Kevin M. Jude

Structural basis for chemokine recognition and activation of a viral G protein–coupled receptor

Molecular “go” signals reveal their secrets Chemokines are proteins that direct how cells move within the body. For instance, chemokines help immune cells locate invading pathogens and ensure that cells position themselves correctly within a developing organ. Cells detect chemokines through G protein–coupled receptors on their surface; however, the molecular details of how these proteins interact remain unclear (see the Perspective by Standfuss). Qin et al. solved the crystal structure of the chemokine receptor CXCR4 bound to the viral chemokine vMIP-II. Burg et al. solved the crystal structure of a viral chemokine receptor bound to the chemokine domain of CX3CL1. Given the role of chemokines in a number of diseases, these results may help in future drug design. Science, this issue p. 1117, p. 1113; see also p. 1071 The crystal structure of a viral chemokine receptor bound to the chemokine CX3CL1 provides insights into chemokine recognition. [Also see Perspective by Standfuss] 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 for Notch1 engagement of Delta-like 4

An interaction that guides cell fate Notch signaling is important in cell fate determination in mammals. Signaling is initiated when the extracellular domain of the transmembrane Notch protein on one cell binds to a surface ligand on another cell. Luca et al. report the crystal structure of the interacting regions of Notch and the Delta-like ligand DLL-4. The Notch protein is modified by O-linked glycan addition, and this is required for signaling. The structure shows two interaction interfaces. A glycan anchors the less conserved interface, which potentially provides a flexible way of regulating Notch interactions during development. Science, this issue p. 847 O-linked glycosylation regulates a receptor-ligand interaction that is important in cell fate determination. Notch receptors guide mammalian cell fate decisions by engaging the proteins Jagged and Delta-like (DLL). The 2.3 angstrom resolution crystal structure of the interacting regions of the Notch1-DLL4 complex reveals a two-site, antiparallel binding orientation assisted by Notch1 O-linked glycosylation. Notch1 epidermal growth factor–like repeats 11 and 12 interact with the DLL4 Delta/Serrate/Lag-2 (DSL) domain and module at the N-terminus of Notch ligands (MNNL) domains, respectively. Threonine and serine residues on Notch1 are functionalized with O-fucose and O-glucose, which act as surrogate amino acids by making specific, and essential, contacts to residues on DLL4. The elucidation of a direct chemical role for O-glycans in Notch1 ligand engagement demonstrates how, by relying on posttranslational modifications of their ligand binding sites, Notch proteins have linked their functional capacity to developmentally regulated biosynthetic pathways.

CD47-blocking immunotherapies stimulate macrophage-mediated destruction of small-cell lung cancer.

Small-cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer with limited treatment options. CD47 is a cell-surface molecule that promotes immune evasion by engaging signal-regulatory protein alpha (SIRPα), which serves as an inhibitory receptor on macrophages. Here, we found that CD47 is highly expressed on the surface of human SCLC cells; therefore, we investigated CD47-blocking immunotherapies as a potential approach for SCLC treatment. Disruption of the interaction of CD47 with SIRPα using anti-CD47 antibodies induced macrophage-mediated phagocytosis of human SCLC patient cells in culture. In a murine model, administration of CD47-blocking antibodies or targeted inactivation of the Cd47 gene markedly inhibited SCLC tumor growth. Furthermore, using comprehensive antibody arrays, we identified several possible therapeutic targets on the surface of SCLC cells. Antibodies to these targets, including CD56/neural cell adhesion molecule (NCAM), promoted phagocytosis in human SCLC cell lines that was enhanced when combined with CD47-blocking therapies. In light of recent clinical trials for CD47-blocking therapies in cancer treatment, these findings identify disruption of the CD47/SIRPα axis as a potential immunotherapeutic strategy for SCLC. This approach could enable personalized immunotherapeutic regimens in patients with SCLC and other cancers.

Structure of a 129Xe-Cryptophane Biosensor Complexed with Human Carbonic Anhydrase II

Cryptophanes represent an exciting class of xenon-encapsulating molecules that can be exploited as probes for nuclear magnetic resonance imaging. The 1.70 A resolution crystal structure of a cryptophane-derivatized benezenesulfonamide complexed with human carbonic anhydrase II shows how an encapsulated xenon atom can be directed to a specific biological target. The crystal structure confirms binding measurements indicating that the cryptophane cage does not strongly interact with the surface of the protein, which may enhance the sensitivity of 129Xe NMR spectroscopic measurements in solution.

