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Seasonal influenza virus infections can cause significant morbidity and mortality, but the threat from the emergence of a new pandemic influenza strain might have potentially even more devastating consequences. As such, there is intense interest in isolating and characterizing potent neutralizing antibodies that target the hemagglutinin (HA) viral surface glycoprotein. Here, we use cryo-electron microscopy (cryoEM) to decipher the mechanism of action of a potent HA head-directed monoclonal antibody (mAb) bound to an influenza H7 HA. The epitope of the antibody is not solvent accessible in the compact, prefusion conformation that typifies all HA structures to date. Instead, the antibody binds between HA head protomers to an epitope that must be partly or transiently exposed in the prefusion conformation. The "breathing" of the HA protomers is implied by the exposure of this epitope, which is consistent with metastability of class I fusion proteins. This structure likely therefore represents an early structural intermediate in the viral fusion process. Understanding the extent of transient exposure of conserved neutralizing epitopes also may lead to new opportunities to combat influenza that have not been appreciated previously.
Members of the orthosomycin family of natural products are decorated polysaccharides with potent antibiotic activity and complex biosynthetic pathways. The defining feature of the orthosomycins is an orthoester linkage between carbohydrate moieties that is necessary for antibiotic activity and is likely formed by a family of conserved oxygenases. Everninomicins are octasaccharide orthosomycins produced by Micromonospora carbonacea that have two orthoester linkages and a methylenedioxy bridge, three features whose formation logically requires oxidative chemistry. Correspondingly, the evd gene cluster encoding everninomicin D encodes two monofunctional nonheme iron, α-ketoglutarate-dependent oxygenases and one bifunctional enzyme with an N-terminal methyltransferase domain and a C-terminal oxygenase domain. To investigate whether the activities of these domains are linked in the bifunctional enzyme EvdMO1, we determined the structure of the N-terminal methyltransferase domain to 1.1 Å and that of the full-length protein to 3.35 Å resolution. Both domains of EvdMO1 adopt the canonical folds of their respective superfamilies and are connected by a short linker. Each domain's active site is oriented such that it faces away from the other domain, and there is no evidence of a channel connecting the two. Our results support EvdMO1 working as a bifunctional enzyme with independent catalytic activities.
Helicobacter pylori is a genetically diverse bacterial species that colonizes the stomach in about half of the human population. Most persons colonized by H. pylori remain asymptomatic, but the presence of this organism is a risk factor for gastric cancer. Multiple populations and subpopulations of H. pylori with distinct geographic distributions are recognized. Genetic differences among these populations might be a factor underlying geographic variation in gastric cancer incidence. Relatively little is known about the genomic features of African H. pylori strains compared to other populations of strains. In this study, we first analyzed the genomes of H. pylori strains from seven globally distributed populations or subpopulations and identified encoded proteins that exhibited the highest levels of sequence divergence. These included secreted proteins, an LPS glycosyltransferase, fucosyltransferases, proteins involved in molybdopterin biosynthesis, and Clp protease adaptor (ClpS). Among proteins encoded by the cag pathogenicity island, CagA and CagQ exhibited the highest levels of sequence diversity. We then identified proteins in strains of Western African origin (classified as hspWAfrica by MLST analysis) with sequences that were highly divergent compared to those in other populations of strains. These included ATP-dependent Clp protease, ClpS, and proteins of unknown function. Three of the divergent proteins sequences identified in West African strains were characterized by distinct insertions or deletions up to 8 amino acids in length. These polymorphisms in rapidly evolving proteins represent robust genetic signatures for H. pylori strains of West African origin.
Pre-mRNA processing protein 40 (Prp40) is a nuclear protein that has a role in pre-mRNA splicing. Prp40 possesses two leucine-rich nuclear export signals, but little is known about the function of Prp40 in the export process. Another protein that has a role in protein export is centrin, a member of the EF-hand superfamily of Ca-binding proteins. Prp40 was found to be a centrin target by yeast-two-hybrid screening using both Homo sapiens centrin 2 (Hscen2) and Chlamydomonas reinhardtii centrin (Crcen). We identified a centrin-binding site within H. sapiens Prp40 homolog A (HsPrp40A), which contains a hydrophobic triad WLL that is known to be important in the interaction with centrin. This centrin-binding site is highly conserved within the first nuclear export signal consensus sequence identified in Saccharomyces cerevisiae Prp40. Here, we examine the interaction of HsPrp40A peptide (HsPrp40Ap) with both Hscen2 and Crcen by isothermal titration calorimetry. We employed the thermodynamic parameterization to estimate the polar and apolar surface area of the interface. In addition, we have defined the molecular mechanism of thermally induced unfolding and dissociation of the Crcen-HsPrp40Ap complex using two-dimensional infrared correlation spectroscopy. These complementary techniques showed for the first time, to our knowledge, that HsPrp40Ap interacts with centrin in vitro, supporting a coupled functional role for these proteins in pre-mRNA splicing.
Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.
Phospholipid flippases in the type IV P-type ATPase (P4-ATPases) family establish membrane asymmetry and play critical roles in vesicular transport, cell polarity, signal transduction, and neurologic development. All characterized P4-ATPases flip glycerophospholipids across the bilayer to the cytosolic leaflet of the membrane, but how these enzymes distinguish glycerophospholipids from sphingolipids is not known. We used a directed evolution approach to examine the molecular mechanisms through which P4-ATPases discriminate substrate backbone. A mutagenesis screen in the yeast Saccharomyces cerevisiae has identified several gain-of-function mutations in the P4-ATPase Dnf1 that facilitate the transport of a novel lipid substrate, sphingomyelin. We found that a highly conserved asparagine (N220) in the first transmembrane segment is a key enforcer of glycerophospholipid selection, and specific substitutions at this site allow transport of sphingomyelin.
