prM-reactive antibodies reveal a role for partially mature virions in dengue virus pathogenesis.

Cleavage of the flavivirus premembrane (prM) structural protein during maturation can be inefficient. The contribution of partially mature flavivirus virions that retain uncleaved prM to pathogenesis during primary infection is unknown. To investigate this question, we characterized the functional properties of newly-generated dengue virus (DENV) prM-reactive monoclonal antibodies (mAbs) in vitro and using a mouse model of DENV disease. Anti-prM mAbs neutralized DENV infection in a virion maturation state-dependent manner. Alanine scanning mutagenesis and cryoelectron microscopy of anti-prM mAbs in complex with immature DENV defined two modes of attachment to a single antigenic site. In vivo, passive transfer of intact anti-prM mAbs resulted in an antibody-dependent enhancement of disease. However, protection against DENV-induced lethality was observed when the transferred mAbs were genetically modified to inhibit their ability to interact with Fcγ receptors. These data establish that in addition to mature forms of the virus, partially mature infectious prM+ virions can also contribute to pathogenesis during primary DENV infections.

Structural perspectives on HCV humoral immune evasion mechanisms.

The molecular mechanisms of hepatitis C virus (HCV) persistence and pathogenesis are poorly understood. The design of an effective HCV vaccine is challenging despite a robust humoral immune response against closely related strains of HCV. This is primarily because of the huge genetic diversity of HCV and the molecular evolution of various virus escape mechanisms. These mechanisms are steered by the presence of a high mutational rate in HCV, structural plasticity of the immunodominant regions on the virion surface of diverse HCV genotypes, and constant amino acid substitutions on key structural components of HCV envelope glycoproteins. Here, we review the molecular basis of neutralizing antibody (nAb)-mediated immune response against diverse HCV variants, HCV-steered humoral immune evasion strategies and explore the essential structural elements to consider for designing a universal HCV vaccine. Structural perspectives on key escape pathways mediated by a point mutation within the epitope, allosteric modulation of the epitope by distant mutations and glycan shift on envelope glycoproteins will be highlighted (abstract graphic).

Structural basis of a potent human monoclonal antibody against Zika virus targeting a quaternary epitope.

Zika virus (ZIKV) is a major human pathogen and member of the Flavivirus genus in the Flaviviridae family. In contrast to most other insect-transmitted flaviviruses, ZIKV also can be transmitted sexually and from mother to fetus in humans. During recent outbreaks, ZIKV infections have been linked to microcephaly, congenital disease, and Guillain-Barré syndrome. Neutralizing antibodies have potential as therapeutic agents. We report here a 4-Å-resolution cryo-electron microscopy structure of the ZIKV virion in complex with Fab fragments of the potently neutralizing human monoclonal antibody ZIKV-195. The footprint of the ZIKV-195 Fab fragment expands across two adjacent envelope (E) protein protomers. ZIKV neutralization by this antibody is presumably accomplished by cross-linking the E proteins, which likely prevents formation of E protein trimers required for fusion of the viral and cellular membranes. A single dose of ZIKV-195 administered 5 days after virus inoculation showed marked protection against lethality in a stringent mouse model of infection.

An exclusive α/ß code directs allostery in TetR-peptide complexes.

The allosteric mechanism of one of the best characterized bacterial transcription regulators, tetracycline repressor (TetR), has recently been questioned. Tetracycline binding induces cooperative folding of TetR, as suggested by recent unfolding studies, rather than switching between two defined conformational states, namely a DNA-binding-competent conformation and a non-DNA-binding conformation. Upon ligand binding, a host of near-native multiconformational structures collapse into a single, highly stabilized protein conformation that is no longer able to bind DNA. Here, structure-function studies performed with four synthetic peptides that bind to TetR and mimic the function of low-molecular-weight effectors, such as tetracyclines, provide new means to discriminate between different allosteric models. Whereas two inducing peptides bind in an extended ß-like conformation, two anti-inducing peptides form an α-helix in the effector binding site of TetR. This exclusive bimodal interaction mode coincides with two distinct overall conformations of TetR, namely one that is identical with induced TetR and one that mirrors the DNA-bound state of TetR. Urea-induced unfolding studies show no increase in thermodynamic stability for any of the peptide complexes, although fluorescence measurements demonstrate peptide binding to TetR. This strongly suggests that, at least for these peptide effectors, a classical two-state allosteric model best describes TetR function.

