only MHC molecules, presenting antigenic peptides. This is opposed to antibodies, which can recognize the universe of potential antigens de novo. This TCR-MHC bias suggests the possible existence of recognition rules. We determined the structure of an autoimmune TCR-pMHC complex (Maynard et al.-left) which suggested that some portions of the TCR-MHC interactions could be highly conserved in other complexes. Subsequently, in Feng et al (right), we found that, indeed, a region of contact between the TCR and MHC is invariant for a particular subset of TCR and MHC molecules, and is, in effect, a recognition codon (see Garcia et al-2009.pdf). Ongoing structural and functional studies in collaboration with Mark Davis, David Kranz, and Paul Allen further explore this issue in both class I and class II MHC systems.
Artemin (ARTN) is a member of the glial cell line derived neurotrophic factor family (GDNF) ligands (GFLs), which regulate the development and maintenance of many neuronal populations in the mammalian nervous system. The structure of the complex formed between ARTN and its receptor GFRalpha3, which is the initiating step in the formation of a ternary signaling complex containing the shared RET receptor, represents a new receptor-ligand interaction mode revealing both conserved and specificity-determining anchor points for all GDNF-R pairs. Cellular studies using receptor chimeras implicate dyad-symmetric composite interfaces for recruitment and dimerization of RET, leading to intracellular signaling. These studies should facilitate the targetting of the GDNF systems for therapeutic applications.
Interleukin-2 (IL-2) is a key immunoregulatory cytokine whose actions are primarily mediated through a high-affinity, quaternary signaling complex found on activated T cells. IL-2 cooperatively assembles this complex (Rickert et al-2004.pdf) by initially engaging the alpha receptor (IL-2Rα) (Rickert et al-2005a.pdf), followed by recruitment of a beta receptor (IL-2Rβ) and the common gamma chain (γc), which also serves as a shared signaling receptor for IL-4, IL-7, IL-9, IL-15, and IL-21. In the structure of the quaternary complex (Wang et al-2005.pdf), IL-2Rα appears to capture IL-2 for presentation to IL-2Rβ and γc. The complex exhibits degenerate features in the γc interface that are ideal for engaging diverse cytokines. Several mutations found in X-linked severe combined immunodeficiency diseases (X-SCID) map to pivotal residues in the γc binding site. This structure provides a framework for all γc-cytokine-receptor complexes.
The pre-B cell receptor (pre-BCR) serves as a checkpoint in B cell development, and we carried out a series of biochemical and structural studies to understand a molecular basis of this event. In Bankovich et al., we showed in the structure of a human pre-BCR Fab-like fragment, consisting of an antibody heavy chain paired with the surrogate light chain, that the Unique regions of VpreB and lambda5 replace the complementarity-determining region 3 (CDR3) loop of an antibody light chain. In this manner the SLC appears to probe the amino acid sequence of heavy chain CDR3, potentially influencing the selection of the antibody repertoire given that heavy chain CDR3 plays the most important role in antigen recognition of all the antibody loops. Biochemical analysis indicates that the pre-BCR is impaired in its ability to recognize antigen, which, together with electron microscopic visualization of a pre-BCR dimer, suggests ligand-independent oligomerization as the likely signaling mechanism.
gamma delta (γδ) T cell receptors, αβ TCR, and antibodies (Ab) are the three lineages of somatically recombined antigen receptors. The potential combinatorial diversity of γδ TCR is greater than that of αβ TCR or antibodies, yet the functional significance of this diversity is unclear given that very few physiologically-relevant ligands for γδ TCR are known. We are engaged in a collaboration with Prof. Y.-h. Chien (Stanford M&I) to further our understanding of γδ TCR antigen recognition. We determined the structure of the first γδ TCR-ligand complex, the G8 γδ TCR in complex with the non-classical MHC T22. We found that G8 binds to T22 at an angle, contrasting with the head-on mode seen for αβ TCR, that G8 primarily uses germline-encoded residues of its CDR3δ for T22 interaction, which the Chien group showed is used by essentially all γδ TCR recognizing T22 ligands (Shin et al-2005.pdf). The details of the interaction suggested a hybrid between innate and adaptive recognition (see also Adams et al-2008.pdf). We are exploring the generality of this finding in other γδ systems.
Molecular architecture of a full-length tall cytokine receptor complex
Small molecule-induced allosteric activation of a bacterial Caspase
Most secreted bacterial toxins are produced as inactive precursors that become proteolytically activated upon entering a eukaryotic cell. A select group of these toxins undergo autoproteolysis, resulting in release of their effector domains. The Vibrio cholerae RTX is a member of the Multifunctional Autoprocessing RTX (MARTX) family of toxins, which all contain a cysteine protease domain (CPD) predicted to mediate autoproteolytic activation of the secreted protoxin upon entry into the eukaryotic cytosol. Through a collaboration with Aimee Shen and Prof. Matt Bogyo, we showed in Lupardus et al-RTX.pdf. that the RTX CPD is efficiently activated by the eukaryote-specific small molecule inositol hexakisphosphate (InsP6), and that InsP6 binds to a basic cleft that is distant from the protease active site, inducing an allosteric switch that leads to autoprocessing and intracellular release of toxin effector domains. Since InsP6 is exclusive to eukaryotes, the responsiveness of bacterial toxins to InsP6 seems an ingenious strategy for regulating the function of a secreted toxin. Recently specific inhibitors have been developed (Shen et al-2009.pdf) and the autoproteolytic activity has been leveraged as a general protein purification tool (Shen et al-2009b.pdf).
