High-resolution crystal structure of LpqH, an immunomodulatory surface lipoprotein of Mycobacterium tuberculosis reveals a distinct fold and a conserved cleft on its surface.

Tuberculosis, caused by Mycobacterium tuberculosis, is predominantly a disease of the lungs acquired by inhaling mycobacteria from infected individuals via airborne droplets. In order to facilitate their entry into the alveolar macrophages, mycobacteria have a collection of pathogen-associated molecular patterns (PAMPs) on their surface that are known to detect certain pattern recognition receptors present on the surface of host cells. A major group of these PAMPs includes mycobacterial lipoproteins, of which, the 19 kDa surface antigen LpqH, has been reported to play a critical role in both host-pathogen interactions as well as pleiotropic immune regulation. Despite its crucial involvement in tuberculosis, the detailed structure-function relationship of this protein remains to be explored. Here, we report the high-resolution crystal structure of the non-acylated LpqH (LpqH48-159) at a resolution of 1.26 Å, which adopts a unique fold. Flow cytometry-based experiments show that the protein can bind and induce apoptosis in PMA-activated human monocytic cell line THP-1, indicative of the preservation of functionality of the protein. Furthermore, analysis of conservation of LpqH sequences from Mycobacterium species reveals a patch of conserved residues on the surface which may play a role in its binding partner recognition and hence in host-pathogen interaction.

HVEM structures and mutants reveal distinct functions of binding to LIGHT and BTLA/CD160.

HVEM is a TNF (tumor necrosis factor) receptor contributing to a broad range of immune functions involving diverse cell types. It interacts with a TNF ligand, LIGHT, and immunoglobulin (Ig) superfamily members BTLA and CD160. Assessing the functional impact of HVEM binding to specific ligands in different settings has been complicated by the multiple interactions of HVEM and HVEM binding partners. To dissect the molecular basis for multiple functions, we determined crystal structures that reveal the distinct HVEM surfaces that engage LIGHT or BTLA/CD160, including the human HVEM-LIGHT-CD160 ternary complex, with HVEM interacting simultaneously with both binding partners. Based on these structures, we generated mouse HVEM mutants that selectively recognized either the TNF or Ig ligands in vitro. Knockin mice expressing these muteins maintain expression of all the proteins in the HVEM network, yet they demonstrate selective functions for LIGHT in the clearance of bacteria in the intestine and for the Ig ligands in the amelioration of liver inflammation.

Cryptic association of B7-2 molecules and its implication for clustering.

T-cell co-stimulation through CD28/CTLA4:B7-1/B7-2 axis is one of the extensively studied pathways that resulted in the discovery of several FDA-approved drugs for autoimmunity and cancer. However, many aspects of the signaling mechanism remain elusive, including oligomeric association and clustering of B7-2 on the cell surface. Here, we describe the structure of the IgV domain of B7-2 and its cryptic association into 1D arrays that appear to represent the pre-signaling state of B7-2 on the cell membrane. Super-resolution microscopy experiments on heterologous cells expressing B7-2 and B7-1 suggest, B7-2 form relatively elongated and larger clusters compared to B7-1. The sequence and structural comparison of other B7 family members, B7-1:CTLA4 and B7-2:CTLA-4 complex structures, support our view that the observed B7-2 1D zipper array is physiologically important. This observed 1D zipper-like array also provides an explanation for its clustering, and upright orientation on the cell surface, and avoidance of spurious signaling.

A structural perspective on the design of decoy immune modulators.

Therapeutic mAbs have dominated the class of immunotherapeutics in general and immune checkpoint inhibitors in particular. The high specificity of mAbs to the target molecule as well as their extended half-life and (or) the effector functions raised by the Fc part are some of the important aspects that contribute to the success of this class of therapeutics. Equally potential candidates are decoys and their fusions that can address some of the inherent limitations of mAbs, like immunogenicity, resistance development, low bio-availability and so on, besides maintaining the advantages of mAbs. The decoys are molecules that trap the ligands and prevent them from interacting with the signaling receptors. Although a few FDA-approved decoy immune modulators are very successful, the potential of this class of drugs is yet to be fully realized. Here, we review various strategies employed in fusion protein therapeutics with a focus on the design of decoy immunomodulators from the structural perspective and discuss how the information on protein structure and function can strategically guide the development of next-generation immune modulators.