Endothelium-protective sphingosine-1-phosphate provided by HDL-associated apolipoprotein M.

Protection of the endothelium is provided by circulating sphingosine-1-phosphate (S1P), which maintains vascular integrity. We show that HDL-associated S1P is bound specifically to both human and murine apolipoprotein M (apoM). Thus, isolated human ApoM(+) HDL contained S1P, whereas ApoM(-) HDL did not. Moreover, HDL in Apom(-/-) mice contains no S1P, whereas HDL in transgenic mice overexpressing human apoM has an increased S1P content. The 1.7-Å structure of the S1P-human apoM complex reveals that S1P interacts specifically with an amphiphilic pocket in the lipocalin fold of apoM. Human ApoM(+) HDL induced S1P(1) receptor internalization, downstream MAPK and Akt activation, endothelial cell migration, and formation of endothelial adherens junctions, whereas apoM(-) HDL did not. Importantly, lack of S1P in the HDL fraction of Apom(-/-) mice decreased basal endothelial barrier function in lung tissue. Our results demonstrate that apoM, by delivering S1P to the S1P(1) receptor on endothelial cells, is a vasculoprotective constituent of HDL.

Crystal structure and functional analysis of the protein disulfide isomerase-related protein ERp29.

The protein disulfide isomerase-related protein ERp29 is a putative chaperone involved in processing and secretion of secretory proteins. Until now, however, both the structure and the exact nature of interacting substrates remained unclear. We provide for the first time a crystal structure of human ERp29, refined to 2.9 A, and show that the protein has considerable structural homology to its Drosophila homolog Wind. We show that ERp29 binds directly not only to thyroglobulin and thyroglobulin-derived peptides in vitro but also to the Wind client protein Pipe and Pipe-derived peptides, although it fails to process Pipe in vivo. A monomeric mutant of ERp29 and a D domain mutant in which the second peptide binding site is inactivated also bind protein substrates, indicating that the monomeric thioredoxin domain is sufficient for client protein binding. Indeed, the b domains of ERp29 or Wind, expressed alone, are sufficient for binding proteins and peptides. Interacting peptides have in common two or more aromatic residues, with stronger binding for sequences with overall basic character. Thus, the data allow a view of the two putative peptide binding sites of ERp29 and indicate that the apparent, different processing activity of the human and Drosophila proteins in vivo does not stem from differences in peptide binding properties.

Kettapeptin: isolation, structure elucidation and activity of a new hexadepsipeptide antibiotic from a terrestrial Streptomyces sp.

The ethyl acetate extract of the Streptomyces sp. isolate GW99/1572 exhibited significant biological activity against Gram-positive bacteria and delivered kettapeptin (1), a new hexadepsipeptide antibiotic of the azinothricin type. The structure was elucidated by various 1D and 2D NMR techniques, mass spectrometry and by comparison of the NMR data with those of closely related antibiotics. The absolute configuration of the compound was derived by crystal structure analysis and by comparison with the optical rotation data of related compounds.

Mapping of a substrate binding site in the protein disulfide isomerase-related chaperone wind based on protein function and crystal structure.

The protein disulfide isomerase (PDI)-related protein Wind is essential in Drosophila melanogaster, and is required for correct targeting of Pipe, an essential Golgi transmembrane 2-O-sulfotransferase. Apart from a thioredoxin fold domain present in all PDI proteins, Wind also has a unique C-terminal D-domain found only in PDI-D proteins. Here, we show that Pipe processing requires dimeric Wind, which interacts directly with the soluble domain of Pipe in vitro, and we map an essential substrate binding site in Wind to the vicinity of an exposed cluster of tyrosines within the thioredoxin fold domain. In vitro, binding occurs to multiple sites within the Pipe polypeptide and shows specificity for two consecutive aromatic residues. A second site in Wind, formed by a cluster of residues within the D-domain, is likewise required for substrate processing. This domain, expressed separately, impairs Pipe processing by the full-length Wind protein, indicating competitive binding to substrate. Our data represent the most accurate map of a peptide binding site in a PDI-related protein available to date and directly show peptide specificity for a naturally occurring substrate.