Kurt Swanson

Solution structure of Vps27 UIM–ubiquitin complex important for endosomal sorting and receptor downregulation

Monoubiquitylation is a well‐characterized signal for the internalization and sorting of integral membrane proteins to distinct cellular organelles. Recognition and transmission of monoubiquitin signals is mediated by a variety of ubiquitin‐binding motifs such as UIM, UBA, UEV, VHS and CUE in endocytic proteins. The yeast Vps27 protein requires two UIMs for efficient interactions with ubiquitin and for sorting cargo into multivesicular bodies. Here we show that the individual UIMs of Vps27 exist as autonomously folded α‐helices that bind ubiquitin independently, non‐cooperatively and with modest affinity. The Vps27 N‐terminal UIM engages the Leu8–Ile44–Val70 hydrophobic patch of ubiquitin through a helical surface conserved in UIMs of diverse proteins, including that of the S5a proteasomal regulatory subunit. The Leu8–Ile44–Val70 ubiquitin surface is also the site of interaction for CUE and UBA domains in endocytic proteins, consistent with the view that ubiquitin‐ binding endocytic proteins act serially on the same monoubiquitylated cargo during transport from cell surface to the lysosome.

Structural basis for immunization with postfusion respiratory syncytial virus fusion F glycoprotein (RSV F) to elicit high neutralizing antibody titers

Respiratory syncytial virus (RSV), the main cause of infant bronchiolitis, remains a major unmet vaccine need despite more than 40 years of vaccine research. Vaccine candidates based on a chief RSV neutralization antigen, the fusion (F) glycoprotein, have foundered due to problems with stability, purity, reproducibility, and potency. Crystal structures of related parainfluenza F glycoproteins have revealed a large conformational change between the prefusion and postfusion states, suggesting that postfusion F antigens might not efficiently elicit neutralizing antibodies. We have generated a homogeneous, stable, and reproducible postfusion RSV F immunogen that elicits high titers of neutralizing antibodies in immunized animals. The 3.2-Å X-ray crystal structure of this substantially complete RSV F reveals important differences from homology-based structural models. Specifically, the RSV F crystal structure demonstrates the exposure of key neutralizing antibody binding sites on the surface of the postfusion RSV F trimer. This unanticipated structural feature explains the engineered RSV F antigens efficiency as an immunogen. This work illustrates how structural-based antigen design can guide the rational optimization of candidate vaccine antigens.

Structure of the Newcastle disease virus hemagglutinin-neuraminidase (HN) ectodomain reveals a four-helix bundle stalk

The paramyxovirus hemagglutinin-neuraminidase (HN) protein plays multiple roles in viral entry and egress, including binding to sialic acid receptors, activating the fusion (F) protein to activate membrane fusion and viral entry, and cleaving sialic acid from carbohydrate chains. HN is an oligomeric integral membrane protein consisting of an N-terminal transmembrane domain, a stalk region, and an enzymatically active neuraminidase (NA) domain. Structures of the HN NA domains have been solved previously; however, the structure of the stalk region has remained elusive. The stalk region contains specificity determinants for F interactions and activation, underlying the requirement for homotypic F and HN interactions in viral entry. Mutations of the Newcastle disease virus HN stalk region have been shown to affect both F activation and NA activities, but a structural basis for understanding these dual affects on HN functions has been lacking. Here, we report the structure of the Newcastle disease virus HN ectodomain, revealing dimers of NA domain dimers flanking the N-terminal stalk domain. The stalk forms a parallel tetrameric coiled-coil bundle (4HB) that allows classification of extensive mutational data, providing insight into the functional roles of the stalk region. Mutations that affect both F activation and NA activities map predominantly to the 4HB hydrophobic core, whereas mutations that affect only F-protein activation map primarily to the 4HB surface. Two of four NA domains interact with the 4HB stalk, and residues at this interface in both the stalk and NA domain have been implicated in HN function.

Structure of the Newcastle disease virus F protein in the post-fusion conformation.

The paramyxovirus F protein is a class I viral membrane fusion protein which undergoes a significant refolding transition during virus entry. Previous studies of the Newcastle disease virus, human parainfluenza virus 3 and parainfluenza virus 5 F proteins revealed differences in the pre- and post-fusion structures. The NDV Queensland (Q) F structure lacked structural elements observed in the other two structures, which are key to the refolding and fusogenic activity of F. Here we present the NDV Australia-Victoria (AV) F protein post-fusion structure and provide EM evidence for its folding to a pre-fusion form. The NDV AV F structure contains heptad repeat elements missing in the previous NDV Q F structure, forming a post-fusion six-helix bundle (6HB) similar to the post-fusion hPIV3 F structure. Electrostatic and temperature factor analysis of the F structures points to regions of these proteins that may be functionally important in their membrane fusion activity.

HBP1 and Mad1 repressors bind the Sin3 corepressor PAH2 domain with opposite helical orientations.

