Hua-Poo Su

The PD-1/PD-L1 complex resembles the antigen-binding Fv domains of antibodies and T cell receptors.

Signaling through the programmed death 1 (PD-1) inhibitory receptor upon binding its ligand, PD-L1, suppresses immune responses against autoantigens and tumors and plays an important role in the maintenance of peripheral immune tolerance. Release from PD-1 inhibitory signaling revives “exhausted” virus-specific T cells in chronic viral infections. Here we present the crystal structure of murine PD-1 in complex with human PD-L1. PD-1 and PD-L1 interact through the conserved front and side of their Ig variable (IgV) domains, as do the IgV domains of antibodies and T cell receptors. This places the loops at the ends of the IgV domains on the same side of the PD-1/PD-L1 complex, forming a surface that is similar to the antigen-binding surface of antibodies and T cell receptors. Mapping conserved residues allowed the identification of residues that are important in forming the PD-1/PD-L1 interface. Based on the structure, we show that some reported loss-of-binding mutations involve the PD-1/PD-L1 interaction but that others compromise protein folding. The PD-1/PD-L1 interaction described here may be blocked by antibodies or by designed small-molecule drugs to lower inhibitory signaling that results in a stronger immune response. The immune receptor-like loops offer a new surface for further study and potentially the design of molecules that would affect PD-1/PD-L1 complex formation and thereby modulate the immune response.

Selective CD4+ T Cell Help for Antibody Responses to a Large Viral Pathogen: Deterministic Linkage of Specificities

Antibody responses are critical components of protective immune responses to many pathogens, but parameters determining which proteins are targeted remain unclear. Vaccination with individual MHC-II-restricted vaccinia virus (VACV, smallpox vaccine) epitopes revealed that CD4(+) T cell help to B cells was surprisingly nontransferable to other virion protein specificities. Many VACV CD4(+) T cell responses identified in an unbiased screen targeted antibody virion protein targets, consistent with deterministic linkage between specificities. We tested the deterministic linkage model by efficiently predicting new vaccinia MHC II epitopes (830% improved efficiency). Finally, we showed CD4(+) T cell help was limiting for neutralizing antibody development and protective immunity in vivo. In contrast to the standard model, these data indicate individual proteins are the unit of B cell-T cell recognition for a large virus. Therefore, MHC restriction is a key selective event for the antiviral antibody response and is probably important for vaccine development to large pathogens.

Structural basis for the inhibition of RNase H activity of HIV-1 reverse transcriptase by RNase H active site-directed inhibitors.

ABSTRACT HIV/AIDS continues to be a menace to public health. Several drugs currently on the market have successfully improved the ability to manage the viral burden in infected patients. However, new drugs are needed to combat the rapid emergence of mutated forms of the virus that are resistant to existing therapies. Currently, approved drugs target three of the four major enzyme activities encoded by the virus that are critical to the HIV life cycle. Although a number of inhibitors of HIV RNase H activity have been reported, few inhibit by directly engaging the RNase H active site. Here, we describe structures of naphthyridinone-containing inhibitors bound to the RNase H active site. This class of compounds binds to the active site via two metal ions that are coordinated by catalytic site residues, D443, E478, D498, and D549. The directionality of the naphthyridinone pharmacophore is restricted by the ordering of D549 and H539 in the RNase H domain. In addition, one of the naphthyridinone-based compounds was found to bind at a second site close to the polymerase active site and non-nucleoside/nucleotide inhibitor sites in a metal-independent manner. Further characterization, using fluorescence-based thermal denaturation and a crystal structure of the isolated RNase H domain reveals that this compound can also bind the RNase H site and retains the metal-dependent binding mode of this class of molecules. These structures provide a means for structurally guided design of novel RNase H inhibitors.

Redundancy and Plasticity of Neutralizing Antibody Responses Are Cornerstone Attributes of the Human Immune Response to the Smallpox Vaccine