Instructive roles for cytokine-receptor binding parameters in determining signaling and functional potency.

Mathematical modeling of the cellular responses to cytokine variants of differing binding affinities may help design better therapies. Modeling cytokine behavior The use of cytokines, such as interleukin-2 (IL-2) or IL-13, as therapies has been hampered by the fact that many cytokines share receptor subunits on different cell types. Moraga et al. generated recombinant variants of IL-13 with a wide range of binding affinities for the IL-13 receptor. Mathematical modeling of the correlation between the receptor binding affinities of the variants and the extents to which they differentially stimulated early and late cellular responses highlighted aspects of receptor-ligand binding properties that should aid in the development of more effective cytokine therapies. Cytokines dimerize cell surface receptors to activate signaling and regulate many facets of the immune response. Many cytokines have pleiotropic effects, inducing a spectrum of redundant and distinct effects on different cell types. This pleiotropy has hampered cytokine-based therapies, and the high doses required for treatment often lead to off-target effects, highlighting the need for a more detailed understanding of the parameters controlling cytokine-induced signaling and bioactivities. Using the prototypical cytokine interleukin-13 (IL-13), we explored the interrelationships between receptor binding and a wide range of downstream cellular responses. We applied structure-based engineering to generate IL-13 variants that covered a spectrum of binding strengths for the receptor subunit IL-13Rα1. Engineered IL-13 variants representing a broad range of affinities for the receptor exhibited similar potencies in stimulating the phosphorylation of STAT6 (signal transducer and activator of transcription 6). Delays in the phosphorylation and nuclear translocation of STAT6 were only apparent for those IL-13 variants with markedly reduced affinities for the receptor. From these data, we developed a mechanistic model that quantitatively reproduced the kinetics of STAT6 phosphorylation for the entire spectrum of binding affinities. Receptor endocytosis played a key role in modulating STAT6 activation, whereas the lifetime of receptor-ligand complexes at the plasma membrane determined the potency of the variant for inducing more distal responses. This complex interrelationship between extracellular ligand binding and receptor function provides the foundation for new mechanism-based strategies that determine the optimal cytokine dose to enhance therapeutic efficacy.

Selective targeting of engineered T cells using orthogonal IL-2 cytokine-receptor complexes

Engineering cytokine-receptor pairs Interleukin-2 (IL-2) is an important cytokine that helps T cells destroy tumors and virus-infected cells. IL-2 has great therapeutic promise but is limited by toxic side effects and its capacity to both activate and repress immune responses. Sockolosky et al. set out to improve IL-2–based immunotherapy by engineering synthetic IL-2–receptor pairs (i.e., IL-2 and its receptor, IL-2R) (see the Perspective by Mackall). Engineered complexes transmitted IL-2 signals but only interacted with each other and not with endogenous IL-2/IL-2R. Treatment of mice with IL-2 improved the ability of engineered T cells to reject tumors with no obvious side effects. This type of approach may provide a way to mitigate toxicities associated with some cytokine-based immunotherapies. Science, this issue p. 1037; see also p. 990 Engineered cytokines are able to improve immunotherapy in mouse tumor models. Interleukin-2 (IL-2) is a cytokine required for effector T cell expansion, survival, and function, especially for engineered T cells in adoptive cell immunotherapy, but its pleiotropy leads to simultaneous stimulation and suppression of immune responses as well as systemic toxicity, limiting its therapeutic use. We engineered IL-2 cytokine-receptor orthogonal (ortho) pairs that interact with one another, transmitting native IL-2 signals, but do not interact with their natural cytokine and receptor counterparts. Introduction of orthoIL-2Rβ into T cells enabled the selective cellular targeting of orthoIL-2 to engineered CD4+ and CD8+ T cells in vitro and in vivo, with limited off-target effects and negligible toxicity. OrthoIL-2 pairs were efficacious in a preclinical mouse cancer model of adoptive cell therapy and may therefore represent a synthetic approach to achieving selective potentiation of engineered cells.