The CC' loop of IgV domains of the immune checkpoint receptors, plays a key role in receptor:ligand affinity modulation.

Antibodies targeting negative regulators of immune checkpoints have shown unprecedented and durable response against variety of malignancies. While the concept of blocking the negative regulators of the immune checkpoints using mAbs appears to be an outstanding approach, their limited effect and several drawbacks, calls for the rational design of next generation of therapeutics. Soluble isoforms of the negative regulators of immune checkpoint pathways are expressed naturally and regulate immune responses. This suggests, affinity-modified versions of these self-molecules could be effective lead molecules for immunotherapy. To obtain better insights on the hotspot regions for modification, we have analysed structures of 18 immune receptor:ligand complexes containing the IgV domain. Interestingly, this analysis reveals that the CC' loop of IgV domain, a loop which is distinct from CDRs of antibodies, plays a pivotal role in affinity modulation, which was previously not highlighted. It is noteworthy that a ~5-residue long CC' loop in a ~120 residue protein makes significant number of hydrophobic and polar interactions with its cognate ligand. The post-interaction movement of CC' loop to accommodate the incoming ligands, seems to provide additional affinity to the interactions. In silico replacement of the CC' loop of TIGIT with that of Nectin-2 and PVR followed by protein docking trials suggests a key role of the CC' loop in affinity modulation in the TIGIT/Nectin pathway. The CC' loop appears to be a hotspot for the affinity modification without affecting the specificity to their cognate receptors.

Structural Basis of CD160:HVEM Recognition.

CD160 is a signaling molecule that interacts with herpes virus entry mediator (HVEM) and contributes to a wide range of immune responses, including T cell inhibition, natural killer cell activation, and mucosal immunity. GPI-anchored and transmembrane isoforms of CD160 share the same ectodomain responsible for HVEM engagement, which leads to bidirectional signaling. Despite the importance of the CD160:HVEM signaling axis and its therapeutic relevance, the structural and mechanistic basis underlying CD160-HVEM engagement has not been described. We report the crystal structures of the human CD160 extracellular domain and its complex with human HVEM. CD160 adopts a unique variation of the immunoglobulin fold and exists as a monomer in solution. The CD160:HVEM assembly exhibits a 1:1 stoichiometry and a binding interface similar to that observed in the BTLA:HVEM complex. Our work reveals the chemical and physical determinants underlying CD160:HVEM recognition and initiation of associated signaling processes.

Comparison of Alicyclobacillus acidocaldarius o-Succinylbenzoate Synthase to Its Promiscuous N-Succinylamino Acid Racemase/ o-Succinylbenzoate Synthase Relatives.

Studying the evolution of catalytically promiscuous enzymes like those from the N-succinylamino acid racemase/ o-succinylbenzoate synthase (NSAR/OSBS) subfamily can reveal mechanisms by which new functions evolve. Some enzymes in this subfamily have only OSBS activity, while others catalyze OSBS and NSAR reactions. We characterized several NSAR/OSBS subfamily enzymes as a step toward determining the structural basis for evolving NSAR activity. Three enzymes were promiscuous, like most other characterized NSAR/OSBS subfamily enzymes. However, Alicyclobacillus acidocaldarius OSBS (AaOSBS) efficiently catalyzes OSBS activity but lacks detectable NSAR activity. Competitive inhibition and molecular modeling show that AaOSBS binds N-succinylphenylglycine with moderate affinity in a site that overlaps its normal substrate. On the basis of possible steric conflicts identified by molecular modeling and sequence conservation within the NSAR/OSBS subfamily, we identified one mutation, Y299I, that increased NSAR activity from undetectable to 1.2 × 102 M-1 s-1 without affecting OSBS activity. This mutation does not appear to affect binding affinity but instead affects kcat, by reorienting the substrate or modifying conformational changes to allow both catalytic lysines to access the proton that is moved during the reaction. This is the first site known to affect reaction specificity in the NSAR/OSBS subfamily. However, this gain of activity was obliterated by a second mutation, M18F. Epistatic interference by M18F was unexpected because a phenylalanine at this position is important in another NSAR/OSBS enzyme. Together, modest NSAR activity of Y299I AaOSBS and epistasis between sites 18 and 299 indicate that additional sites influenced the evolution of NSAR reaction specificity in the NSAR/OSBS subfamily.