The S100 proteins are a unique class of EF-hand Ca(2+) binding proteins distributed in a cell-specific, tissue-specific, and cell cycle-specific manner in humans and other vertebrates. These proteins are distinguished by their distinctive homodimeric structure, both intracellular and extracellular functions, and the ability to bind transition metals at the dimer interface. Here we summarize current knowledge of S100 protein binding of Zn(2+), Cu(2+) and Mn(2+) ions, focusing on binding affinities, conformational changes that arise from metal binding, and the roles of transition metal binding in S100 protein function.
Upon Notch pathway activation, the receptor is cleaved to release the Notch intracellular domain (NICD), which translocates to the nucleus to activate gene transcription. Using Xenopus egg extracts, we have identified a Notch1-specific destruction signal (N1-Box). We show that mutations in the N1-Box inhibit NICD1 degradation and that the N1-Box is transferable for the promotion of degradation of heterologous proteins in Xenopus egg extracts and in cultured human cells. Mutation of the N1-Box enhances Notch1 activity in cultured human cells and zebrafish embryos. Human cancer mutations within the N1-Box enhance Notch1 signaling in transgenic zebrafish, highlighting the physiological relevance of this destruction signal. We find that binding of the Notch nuclear factor, CSL, to the N1-Box blocks NICD1 turnover. Our studies reveal a mechanism by which degradation of NICD1 is regulated by the N1-Box to minimize stochastic flux and to establish a threshold for Notch1 pathway activation.
Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.
The MYC family of oncogenes encodes a set of three related transcription factors that are overexpressed in many human tumors and contribute to the cancer-related deaths of more than 70,000 Americans every year. MYC proteins drive tumorigenesis by interacting with co-factors that enable them to regulate the expression of thousands of genes linked to cell growth, proliferation, metabolism and genome stability. One effective way to identify critical co-factors required for MYC function has been to focus on sequence motifs within MYC that are conserved throughout evolution, on the assumption that their conservation is driven by protein-protein interactions that are vital for MYC activity. In addition to their DNA-binding domains, MYC proteins carry five regions of high sequence conservation known as Myc boxes (Mb). To date, four of the Mb motifs (MbI, MbII, MbIIIa and MbIIIb) have had a molecular function assigned to them, but the precise role of the remaining Mb, MbIV, and the reason for its preservation in vertebrate Myc proteins, is unknown. Here, we show that MbIV is required for the association of MYC with the abundant transcriptional coregulator host cell factor-1 (HCF-1). We show that the invariant core of MbIV resembles the tetrapeptide HCF-binding motif (HBM) found in many HCF-interaction partners, and demonstrate that MYC interacts with HCF-1 in a manner indistinguishable from the prototypical HBM-containing protein VP16. Finally, we show that rationalized point mutations in MYC that disrupt interaction with HCF-1 attenuate the ability of MYC to drive tumorigenesis in mice. Together, these data expose a molecular function for MbIV and indicate that HCF-1 is an important co-factor for MYC.
Next-generation sequencing has transformed the ability to link genotypes to phenotypes and facilitates the dissection of genetic contribution to complex traits. However, it is challenging to link genetic variants with the perturbed functional effects on proteins encoded by such genes. Here we show how RNA sequencing can be exploited to construct genotype-specific protein sequence databases to assess natural variation in proteins, providing information about the molecular toolbox driving cellular processes. For this study, we used two natural genotypes selected from a recent genome-wide association study of Populus trichocarpa, an obligate outcrosser with tremendous phenotypic variation across the natural population. This strategy allowed us to comprehensively catalogue proteins containing single amino acid polymorphisms (SAAPs), as well as insertions and deletions. We profiled the frequency of 128 types of naturally occurring amino acid substitutions, including both expected (neutral) and unexpected (non-neutral) SAAPs, with a subset occurring in regions of the genome having strong polymorphism patterns consistent with recent positive and/or divergent selection. By zeroing in on the molecular signatures of these important regions that might have previously been uncharacterized, we now provide a high-resolution molecular inventory that should improve accessibility and subsequent identification of natural protein variants in future genotype-to-phenotype studies.
Antigenic drift of circulating seasonal influenza viruses necessitates an international vaccine effort to reduce the impact on human health. A critical feature of the seasonal vaccine is that it stimulates an already primed immune system to diversify memory B cells to recognize closely related, but antigenically distinct, influenza glycoproteins (hemagglutinins). Influenza pandemics arise when hemagglutinins to which no preexisting adaptive immunity exists acquire the capacity to infect humans. Hemagglutinin 5 is one subtype to which little preexisting immunity exists and is only a few acquired mutations away from the ability to transmit efficiently between ferrets, and possibly humans. Here, we describe the structure and molecular mechanism of neutralization by H5.3, a vaccine-elicited antibody that neutralizes hemagglutinin 5 viruses and variants with expanded host range. H5.3 binds in the receptor-binding site, forming contacts that recapitulate many of the sialic acid interactions, as well as multiple peripheral interactions, yet is not sensitive to mutations that alter sialic acid binding. H5.3 is highly specific for a subset of H5 strains, and this specificity arises from interactions to the periphery of the receptor-binding site. H5.3 is also extremely potent, despite retaining germ line-like conformational flexibility.