Assembly and architecture of the EBV B cell entry triggering complex.

Epstein-Barr Virus (EBV) is an enveloped double-stranded DNA virus of the gammaherpesvirinae sub-family that predominantly infects humans through epithelial cells and B cells. Three EBV glycoproteins, gH, gL and gp42, form a complex that targets EBV infection of B cells. Human leukocyte antigen (HLA) class II molecules expressed on B cells serve as the receptor for gp42, triggering membrane fusion and virus entry. The mechanistic role of gHgL in herpesvirus entry has been largely unresolved, but it is thought to regulate the activation of the virally-encoded gB protein, which acts as the primary fusogen. Here we study the assembly and function of the reconstituted B cell entry complex comprised of gHgL, gp42 and HLA class II. The structure from negative-stain electron microscopy provides a detailed snapshot of an intermediate state in EBV entry and highlights the potential for the triggering complex to bring the two membrane bilayers into proximity. Furthermore, gHgL interacts with a previously identified, functionally important hydrophobic pocket on gp42, defining the overall architecture of the complex and playing a critical role in membrane fusion activation. We propose a macroscopic model of the initiating events in EBV B cell fusion centered on the formation of the triggering complex in the context of both viral and host membranes. This model suggests how the triggering complex may bridge the two membrane bilayers, orienting critical regions of the N- and C- terminal ends of gHgL to promote the activation of gB and efficient membrane fusion

Charge Detection Mass Spectrometry Reveals Conformational Heterogeneity in Megadalton-Sized Monoclonal Antibody Aggregates

Aggregation of protein-based therapeutics can occur during development, production, or storage and can lead to loss of efficacy and potential toxicity. Native mass spectrometry of a covalently linked pentameric monoclonal antibody complex with a mass of ∼800 kDa reveals several distinct conformations, smaller complexes, and abundant higher-order aggregates of the pentameric species. Charge detection mass spectrometry (CDMS) reveals individual oligomers up to the pentamer mAb trimer (15 individual mAb molecules; ∼2.4 MDa) whereas intermediate aggregates composed of 6-9 mAb molecules and aggregates larger than the pentameric dimer (1.6 MDa) were not detected/resolved by standard mass spectrometry, size exclusion chromatography (SEC), capillary electrophoresis (CE-SDS), or by mass photometry. Conventional quadrupole time-of-flight mass spectrometry (QTOF MS), mass photometry, SEC, and CE-SDS did not resolve partially or more fully unfolded conformations of each oligomer that were readily identified using CDMS by their significantly higher extents of charging. Trends in the charge-state distributions of individual oligomers provides detailed insight into how the structures of compact and elongated mAb aggregates change as a function of aggregate size. These results demonstrate the advantages of CDMS for obtaining accurate masses and information about the conformations of large antibody aggregates despite extensive overlapping m/z values. These results open up the ability to investigate structural changes that occur in small, soluble oligomers during the earliest stages of aggregation for antibodies or other proteins

Cycling of Etk and Etp Phosphorylation States Is Involved in Formation of Group 4 Capsule by Escherichia coli

Capsules frequently play a key role in bacterial interactions with their environment. Escherichia coli capsules were categorized as groups 1 through 4, each produced by a distinct mechanism. Etk and Etp are members of protein families required for the production of group 1 and group 4 capsules. These members function as a protein tyrosine kinase and protein tyrosine phosphatase, respectively. We show that Etp dephosphorylates Etk in vivo, and mutations rendering Etk or Etp catalytically inactive result in loss of group 4 capsule production, supporting the notion that cyclic phosphorylation and dephosphorylation of Etk is required for capsule formation. Notably, Etp also becomes tyrosine phosphorylated in vivo and catalyzes rapid auto-dephosphorylation. Further analysis identified Tyr121 as the phosphorylated residue of Etp. Etp containing Phe, Glu or Ala in place of Tyr121 retained phosphatase activity and catalyzed dephosphorylation of Etp and Etk. Although EtpY121E and EtpY121A still supported capsule formation, EtpY121F failed to do so. These results suggest that cycles of phosphorylation and dephosphorylation of Etp, as well as Etk, are involved in the formation of group 4 capsule, providing an additional regulatory layer to the complex control of capsule production.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/92200/1/journal_pone_0037984.pd

Crystal Structures of GfcC, a Group 4 Capsule Operon Protein from Escherichia coli, and YraM, an Outer Membrane Lipoprotein from Haemophilus influenzae.