In an effort to begin to understand how extracellular cytokine receptor engagement leads to intracellular signaling, as well as elucidate the structural organization of a quaternary tall receptor complex containing both gp130 and LIFR, Skiniotis et al. analyzed the structure of a full-length signaling complex using a combination of Electron Microscopy, X-ray crystallography, NMR, and calorimetry. Thermodynamic analysis indicates that CNTF/CNTFR heterodimerizes gp130 and LIF-R non-cooperatively to form an asymmetric 1:1:1:1 complex. A crystal structure of the unliganded cytokine-binding fragment of the LIF-R ectodomain, combined with single particle electron microscopic (EM) analysis of the full-length gp130/LIF-R/CNTFR/CNTF complex, elucidates the asymmetric structural arrangement of the quaternary complex. The EM analysis also reveals that the receptor extracellular regions are connected to the transmembrane regions as a continuous unit, with apparently mimimal segmental flexibility, and are therefore poised to sensitively transduce extracellular ligand engagement signals to the intracellular regions. Finally, these data elucidate the organizing principles for assembly of all tall class of gp130-family cytokine receptors, complexes including LIF, Leptin, IL-27, IL-23, IL-12, and others.
T helper (Th) cell have a central role in modulating immune responses. Their development and homeostasis is largely controlled by type I cytokines. However, while Th1 and Th2 cells have long been known to regulate cellular and humoral immunity, Th17 cells have been recently identified as a Th lineage that regulates inflammation via production of distinct cytokines such as interleukin (IL)-17. There is growing evidence that Th17 cells are pathological in many human diseases, leading to intense interest in defining their receptor biology and developing strategies to block their pathological effects. In Ely et al., we determined the crystal structure of a 1:2 complex of IL-17RA bound to IL-17F. The manner of complex formation is structurally unique for cytokines. We also find that the first receptor-binding event to the IL-17 cytokines modulates the affinity and specificity for the second receptor in order to promote hetero- versus homo-dimeric complex formation. IL-17RA utilizes a common recognition strategy to bind IL-17 cytokines, allowing it to act as a shared receptor within multiple different heteromeric signaling complexes. Together with a cytokine called Interleukin-23, whose structure we also determined (Lupardus et al-2009.pdf), the IL-17/IL-23 axis appears to provide a linkage between innate and cellular immunity.
Linking innate and adaptive Immunity: the Interleukin-17/Interleukin-23 axis
Structural snapshots of full-length JAK1 and a cytokine receptor-JAK1 holocomplex
Images to the left are linked to PDF of the relevant publication.
Structural elucidation of TCR specificity for MHC class II IE-k presenting MCC
T cells specific for the cytochrome c Ag are widely used to investigate many aspects of TCR specificity and interactions with peptide-MHC, but structural information has long been elusive. In this study, we present structures for the well-studied 2B4 TCR, as well as a naturally occurring variant of the 5c.c7 TCR, 226, which is cross-reactive with more than half of possible substitutions at all three TCR-sensitive residues on the peptide Ag. These structures alone and in complex with peptide-MHC ligands allow us to reassess many prior mutagenesis results. In addition, the structure of 226 bound to one peptide variant, p5E, shows major changes in the CDR3 contacts compared with wild-type, yet the TCR V-region contacts with MHC are conserved. These and other data illustrate the ability of TCRs to accommodate large variations in CDR3 structure and peptide contacts within the constraints of highly conserved TCR-MHC interactions.
While there is a great deal known about the structural basis of extracellular cytokine receptor activation, there is nearly nothing known about the structutres and intracellular mechanisms of cytokine receptor signaling through the Janus kinases (JAKSs). We are attempting to obtain structural information to understand the nature of JAK coupling to extracellular ligand bidning. Here, we reconstitute the full-length gp130 homodimer in complex with the cytokine interleukin-6 (IL-6), its alpha receptor (IL-6Rα) and Jak1, for electron microscopy imaging. We find that the full-length gp130 homodimer complex has intimate interactions between the trans- and juxtamembrane segments of the two receptors, appearing to form a continuous connection between the extra- and intracellular regions. 2D averages and 3D reconstructions of full-length Jak1 reveal a three lobed structure possessing extensive intersegmental flexibility that likely facilitates allosteric activation. Single-particle imaging of the gp130/IL-6/IL-6Rα/Jak1 holocomplex shows Jak1 associated with the membrane proximal intracellular regions of gp130, abutting the would-be inner leaflet of the cell membrane. This first snapshot of an intact JAK molecule has been very informative about the potential for conformational change in JAK, and we are continuing on this line of study.
