Significance: Biologics represent a major class of therapeutics for the treatment of malignancies, autoimmune diseases, and infectious diseases. Ipilimumab is the first-in-class immunotherapeutic for blockade of CTLA-4 and significantly benefits overall survival of patients with metastatic melanoma. The X-ray crystal structure of the ipilimumab:CTLA-4 complex defines the atomic interactions responsible for affinity and selectivity and demonstrates that the therapeutic action of ipilimumab is due to direct steric competition with the B7 ligands for binding to CTLA-4.

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.

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.

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 heavy chain 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.

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. C. neoformans is a pathogenic yeast that is the causative agent of cryptococcal meningitis. This fungal pathogen causes disease in immune compromised hosts, primarily AIDS patients in developing countries, though it also afflicts organ transplant patients and patients undergoing chemotherapy. There are >600,000 deaths per year and >1 million new infections. Unfortunately, treatment options for C. neoformans are limited and cause high kidney and liver toxicity. Thus, understanding specific steps in pathogenesis may help with design of new therapeutics. We have identified a gene (HVA1) whose absence is associated with a hypervirulent phenotype in mice. Metabolomics analysis suggests that when HVA1 is absent there is a block in the citric acid cycle, while structural analysis of the Hva1 protein suggests a potential interaction with NADPH. Fungal burden experiments in macrophages recapitulate the hypervirulent phenotype in mice only in the presence of exogenous NADPH, suggesting that modulation of NADPH affects virulence. This work adds to the growing list of genes involved in pathogen metabolism that also contribute to virulence and pathogenesis, underscoring the need to better understand the mechanisms of how pathogen metabolism affects virulence.