Crystal structures of APRT from Francisella tularensis - an N-H···N hydrogen bond imparts adenine specificity in adenine phosporibosyltransferases.

Francisella tularensisis, the causative agent of tularemia has been classified as a category A bioterrorism agent. Here, we present the crystal structure of apo and adenine bound form of the adenine phosphoribosyltransferase (APRT) from Francisella tularensis. APRT is an enzyme involved in the salvage of adenine (a 6-aminopurine), converting it to AMP. The purine salvage pathway relies on two essential and distinct enzymes to convert 6-aminopurine and 6-oxopurines into corresponding nucleotides. The mechanism by which these enzymes differentiate different purines is not clearly understood. Analysis of the structures of apo and adenine-bound APRT from F. tularensis, together with all other available structures of APRTs, suggests that (a) the base-binding loop is stabilized by a cluster of aromatic and conformation-restricting proline residues, and (b) an N-H···N hydrogen bond between the base-binding loop and the N1 atom of adenine is the key interaction that differentiates adenine from 6-oxopurines. These observations were corroborated by bioinformatics analysis of ~ 4000 sequences of APRTs (with 80% identity cutoff), which confirmed that the residues conferring rigidity to the base-binding loop are highly conserved. Furthermore, an F23A mutation on the base-binding loop severely affects the efficiency of the enzyme. We extended our analysis to the structure and sequences of APRTs from the Trypanosomatidae family with a destabilizing insertion on the base-binding loop and propose the mechanism by which these evolutionarily divergent enzymes achieve base specificity. Our results suggest that the base-binding loop not only confers appropriate affinity but also provides defined specificity for adenine. ENZYME: EC 2.4.2.7 DATABASE: Structural data are available in Protein Data Bank (PDB) under the accession numbers 5YW2 and 5YW5.

Structures of the L27 Domain of Disc Large Homologue 1 Protein Illustrate a Self-Assembly Module.

Disc large 1 (Dlg1) proteins, members of the MAGUK protein family, are linked to cell polarity via their participation in multiprotein assemblies. At their N-termini, Dlg1 proteins contain a L27 domain. Typically, the L27 domains participate in the formation of obligate hetero-oligomers with the L27 domains from their cognate partners. Among the MAGUKs, Dlg1 proteins exist as homo-oligomers, and the oligomerization is solely dependent on the L27 domain. Here we provide biochemical and structural evidence of homodimerization via the L27 domain of Dlg1 from Drosophila melanogaster. The structure reveals that the core of the dimer is formed by a distinctive six-helix assembly, involving all three conserved helices from each subunit (monomer). The homodimer interface is extended by the C-terminal tail of the L27 domain of Dlg1, which forms a two-stranded antiparallel ß-sheet. The structure reconciles and provides a structural context for a large body of available mutational data. From our analyses, we conclude that the observed L27 homodimerization is most likely a feature unique to the Dlg1 orthologs within the MAGUK family.

Can the propensity of protein crystallization be increased by using systematic screening with metals?

Protein crystallization is one of the major bottlenecks in protein structure elucidation with new strategies being constantly developed to improve the chances of crystallization. Generally, well-ordered epitopes possessing complementary surface and capable of producing stable inter-protein interactions generate a regular three-dimensional arrangement of protein molecules which eventually results in a crystal lattice. Metals, when used for crystallization, with their various coordination numbers and geometries, can generate such epitopes mediating protein oligomerization and/or establish crystal contacts. Some examples of metal-mediated oligomerization and crystallization together with our experience on metal-mediated crystallization of a putative rRNA methyltransferase from Sinorhizobium meliloti are presented. Analysis of crystal structures from protein data bank (PDB) using a non-redundant data set with a 90% identity cutoff, reveals that around 67% of proteins contain at least one metal ion, with ∼14% containing combination of metal ions. Interestingly, metal containing conditions in most commercially available and popular crystallization kits generally contain only a single metal ion, with combinations of metals only in a very few conditions. Based on the results presented in this review, it appears that the crystallization screens need expansion with systematic screening of metal ions that could be crucial for stabilizing the protein structure or for establishing crystal contact and thereby aiding protein crystallization.

Structural basis for cancer immunotherapy by the first-in-class checkpoint inhibitor ipilimumab.