Recruitment of the histone deacetylase (HDAC)-associated Sin3 corepressor is an obligatory step in many eukaryotic gene silencing pathways. Here we show that HBP1, a cell cycle inhibitor and regulator of differentiation, represses transcription in a HDAC/Sin3-dependent manner by targeting the mammalian Sin3A (mSin3A) PAH2 domain. HBP1 is unrelated to the Mad1 repressor for which high-resolution structures in complex with PAH2 have been described. We show that like Mad1, the HBP1 transrepression domain binds through a helical structure to the hydrophobic cleft of mSin3A PAH2. Notably, the HBP1 helix binds PAH2 in a reversed orientation relative to Mad1 and, equally unexpectedly, this is correlated with a chain reversal of the minimal Sin3 interaction motifs. These results not only provide insights into how multiple, unrelated transcription factors recruit the same coregulator, but also have implications for how sequence similarity searches are conducted.

A Monomeric Uncleaved Respiratory Syncytial Virus F Antigen Retains Prefusion-Specific Neutralizing Epitopes

ABSTRACT Respiratory syncytial virus (RSV) is the leading infectious cause of severe respiratory disease in infants and a major cause of respiratory illness in the elderly. There remains an unmet vaccine need despite decades of research. Insufficient potency, homogeneity, and stability of previous RSV fusion protein (F) subunit vaccine candidates have hampered vaccine development. RSV F and related parainfluenza virus (PIV) F proteins are cleaved by furin during intracellular maturation, producing disulfide-linked F1 and F2 fragments. During cell entry, the cleaved Fs rearrange from prefusion trimers to postfusion trimers. Using RSV F constructs with mutated furin cleavage sites, we isolated an uncleaved RSV F ectodomain that is predominantly monomeric and requires specific cleavage between F1 and F2 for self-association and rearrangement into stable postfusion trimers. The uncleaved RSV F monomer is folded and homogenous and displays at least two key RSV-neutralizing epitopes shared between the prefusion and postfusion conformations. Unlike the cleaved trimer, the uncleaved monomer binds the prefusion-specific monoclonal antibody D25 and human neutralizing immunoglobulins that do not bind to postfusion F. These observations suggest that the uncleaved RSV F monomer has a prefusion-like conformation and is a potential prefusion subunit vaccine candidate. IMPORTANCE RSV is the leading infectious cause of severe respiratory disease in infants and a major cause of respiratory illness in the elderly. Development of an RSV vaccine was stymied when a clinical trial using a formalin-inactivated RSV virus made disease, following RSV infection, more severe. Recent studies have defined the structures that the RSV F envelope glycoprotein adopts before and after virus entry (prefusion and postfusion conformations, respectively). Key neutralization epitopes of prefusion and postfusion RSV F have been identified, and a number of current vaccine development efforts are focused on generating easily produced subunit antigens that retain these epitopes. Here we show that a simple modification in the F ectodomain results in a homogeneous protein that retains critical prefusion neutralizing epitopes. These results improve our understanding of RSV F protein folding and structure and can guide further vaccine design efforts.

Identification and characterization of modular domains that bind ubiquitin

To receive and transmit the information carried by ubiquitin signals, cells have evolved an array of modular ubiquitin-binding domains. These domains bind directly and noncovalently to monoubiquitin and polyubiquitin chains and are found within proteins that function in diverse biological processes. Ubiquitin-binding domains characterized thus far are generally small and structurally diverse, yet they all interact with the same hydrophobic patch on the surface of ubiquitin. The rapid identification and characterization of ubiquitin-binding domains has been accomplished through the extensive use of bioinformatics, biochemistry, molecular biology, and biophysics. Here, we discuss the strategies and tools that have been most successful in the identification and characterization of ubiquitin-binding domains.

Structure of the Newcastle Disease Virus F Protein in the Post-Fusion Conformation

Newcastle disease virus (NDV) is a member of the Paramyxoviridae family. The NDV fusion (F) glycoprotein, which is responsible for merging the viral and cellular bilayers during entry. The X-ray crystal structures have been solved of F proteins in the post-fusion and the pre-fusion conformations, providing atomic level information regarding the conformational transitions accompanying fusion. However, our understanding of the similarities between different F glycoproteins in these two conformational states remains incomplete.Here, we present the crystal structure of the secreted, uncleaved ectodomain of the NDV F protein. Previous structural analysis of a related NDV F protein was missing key elements of the functional regions of the protein, including two helical segments (HRA and HRB) that assemble into a stable six helix bundle (6HB) in the post-fusion form. We have produced the NDV F protein in pre-and post-fusion conformations, using analogous constructs that produced a pre-fusion PIV5 F structure and a post-fusion HPIV3 F structure. We demonstrate that the two NDV F proteins exhibit the pre-and post-fusion forms through EM analysis and we have solved the crystal structure of the post-fusion form of the NDV F protein. In contrast to the previously determined NDV F structure, our new crystal structure contains the 6HB at the base of the stalk region, consistent with the EM observations and the previously determined HPIV3 F structure. Global superposition of the NDV and HPIV3 structures demonstrates maximum correspondence between distal portions of the structures, with orientation or adjustments in linking domains and the extended HRA stalk. Electrostatic profiles of the NDV, HPIV3, and PIV5 F structures show elements of conserved charge distributions despite significant sequence differences in these glycoproteins, which may be important for their common functionality.