Theodore S. Jardetzky

Structural Basis for Paramyxovirus-Mediated Membrane Fusion

Paramyxoviruses are responsible for significant human mortality and disease worldwide, but the molecular mechanisms underlying their entry into host cells remain poorly understood. We have solved the crystal structure of a fragment of the simian parainfluenza virus 5 fusion protein (SV5 F), revealing a 96 A long coiled coil surrounded by three antiparallel helices. This structure places the fusion and transmembrane anchor of SV5 F in close proximity with a large intervening domain at the opposite end of the coiled coil. Six amino acids, potentially part of the fusion peptide, form a segment of the central coiled coil, suggesting that this structure extends into the membrane. Deletion mutants of SV5 F indicate that putative flexible tethers between the coiled coil and the viral membrane are dispensable for fusion. The lack of flexible tethers may couple a final conformational change in the F protein directly to the fusion of two bilayers.

Structure of the parainfluenza virus 5 F protein in its metastable, prefusion conformation

Enveloped viruses have evolved complex glycoprotein machinery that drives the fusion of viral and cellular membranes, permitting entry of the viral genome into the cell. For the paramyxoviruses, the fusion (F) protein catalyses this membrane merger and entry step, and it has been postulated that the F protein undergoes complex refolding during this process. Here we report the crystal structure of the parainfluenza virus 5 F protein in its prefusion conformation, stabilized by the addition of a carboxy-terminal trimerization domain. The structure of the F protein shows that there are profound conformational differences between the pre- and postfusion states, involving transformations in secondary and tertiary structure. The positions and structural transitions of key parts of the fusion machinery, including the hydrophobic fusion peptide and two helical heptad repeat regions, clarify the mechanism of membrane fusion mediated by the F protein.

Structure of the Fc fragment of human IgE bound to its high-affinityreceptor FcεRIα

The initiation of immunoglobulin-E (IgE)-mediated allergic responses requires the binding of IgE antibody to its high-affinity receptor, FcεRI. Crosslinking of FcεRI initiates an intracellular signal transduction cascade that triggers the release of mediators of the allergic response. The interaction of the crystallizable fragment (Fc) of IgE (IgE-Fc) with FcεRI is a key recognition event of this process and involves the extracellular domains of the FcεRI α-chain. To understand the structural basis for this interaction, we have solved the crystal structure of the human IgE-Fc–FcεRIα complex to 3.5-Å resolution. The crystal structure reveals that one receptor binds one dimeric IgE-Fc molecule asymmetrically through interactions at two sites, each involving one Cε3 domain of the IgE-Fc. The interaction of one receptor with the IgE-Fc blocks the binding of a second receptor, and features of this interaction are conserved in other members of the Fc receptor family. The structure suggests new approaches to inhibiting the binding of IgE to FcεRI for the treatment of allergy and asthma.

Fusing structure and function: a structural view of the herpesvirus entry machinery

Herpesviruses are double-stranded DNA, enveloped viruses that infect host cells through fusion with either the host cell plasma membrane or endocytic vesicle membranes. Efficient infection of host cells by herpesviruses is remarkably more complex than infection by other viruses, as it requires the concerted effort of multiple glycoproteins and involves multiple host receptors. The structures of the major viral glycoproteins and a number of host receptors involved in the entry of the prototypical herpesviruses, the herpes simplex viruses (HSVs) and Epstein–Barr virus (EBV), are now known. These structural studies have accelerated our understanding of HSV and EBV binding and fusion by revealing the conformational changes that occur on virus–receptor binding, depicting potential sites of functional protein and lipid interactions, and identifying the probable viral fusogen.

Membrane fusion machines of paramyxoviruses: capture of intermediates of fusion

Peptides derived from heptad repeat regions adjacent to the fusion peptide and transmembrane domains of many viral fusion proteins form stable helical bundles and inhibit fusion specifically. Paramyxovirus SV5 fusion (F) protein‐mediated fusion and its inhibition by the peptides N‐1 and C‐1 were analyzed. The temperature dependence of fusion by F suggests that thermal energy, destabilizing proline residues and receptor binding by the hemagglutinin–neuraminidase (HN) protein collectively contribute to F activation from a metastable native state. F‐mediated fusion was reversibly arrested by low temperature or membrane‐incorporated lipids, and the resulting F intermediates were characterized. N‐1 inhibited an earlier F intermediate than C‐1. Co‐expression of HN with F lowered the temperature required to attain the N‐1‐inhibited intermediate, consistent with HN binding to its receptor stimulating a conformational change in F. C‐1 bound and inhibited an intermediate of F that could be detected until a point directly preceding membrane merger. The data are consistent with C‐1 binding a pre‐hairpin intermediate of F and with helical bundle formation being coupled directly to membrane fusion.