Rational modulation of the immune response with biologics represents one of the most promising and active areas for the realization of new therapeutic strategies. In particular, the use of function blocking monoclonal antibodies targeting checkpoint inhibitors such as CTLA-4 and PD-1 have proven to be highly effective for the systemic activation of the human immune system to treat a wide range of cancers. Ipilimumab is a fully human antibody targeting CTLA-4 that received FDA approval for the treatment of metastatic melanoma in 2011. Ipilimumab is the first-in-class immunotherapeutic for blockade of CTLA-4 and significantly benefits overall survival of patients with metastatic melanoma. Understanding the chemical and physical determinants recognized by these mAbs provides direct insight into the mechanisms of pathway blockade, the organization of the antigen-antibody complexes at the cell surface, and opportunities to further engineer affinity and selectivity. Here, we report the 3.0 Å resolution X-ray crystal structure of the complex formed by ipilimumab with its human CTLA-4 target. This structure reveals that ipilimumab contacts the front ß-sheet of CTLA-4 and intersects with the CTLA-4:Β7 recognition surface, indicating that direct steric overlap between ipilimumab and the B7 ligands is a major mechanistic contributor to ipilimumab function. The crystallographically observed binding interface was confirmed by a comprehensive cell-based binding assay against a library of CTLA-4 mutants and by direct biochemical approaches. This structure also highlights determinants responsible for the selectivity exhibited by ipilimumab toward CTLA-4 relative to the homologous and functionally related CD28.

The hidden treasure in your data: phasing with unexpected weak anomalous scatterers from routine data sets.

Single-wavelength anomalous dispersion (SAD) utilizing anomalous signal from native S atoms, or other atoms with Z ≤ 20, generally requires highly redundant data collected using relatively long-wavelength X-rays. Here, the results from two proteins are presented where the anomalous signal from serendipitously acquired surface-bound Ca atoms with an anomalous data multiplicity of around 10 was utilized to drive de novo structure determination. In both cases, the Ca atoms were acquired from the crystallization solution, and the data-collection strategy was not optimized to exploit the anomalous signal from these scatterers. The X-ray data were collected at 0.98 Šwavelength in one case and at 1.74 Šin the other (the wavelength was optimized for sulfur, but the anomalous signal from calcium was exploited for structure solution). Similarly, using a test case, it is shown that data collected at ∼1.0 Šwavelength, where the f'' value for sulfur is 0.28 e, are sufficient for structure determination using intrinsic S atoms from a strongly diffracting crystal. Interestingly, it was also observed that SHELXD was capable of generating a substructure solution from high-exposure data with a completeness of 70% for low-resolution reflections extending to 3.5 Šresolution with relatively low anomalous multiplicity. Considering the fact that many crystallization conditions contain anomalous scatterers such as Cl, Ca, Mn etc., checking for the presence of fortuitous anomalous signal in data from well diffracting crystals could prove useful in either determining the structure de novo or in accurately assigning surface-bound atoms.

Structure-guided development of a high-affinity human Programmed Cell Death-1: Implications for tumor immunotherapy.

Programmed Cell Death-1 (PD-1) is an inhibitory immune receptor, which plays critical roles in T cell co-inhibition and exhaustion upon binding to its ligands PD-L1 and PD-L2. We report the crystal structure of the human PD-1 ectodomain and the mapping of the PD-1 binding interface. Mutagenesis studies confirmed the crystallographic interface, and resulted in mutant PD-1 receptors with altered affinity and ligand-specificity. In particular, a high-affinity mutant PD-1 (HA PD-1) exhibited 45 and 30-fold increase in binding to PD-L1 and PD-L2, respectively, due to slower dissociation rates. This mutant (A132L) was used to engineer a soluble chimeric Ig fusion protein for cell-based and in vivo studies. HA PD-1 Ig showed enhanced binding to human dendritic cells, and increased T cell proliferation and cytokine production in a mixed lymphocyte reaction (MLR) assay. Moreover, in an experimental model of murine Lewis lung carcinoma, HA PD-1 Ig treatment synergized with radiation therapy to decrease local and metastatic tumor burden, as well as in the establishment of immunological memory responses. Our studies highlight the value of structural considerations in guiding the design of a high-affinity chimeric PD-1 Ig fusion protein with robust immune modulatory properties, and underscore the power of combination therapies to selectively manipulate the PD-1 pathway for tumor immunotherapy.