ABSTRACT The smallpox vaccine is widely considered the gold standard for human vaccines, yet the key antibody targets in humans remain unclear. We endeavored to identify a stereotypic, dominant, mature virion (MV) neutralizing antibody target in humans which could be used as a diagnostic serological marker of protective humoral immunity induced by the smallpox vaccine (vaccinia virus [VACV]). We have instead found that diversity is a defining characteristic of the human antibody response to the smallpox vaccine. We show that H3 is the most immunodominant VACV neutralizing antibody target, as determined by correlation analysis of immunoglobulin G (IgG) specificities to MV neutralizing antibody titers. It was determined that purified human anti-H3 IgG is sufficient for neutralization of VACV; however, depletion or blockade of anti-H3 antibodies revealed no significant reduction in neutralization activity, showing anti-H3 IgG is not required in vaccinated humans (or mice) for neutralization of MV. Comparable results were obtained for human (and mouse) anti-L1 IgG and even for anti-H3 and anti-L1 IgG in combination. In addition to H3 and L1, human antibody responses to D8, A27, D13, and A14 exhibited statistically significant correlations with virus neutralization. Altogether, these data indicate the smallpox vaccine succeeds in generating strong neutralizing antibody responses not by eliciting a stereotypic response to a single key antigen but instead by driving development of neutralizing antibodies to multiple viral proteins, resulting in a “safety net” of highly redundant neutralizing antibody responses, the specificities of which can vary from individual to individual. We propose that this is a fundamental attribute of the smallpox vaccine.

Discovery of 1-Amino-5H-pyrido[4,3-b]indol-4-carboxamide Inhibitors of Janus Kinase 2 (JAK2) for the Treatment of Myeloproliferative Disorders

The JAK-STAT pathway mediates signaling by cytokines, which control survival, proliferation, and differentiation of a variety of cells. In recent years, a single point mutation (V617F) in the tyrosine kinase JAK2 was found to be present with a high incidence in myeloproliferative disorders (MPDs). This mutation led to hyperactivation of JAK2, cytokine-independent signaling, and subsequent activation of downstream signaling networks. The genetic, biological, and physiological evidence suggests that JAK2 inhibitors could be effective in treating MPDs. De novo design efforts of new scaffolds identified 1-amino-5H-pyrido[4,3-b]indol-4-carboxamides as a new viable lead series. Subsequent optimization of cell potency, metabolic stability, and off-target activities of the leads led to the discovery of 7-(2-aminopyrimidin-5-yl)-1-{[(1R)-1-cyclopropyl-2,2,2-trifluoroethyl]amino}-5H-pyrido[4,3-b]indole-4-carboxamide (65). Compound 65 is a potent, orally active inhibitor of JAK2 with excellent selectivity, PK profile, and in vivo efficacy in animal models.

The essential mosquito-stage P25 and P28 proteins from Plasmodium form tile-like triangular prisms.

P25 and P28 proteins are essential for Plasmodium parasites to infect mosquitoes and are leading candidates for a transmission-blocking malaria vaccine. The Plasmodium vivax P25 is a triangular prism that could tile the parasite surface. The residues forming the triangle are conserved in P25 and P28 from all Plasmodium species. A cocrystal structure shows that a transmission-blocking antibody uses only its heavy chain to bind Pvs25 at a vertex of the triangle.

The Cysteine-Rich Interdomain Region from the Highly Variable Plasmodium falciparum Erythrocyte Membrane Protein-1 Exhibits a Conserved Structure

Plasmodium falciparum malaria parasites, living in red blood cells, express proteins of the erythrocyte membrane protein-1 (PfEMP1) family on the red blood cell surface. The binding of PfEMP1 molecules to human cell surface receptors mediates the adherence of infected red blood cells to human tissues. The sequences of the 60 PfEMP1 genes in each parasite genome vary greatly from parasite to parasite, yet the variant PfEMP1 proteins maintain receptor binding. Almost all parasites isolated directly from patients bind the human CD36 receptor. Of the several kinds of highly polymorphic cysteine-rich interdomain region (CIDR) domains classified by sequence, only the CIDR1α domains bind CD36. Here we describe the CD36-binding portion of a CIDR1α domain, MC179, as a bundle of three α-helices that are connected by a loop and three additional helices. The MC179 structure, containing seven conserved cysteines and 10 conserved hydrophobic residues, predicts similar structures for the hundreds of CIDR sequences from the many genome sequences now known. Comparison of MC179 with the CIDR domains in the genome of the P. falciparum 3D7 strain provides insights into CIDR domain structure. The CIDR1α three-helix bundle exhibits less than 20% sequence identity with the three-helix bundles of Duffy-binding like (DBL) domains, but the two kinds of bundles are almost identical. Despite the enormous diversity of PfEMP1 sequences, the CIDR1α and DBL protein structures, taken together, predict that a PfEMP1 molecule is a polymer of three-helix bundles elaborated by a variety of connecting helices and loops. From the structures also comes the insight that DBL1α domains are approximately 100 residues larger and that CIDR1α domains are approximately 100 residues smaller than sequence alignments predict. This new understanding of PfEMP1 structure will allow the use of better-defined PfEMP1 domains for functional studies, for the design of candidate vaccines, and for understanding the molecular basis of cytoadherence.