GfcC is a small (26 kDa) periplasmic protein encoded within the seven gene gfc operon comprised of gfcABCDE, etp and etk of E. coli. This operon in enteropathogenic strains is essential for the expression of a group 4 polysaccharide capsule whose oligosaccharide repeat units are identical to those in lipopolysaccharide . The GfcC structure shows striking similarities with the outer membrane lipoprotein Wza from group 1 polysaccharide export. Both have two β-grasp domains for each monomer and an amphipathic C-terminal helical region. GfcC and its homologs also have a 40-residue long helical hairpin inserted into the first β-grasp domain not found in Wza. The C-terminal helices of Wza form a novel octameric alpha-helical barrel in the outer membrane. This barrel most likely exports the polysaccharide from the periplasm where it is synthesized. Although GfcC has a similar helix, it is shorter and folded back onto a β-grasp domain and hence GfcC exists as a soluble monomer in solution. Since the group 4 operon also has a wza homolog, gfcE, the function of GfcC is not clear. The following gene encodes GfcD, predicted to be a large β-barrel outer membrane lipoprotein. Homologs of gfcC and gfcD are fused in several species, notably in Burkholderia sp., suggesting an interaction in vivo between the two encoded proteins. YraM, a 575-residue outer membrane lipoprotein essential for growth in H. influenzae, has two distinct domains, an N-terminal domain with tetratricopeptide repeats and a C-terminal periplasmic binding protein-like domain. The C-domain has conserved residues clustered in the cleft where other binding proteins bind small molecule ligands. The N-domain resembles other proteins that bind protein or peptidoglycan ligands. The two domains are arranged adjacent connected by a 6-residue linker. Normal Mode analysis (specifically Elastic network modeling) suggests flexibility in the overall conformation between the two domains with the linker acting as a hinge. The function of YraM is unknown but it represents a novel fusion of domains with two distinct binding capabilities –one a small molecule ligand (to the C-domain) and the other possibly a protein (to the N-domain), both binding partners are yet to be identified.Ph.D.BiophysicsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/84569/1/karthiks_1.pd

Inhibition of EBV-mediated membrane fusion by anti-gHgL antibodies

Herpesvirus entry into cells requires the coordinated action of multiple virus envelope glycoproteins, including gH, gL, and gB. For EBV, the gp42 protein assembles into complexes with gHgL heterodimers and binds HLA class II to activate gB-mediated membrane fusion with B cells. EBV tropism is dictated by gp42 levels in the virion, as it inhibits entry into epithelial cells while promoting entry into B cells. The gHgL and gB proteins are targets of neutralizing antibodies and potential candidates for subunit vaccine development, but our understanding of their neutralizing epitopes and the mechanisms of inhibition remain relatively unexplored. Here we studied the structures and mechanisms of two anti-gHgL antibodies, CL40 and CL59, that block membrane fusion with both B cells and epithelial cells. We determined the structures of the CL40 and CL59 complexes with gHgL using X-ray crystallography and EM to identify their epitope locations. CL59 binds to the C-terminal domain IV of gH, while CL40 binds to a site occupied by the gp42 receptor binding domain. CL40 binding to gHgL/gp42 complexes is not blocked by gp42 and does not interfere with gp42 binding to HLA class II, indicating that its ability to block membrane fusion with B cells represents a defect in gB activation. These data indicate that anti-gHgL neutralizing antibodies can block gHgL-mediated activation of gB through different surface epitopes and mechanisms

Structural basis of omalizumab therapy and omalizumab-mediated IgE exchange.

Omalizumab is a widely used therapeutic anti-IgE antibody. Here we report the crystal structure of the omalizumab-Fab in complex with an IgE-Fc fragment. This structure reveals the mechanism of omalizumab-mediated inhibition of IgE interactions with both high- and low-affinity IgE receptors, and explains why omalizumab selectively binds free IgE. The structure of the complex also provides mechanistic insight into a class of disruptive IgE inhibitors that accelerate the dissociation of the high-affinity IgE receptor from IgE. We use this structural data to generate a mutant IgE-Fc fragment that is resistant to omalizumab binding. Treatment with this omalizumab-resistant IgE-Fc fragment, in combination with omalizumab, promotes the exchange of cell-bound full-length IgE with omalizumab-resistant IgE-Fc fragments on human basophils. This combination treatment also blocks basophil activation more efficiently than either agent alone, providing a novel approach to probe regulatory mechanisms underlying IgE hypersensitivity with implications for therapeutic interventions

Plasmids.

<p>Plasmids.</p

Etk and Wzc are not essential for Etp phosphorylation.

<p>EtpD119A was expressed in different EPEC strains with deletions in the <i>etp</i> gene and the kinase genes <i>etk</i> and <i>wzc</i>. Proteins were extracted and the amount and phosphorylation state of Etp were tested by Western blot using anti-6His and anti-PY antibodies, respectively. The presence of intact genes is indicated above the lanes.</p

EtpY121F retained phosphatase activity but failed to support capsule formation.

<p>(A) Proteins were extracted from wild-type EPEC, the Δ<i>etp::kan</i> mutant, or mutant complemented with plasmids derived from pEtp (pCNY506) and expressing different Etp variants with C-terminal 6His tags. The levels of Etk, phosphorylated Etk, recombinant Etp, and phosphorylated Etp were determined by Western blot analysis using anti-Etk, anti-6His, and anti-PY antibodies. The strain and the complementing mutant of Etp are indicated above the blots and the antibodies used in the immunoblots are indicated on the left side. The levels of Etk dephosphorylation and Etp autodephosphorylation are indicated below the blots. (B) Capsule polysaccharide was extracted and purified from the same strains presented in (A). Two-fold serial dilutions of purified capsule were dotted on PVDF and developed with anti-O127 antibody. The identity of the strain and complementing plasmid are indicated above the blot and direction of capsule polysaccharide dilution is indicated at the right side of the blot.</p