Structural, mutational and biophysical studies reveal a canonical mode of molecular recognition between immune receptor TIGIT and nectin-2.

In addition to antigen-specific stimulation of T cell receptor (TCR) by a peptide-MHC complex, the functional outcome of TCR engagement is regulated by antigen-independent costimulatory signals. Costimulatory signals are provided by an array of interactions involving activating and inhibitory receptors expressed on T cells and their cognate ligands on antigen presenting cells. T cell immunoglobulin and ITIM domain (TIGIT), a recently identified immune receptor expressed on T and NK cells, upon interaction with either of its two ligands, nectin-2 or poliovirus receptor (PVR), inhibits activation of T and NK cells. Here we report the crystal structure of the human TIGIT ectodomain, which exhibits the classic two-layer ß-sandwich topology observed in other immunoglobulin super family (IgSF) members. Biophysical studies indicate that TIGIT is monomeric in solution but can form a dimer at high concentrations, consistent with the observation of a canonical immunoglobulin-like dimer interface in the crystalline state. Based on existing structural data, we present a model of the TIGIT:nectin-2 complex and utilized complementary biochemical studies to map the nectin-binding interface on TIGIT. Our data provide important structural and biochemical determinants responsible for the recognition of nectin-2 by TIGIT. Defining the TIGIT:nectin-2 binding interface provides the basis for rational manipulation of this molecular interaction for the development of immunotherapeutic reagents in autoimmunity and cancer.

A Small Protein Associated with Fungal Energy Metabolism Affects the Virulence of Cryptococcus neoformans in Mammals.

The pathogenic yeast Cryptococcus neoformans causes cryptococcosis, a life-threatening fungal disease. C. neoformans has multiple virulence mechanisms that are non-host specific, induce damage and interfere with immune clearance. Microarray analysis of C. neoformans strains serially passaged in mice associated a small gene (CNAG_02591) with virulence. This gene, hereafter identified as HVA1 (hypervirulence-associated protein 1), encodes a protein that has homologs of unknown function in plant and animal fungi, consistent with a conserved mechanism. Expression of HVA1 was negatively correlated with virulence and was reduced in vitro and in vivo in both mouse- and Galleria-passaged strains of C. neoformans. Phenotypic analysis in hva1Δ and hva1Δ+HVA1 strains revealed no significant differences in established virulence factors. Mice infected intravenously with the hva1Δ strain had higher fungal burden in the spleen and brain, but lower fungal burden in the lungs, and died faster than mice infected with H99W or the hva1Δ+HVA1 strain. Metabolomics analysis demonstrated a general increase in all amino acids measured in the disrupted strain and a block in the TCA cycle at isocitrate dehydrogenase, possibly due to alterations in the nicotinamide cofactor pool. Macrophage fungal burden experiments recapitulated the mouse hypervirulent phenotype of the hva1Δ strain only in the presence of exogenous NADPH. The crystal structure of the Hva1 protein was solved, and a comparison of structurally similar proteins correlated with the metabolomics data and potential interactions with NADPH. We report a new gene that modulates virulence through a mechanism associated with changes in fungal metabolism.

Structure and cancer immunotherapy of the B7 family member B7x.

B7x (B7-H4 or B7S1) is a member of the B7 family that can inhibit T cell function. B7x protein is absent in most normal human tissues and immune cells, but it is overexpressed in human cancers and often correlates with negative clinical outcome. The expression pattern and function of B7x suggest that it may be a potent immunosuppressive pathway in human cancers. Here, we determined the crystal structure of the human B7x immunoglobulin variable (IgV) domain at 1.59 Å resolution and mapped the epitopes recognized by monoclonal antibodies. We developed an in vivo system to screen therapeutic monoclonal antibodies against B7x and found that the clone 1H3 significantly inhibited growth of B7x-expressing tumors in vivo via multiple mechanisms. Furthermore, the surviving mice given 1H3 treatment were resistant to tumor rechallenge. Our data suggest that targeting B7x on tumors is a promising cancer immunotherapy and humanized 1H3 may be efficacious for immunotherapy of human cancers.

Targeting Mycobacterium tuberculosis nucleoid-associated protein HU with structure-based inhibitors.