The Structure of the Poxvirus A33 Protein Reveals a Dimer of Unique C-Type Lectin-Like Domains

ABSTRACT The current vaccine against smallpox is an infectious form of vaccinia virus that has significant side effects. Alternative vaccine approaches using recombinant viral proteins are being developed. A target of subunit vaccine strategies is the poxvirus protein A33, a conserved protein in the Chordopoxvirinae subfamily of Poxviridae that is expressed on the outer viral envelope. Here we have determined the structure of the A33 ectodomain of vaccinia virus. The structure revealed C-type lectin-like domains (CTLDs) that occur as dimers in A33 crystals with five different crystal lattices. Comparison of the A33 dimer models shows that the A33 monomers have a degree of flexibility in position within the dimer. Structural comparisons show that the A33 monomer is a close match to the Link module class of CTLDs but that the A33 dimer is most similar to the natural killer (NK)-cell receptor class of CTLDs. Structural data on Link modules and NK-cell receptor-ligand complexes suggest a surface of A33 that could interact with viral or host ligands. The dimer interface is well conserved in all known A33 sequences, indicating an important role for the A33 dimer. The structure indicates how previously described A33 mutations disrupt protein folding and locates the positions of N-linked glycosylations and the epitope of a protective antibody.

The Structure of G4, the Poxvirus Disulfide Oxidoreductase Essential for Virus Maturation and Infectivity

ABSTRACT The possibility of the release of smallpox virus into a predominantly nonimmunized population highlights the importance of understanding poxvirus biology. Poxviruses encode a conserved pathway that is required to oxidize disulfide bonds in nascent viral proteins that fold in the reducing environment of the eukaryotic host cytoplasm. We present the structure of the last enzyme of the vaccinia virus pathway, G4, which is almost identical in smallpox virus. G4 catalyzes the formation of disulfide bonds in proteins that are critical for virus maturation and host cell infection. G4 contains a thioredoxin fold and a Cys-X-X-Cys active site. In solution, G4 monomers and dimers are observed. In the crystal, G4 is found as a dimer that buries 4,500 Å2 in the interface and occludes the active site, which could protect the reactive disulfide from reduction in the cytoplasm. The structure serves as a model for drug design targeting viral disulfide bond formation.

The Vaccinia Virus H3 Envelope Protein, a Major Target of Neutralizing Antibodies, Exhibits a Glycosyltransferase Fold and Binds UDP-Glucose.

ABSTRACT The highly conserved H3 poxvirus protein is a major target of the human antibody response against poxviruses and is likely a key contributor to protection against infection. Here, we present the crystal structure of H3 from vaccinia virus at a 1.9-Å resolution. H3 looks like a glycosyltransferase, a family of enzymes that transfer carbohydrate molecules to a variety of acceptor substrates. Like glycosyltransferases, H3 binds UDP-glucose, as shown by saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy, and this binding requires Mg2+. Mutation of the glycosyltransferase-like metal ion binding motif in H3 greatly diminished its binding to UDP-glucose. We found by flow cytometry that H3 binds to the surface of human cells but does not bind well to cells that are deficient in surface glycosaminoglycans. STD NMR experiments using a heparin sulfate decasaccharide confirmed that H3 binds heparin sulfate. We propose that a surface of H3 with an excess positive charge may be the binding site for heparin. Heparin binding and glycosyltransferase activity may be involved in the function of H3 in the poxvirus life cycle. IMPORTANCE Poxviruses are under intense research because of bioterrorism concerns, zoonotic infections, and the side effects of existing smallpox vaccines. The smallpox vaccine using vaccinia virus has been highly successful, but it is still unclear why the vaccine is so effective. Studying the antigens that the immune system recognizes may allow a better understanding of how the vaccine elicits immunity and how improved vaccines can be developed. Poxvirus protein H3 is a major target of the immune system. The H3 crystal structure shows that it has a glycosyltransferase protein fold. We demonstrate that H3 binds the sugar nucleotide UDP-glucose, as do glycosyltransferases. Our experiments also reveal that H3 binds cell surface molecules that are involved in the attachment of poxviruses to cells. These structural and functional studies of H3 will help in designing better vaccines and therapeutics.