Real-time detection of DNA topological changes with a fluorescently labeled cruciform

Topoisomerases are essential cellular enzymes that maintain the appropriate topological status of DNA and are the targets of several antibiotic and chemotherapeutic agents. High-throughput (HT) analysis is desirable to identify new topoisomerase inhibitors, but standard in vitro assays for DNA topology, such as gel electrophoresis, are time-consuming and are not amenable to HT analysis. We have exploited the observation that closed-circular DNA containing an inverted repeat can release the free energy stored in negatively supercoiled DNA by extruding the repeat as a cruciform. We inserted an inverted repeat containing a fluorophore-quencher pair into a plasmid to enable real-time monitoring of plasmid supercoiling by a bacterial topoisomerase, Escherichia coli gyrase. This substrate produces a fluorescent signal caused by the extrusion of the cruciform and separation of the labels as gyrase progressively underwinds the DNA. Subsequent relaxation by a eukaryotic topoisomerase, human topo IIα, causes reintegration of the cruciform and quenching of fluorescence. We used this approach to develop a HT screen for inhibitors of gyrase supercoiling. This work demonstrates that fluorescently labeled cruciforms are useful as general real-time indicators of changes in DNA topology that can be used to monitor the activity of DNA-dependent motor proteins.

The IFN-λ-IFN-λR1-IL-10Rβ Complex Reveals Structural Features Underlying Type III IFN Functional Plasticity

SUMMARY Type III interferons (IFN‐&lgr;s) signal through a heterodimeric receptor complex composed of the IFN‐&lgr;R1 subunit, specific for IFN‐&lgr;s, and interleukin‐10R&bgr; (IL‐10R&bgr;), which is shared by multiple cytokines in the IL‐10 superfamily. Low affinity of IL‐10R&bgr; for cytokines has impeded efforts aimed at crystallizing cytokine‐receptor complexes. We used yeast surface display to engineer a higher‐affinity IFN‐&lgr; variant, H11, which enabled crystallization of the ternary complex. The structure revealed that IL‐10R&bgr; uses a network of tyrosine residues as hydrophobic anchor points to engage IL‐10 family cytokines that present complementary hydrophobic binding patches, explaining its role as both a cross‐reactive but cytokine‐specific receptor. H11 elicited increased anti‐proliferative and antiviral activities in vitro and in vivo. In contrast, engineered higher‐affinity type I IFNs did not increase antiviral potency over wild‐type type I IFNs. Our findings provide insight into cytokine recognition by the IL‐10R family and highlight the plasticity of type III interferon signaling and its therapeutic potential. HIGHLIGHTSThe IFN‐&lgr; ternary complex provides insight into the mechanism of IL‐10R&bgr; engagementHigh‐affinity IFN‐&lgr;3 elicits greater antiproliferative and antiviral activitiesIn vivo, an engineered IFN‐&lgr;3 has enhanced antiviral activity over the wild‐typeIn contrast to IFN‐&lgr;s, high‐affinity type I IFNs do not improve antiviral activity &NA; Using an engineered high‐affinity IFN‐&lgr;, Mendoza et al. solve the structure of the IFN‐&lgr;/IFN‐&lgr;R1/IL‐10R&bgr; ternary signaling complex. The structure reveals how IL‐10R&bgr; can act as both a cross‐reactive but cytokine‐specific receptor. Structure‐activity relationships of engineered type I and III IFNs provide insights into enhancing interferon functional potency.

A human anti-IL-2 antibody that potentiates regulatory T cells by a structure-based mechanism

Interleukin-2 (IL-2) has been shown to suppress immune pathologies by preferentially expanding regulatory T cells (Tregs). However, this therapy has been limited by off-target complications due to pathogenic cell expansion. Recent efforts have been focused on developing a more selective IL-2. It is well documented that certain anti-mouse IL-2 antibodies induce conformational changes that result in selective targeting of Tregs. We report the generation of a fully human anti-IL-2 antibody, F5111.2, that stabilizes IL-2 in a conformation that results in the preferential STAT5 phosphorylation of Tregs in vitro and selective expansion of Tregs in vivo. When complexed with human IL-2, F5111.2 induced remission of type 1 diabetes in the NOD mouse model, reduced disease severity in a model of experimental autoimmune encephalomyelitis and protected mice against xenogeneic graft-versus-host disease. These results suggest that IL-2–F5111.2 may provide an immunotherapy to treat autoimmune diseases and graft-versus-host disease.A human anti-IL-2 antibody that selectively expands regulatory T cells is developed in this study for clinical applications aiming to mitigate autoimmune and inflammatory disorders and to promote transplant tolerance.

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.