The nucleoid-associated protein HU plays an important role in maintenance of chromosomal architecture and in global regulation of DNA transactions in bacteria. Although HU is essential for growth in Mycobacterium tuberculosis (Mtb), there have been no reported attempts to perturb HU function with small molecules. Here we report the crystal structure of the N-terminal domain of HU from Mtb. We identify a core region within the HU-DNA interface that can be targeted using stilbene derivatives. These small molecules specifically inhibit HU-DNA binding, disrupt nucleoid architecture and reduce Mtb growth. The stilbene inhibitors induce gene expression changes in Mtb that resemble those induced by HU deficiency. Our results indicate that HU is a potential target for the development of therapies against tuberculosis.

Structure of the S100A4/myosin-IIA complex.

BACKGROUND: S100A4, a member of the S100 family of Ca2+-binding proteins, modulates the motility of both non-transformed and cancer cells by regulating the localization and stability of cellular protrusions. Biochemical studies have demonstrated that S100A4 binds to the C-terminal end of the myosin-IIA heavy chain coiled-coil and disassembles myosin-IIA filaments; however, the mechanism by which S100A4 mediates myosin-IIA depolymerization is not well understood. RESULTS: We determined the X-ray crystal structure of the S100A4Δ8C/MIIA(1908-1923) peptide complex, which showed an asymmetric binding mode for the myosin-IIA peptide across the S100A4 dimer interface. This asymmetric binding mode was confirmed in NMR studies using a spin-labeled myosin-IIA peptide. In addition, our NMR data indicate that S100A4Δ8C binds the MIIA(1908-1923) peptide in an orientation very similar to that observed for wild-type S100A4. Studies of complex formation using a longer, dimeric myosin-IIA construct demonstrated that S100A4 binding dissociates the two myosin-IIA polypeptide chains to form a complex composed of one S100A4 dimer and a single myosin-IIA polypeptide chain. This interaction is mediated, in part, by the instability of the region of the myosin-IIA coiled-coil encompassing the S100A4 binding site. CONCLUSION: The structure of the S100A4/MIIA(1908-1923) peptide complex has revealed the overall architecture of this assembly and the detailed atomic interactions that mediate S100A4 binding to the myosin-IIA heavy chain. These structural studies support the idea that residues 1908-1923 of the myosin-IIA chain heavy represent a core sequence for the S100A4/myosin-IIA complex. In addition, biophysical studies suggest that structural fluctuations within the myosin-IIA coiled-coil may facilitate S100A4 docking onto a single myosin-IIA polypeptide chain.

Functional classification of immune regulatory proteins.

The members of the immunoglobulin superfamily (IgSF) control innate and adaptive immunity and are prime targets for the treatment of autoimmune diseases, infectious diseases, and malignancies. We describe a computational method, termed the Brotherhood algorithm, which utilizes intermediate sequence information to classify proteins into functionally related families. This approach identifies functional relationships within the IgSF and predicts additional receptor-ligand interactions. As a specific example, we examine the nectin/nectin-like family of cell adhesion and signaling proteins and propose receptor-ligand interactions within this family. Guided by the Brotherhood approach, we present the high-resolution structural characterization of a homophilic interaction involving the class-I MHC-restricted T-cell-associated molecule, which we now classify as a nectin-like family member. The Brotherhood algorithm is likely to have a significant impact on structural immunology by identifying those proteins and complexes for which structural characterization will be particularly informative.

Structure and T cell inhibition properties of B7 family member, B7-H3.

T cell activity is controlled by a combination of antigen-dependent signaling through the T cell receptor and a set of auxiliary signals delivered through antigen-independent interactions, including the recognition of the B7 family of ligands. B7-H3 is a recently identified B7 family member that is strongly overexpressed in a range of cancers and correlates with poor prognosis. We report the crystal structure of murine B7-H3 at a 3 Å resolution, which provides a model for the organization of the IgV and IgC domains within the ectodomain. We demonstrate that B7-H3 inhibits T cell proliferation and show that the FG loop of the IgV domain plays a critical role in this function. B7-H3 crystallized as an unusual dimer arising from the exchange of the G strands in the IgV domains of partner molecules. This arrangement, in combination with previous reports, highlights the dynamic nature and plasticity of the immunoglobulin fold.