Type I Interferons (IFNs) are important cytokines for innate immunity against viruses and cancer. Sixteen human type I IFN variants signal through the same cell-surface receptors, IFNAR1 and IFNAR2, yet they can evoke markedly different physiological effects. We collaborated with the Schreiber and Piehler groups to explore the structural and mechanistic basis of this perplexing issue. The crystal structures of two human type I IFN ternary signaling complexes containing IFNalpha2 and IFNomega reveal recognition modes and heterotrimeric architectures that are unique among the cytokine receptor superfamily but conserved between different type I IFNs. Receptor-ligand cross-reactivity is enabled by conserved receptor-ligand "anchor points" interspersed among ligand-specific interactions that "tune" the relative IFN-binding affinities, in an apparent extracellular "ligand proofreading" mechanism that modulates biological activity. Functional differences between IFNs are linked to their respective receptor recognition chemistries, in concert with a ligand-induced conformational change in IFNAR1, that collectively control signal initiation and complex stability, ultimately regulating differential STAT phosphorylation profiles, receptor internalization rates, and downstream gene expression patterns. A video tutorial of these results can be found at: http://www.youtube.com/watch?v=czUaVG7I4yA
The architecture of the T cell receptor/MHC complex impacts signaling
T cell receptor (TCR) engagement of peptide-major histocompatibility complex (pMHC) is essential to adaptive immunity, but it is unknown whether TCR signaling responses are influenced by the binding topology of the TCR-peptide-MHC complex. We developed yeast-displayed pMHC libraries that enabled us to identify new peptide sequences reactive with a single TCR. Structural analysis showed that four peptides bound to the TCR with distinct 3D and 2D affinities using entirely different binding chemistries. Three of the peptides that shared a common docking mode, where key TCR-MHC germline interactions are preserved, induced TCR signaling. The fourth peptide failed to induce signaling and was recognized in a substantially different TCR-MHC binding mode that apparently exceeded geometric tolerances compatible with signaling. We suggest that the stereotypical TCR-MHC docking paradigm evolved from productive signaling geometries and that TCR signaling can be modulated by peptides that are recognized in alternative TCR-pMHC binding orientations. An accompanying essay by Wang & Reinherz.pdf discusses the implications of this study.
Engineering “superkines” to tune immune cellular responsiveness
Two recent publications from the lab exemplify our efforts to exploit the wealth of structural information we have acquired about cytokine receptor complexes, to engineer cytokines with new, or altered activities that might be therapeutically useful. In Levin-2012.pdf. we reported the creation of “super-2” using in vitro evolution to stabilize Interleukin-2 such that it binds to one of its receptors with greater than 300-fold higher affinity. Super-2 has greatly increased potency on certain T cell subsets and appears to be more efficacious and less toxic in several in vivo tumor models. In Juntilla-2012.pdf., we reported the creation of a “super-4” that exhibits both higher affinity for one of its two receptors, but altered cell subset selectivity. Here we tested the concept that a highly pleiotropic cytokine could be engineered to exploit differences in its receptor expression levels on different cell types to achieve some measure of selectivity. Together, super-2 and super-4 constitute the first examples from our lab at attempts to “tune” the immune system by combining structure, protein engineering, cell signaling, and in vivo disease models. See also the perspectives on super-2 (Boder-2012.pdf) and super-4 (Sidhu-N&V.pdf) accompanying these papers.
In this study, we combine structural, biochemical, computational, cell signaling and gene expression approaches to ask how functional specification can be achieved by two different ligands (IL-2 and IL-15) that act through shared receptors. Interleukin 15 (IL-15) and IL-2 have distinct immunological functions even though both signal through the receptor subunit IL-2Rβ and the common γ-chain (γ(c)). Here, in Ring-2012.pdf we found that in the structure of the IL-15-IL-15Rα-IL-2Rβ-γ(c) quaternary complex, IL-15 binds to IL-2Rβ and γ(c) in a heterodimer nearly indistinguishable from that of the IL-2-IL-2Rα-IL-2Rβ-γ(c) complex, despite their different receptor-binding chemistries. IL-15Rα substantially increased the affinity of IL-15 for IL-2Rβ, and this allostery was required for IL-15 trans signaling. Consistent with their identical IL-2Rβ-γ(c) dimer geometries, IL-2 and IL-15 showed similar signaling properties in lymphocytes, with any differences resulting from disparate receptor affinities. Thus, IL-15 and IL-2 induced similar signals, and the cytokine specificity of IL-2Rα versus IL-15Rα determined cellular responsiveness. Our results provide new insights for the development of specific immunotherapeutics based on IL-15 or IL-2. See also a perspective written on this paper Ring-N&V.pdf
The Wnt/Frizzled system is an incredibly pleiotropic receptor-ligand axis that plays critical roles in cell fate decisions. Wnts are lipid-modified morphogens that play critical roles in development principally through engagement of Frizzled receptors. In Janda-2012.pdf, 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. We recently found that the unusual Wnt structure evolved by fusing a saponin lipid binding domain to a degenerate cytokine fold as explained in Bazan-Wnt.pdf
How do two different cytokines signal through the same receptor heterodimer ?
The structure of a Wnt and how it engages Frizzled