Structures of an ActRIIB:activin A complex reveal a novel binding mode for TGF‐β ligand:receptor interactions

The TGF‐β superfamily of ligands and receptors stimulate cellular events in diverse processes ranging from cell fate specification in development to immune suppression. Activins define a major subgroup of TGF‐β ligands that regulate cellular differentiation, proliferation, activation and apoptosis. Activins signal through complexes formed with type I and type II serine/threonine kinase receptors. We have solved the crystal structure of activin A bound to the extracellular domain of a type II receptor, ActRIIB, revealing the details of this interaction. ActRIIB binds to the outer edges of the activin finger regions, with the two receptors juxtaposed in close proximity, in a mode that differs from TGF‐β3 binding to type II receptors. The dimeric activin A structure differs from other known TGF‐β ligand structures, adopting a compact folded‐back conformation. The crystal structure of the complex is consistent with recruitment of two type I receptors into a close packed arrangement at the cell surface and suggests that diversity in the conformational arrangements of TGF‐β ligand dimers could influence cellular signaling processes.

Peptide binding to HLA-DR1: a peptide with most residues substituted to alanine retains MHC binding.

Major histocompatibility complex (MHC) glycoproteins play an important role in the development of an effective immune response. An important MHC function is the ability to bind and present ‘processed antigens’ (peptides) to T cells. We show here that the purified human class II MHC molecule, HLA‐DR1, binds peptides that have been shown to be immunogenic in vivo. Detergent‐solubilized HLA‐DR1 and a papain‐cleaved form of the protein lacking the transmembrane and intracellular regions have similar peptide binding properties. A total of 39 single substitutions were made throughout an HLA‐DR1 restricted hemagglutinin epitope and the results determine one amino acid in this peptide which is crucial to binding. Based on this analysis, a synthetic peptide was designed containing two residues from the original hemagglutinin epitope embedded in a chain of polyalanine. This peptide binds to HLA‐DR1, indicating that the majority of peptide side chains are not required for high affinity peptide binding.

Paramyxovirus membrane fusion: Lessons from the F and HN atomic structures

Abstract Paramyxoviruses enter cells by fusion of their lipid envelope with the target cell plasma membrane. Fusion of the viral membrane with the plasma membrane allows entry of the viral genome into the cytoplasm. For paramyxoviruses, membrane fusion occurs at neutral pH, but the trigger mechanism that controls the viral entry machinery such that it occurs at the right time and in the right place remains to be elucidated. Two viral glycoproteins are key to the infection process—an attachment protein that varies among different paramyxoviruses and the fusion (F) protein, which is found in all paramyxoviruses. For many of the paramyxoviruses (parainfluenza viruses 1–5, mumps virus, Newcastle disease virus and others), the attachment protein is the hemagglutinin/neuraminidase (HN) protein. In the last 5 years, atomic structures of paramyxovirus F and HN proteins have been reported. The knowledge gained from these structures towards understanding the mechanism of viral membrane fusion is described.

Crystal Structure of the Human High-Affinity IgE Receptor

Allergic responses result from the activation of mast cells by the human high-affinity IgE receptor. IgE-mediated allergic reactions may develop to a variety of environmental compounds, but the initiation of a response requires the binding of IgE to its high-affinity receptor. We have solved the X-ray crystal structure of the antibody-binding domains of the human IgE receptor at 2.4 A resolution. The structure reveals a highly bent arrangement of immunoglobulin domains that form an extended convex surface of interaction with IgE. A prominent loop that confers specificity for IgE molecules extends from the receptor surface near an unusual arrangement of four exposed tryptophans. The crystal structure of the IgE receptor provides a foundation for the development of new therapeutic approaches to allergy treatment.

Structure of a trimeric variant of the Epstein–Barr virus glycoprotein B

Epstein–Barr virus (EBV) is a herpesvirus that is associated with development of malignancies of lymphoid tissue. EBV infections are life-long and occur in >90% of the population. Herpesviruses enter host cells in a process that involves fusion of viral and cellular membranes. The fusion apparatus is comprised of envelope glycoprotein B (gB) and a heterodimeric complex made of glycoproteins H and L. Glycoprotein B is the most conserved envelope glycoprotein in human herpesviruses, and the structure of gB from Herpes simplex virus 1 (HSV-1) is available. Here, we report the crystal structure of the secreted EBV gB ectodomain, which forms 16-nm long spike-like trimers, structurally homologous to the postfusion trimers of the fusion protein G of vesicular stomatitis virus (VSV). Comparative structural analyses of EBV gB and VSV G, which has been solved in its pre and postfusion states, shed light on gB residues that may be involved in conformational changes and membrane fusion. Also, the EBV gB structure reveals that, despite the high sequence conservation of gB in herpesviruses, the relative orientations of individual domains, the surface charge distributions, and the structural details of EBV gB differ from the HSV-1 protein, indicating regions and residues that may have important roles in virus-specific entry.