Protein secretion is a common property of pathogenic microbes. Gram-negative bacterial pathogens use at least 6 distinct extracellular protein secretion systems to export proteins through their ...multilayered cell envelope and in some cases into host cells. Among the most widespread is the newly recognized Type VI secretion system (T6SS) which is composed of 15-20 proteins whose biochemical functions are not well understood. Using crystallographic, biochemical, and bioinformatic analyses, we identified 3 T6SS components, which are homologous to bacteriophage tail proteins. These include the tail tube protein; the membrane-penetrating needle, situated at the distal end of the tube; and another protein associated with the needle and tube. We propose that T6SS is a multicomponent structure whose extracellular part resembles both structurally and functionally a bacteriophage tail, an efficient machine that translocates proteins and DNA across lipid membranes into cells.
Crystal structure of the human PRMT5:MEP50 complex Antonysamy, Stephen; Bonday, Zahid; Campbell, Robert M ...
Proceedings of the National Academy of Sciences,
10/2012, Letnik:
109, Številka:
44
Journal Article
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Protein arginine methyltransferases (PRMTs) play important roles in several cellular processes, including signaling, gene regulation, and transport of proteins and nucleic acids, to impact growth, ...differentiation, proliferation, and development. PRMT5 symmetrically di-methylates the two-terminal ω-guanidino nitrogens of arginine residues on substrate proteins. PRMT5 acts as part of a multimeric complex in concert with a variety of partner proteins that regulate its function and specificity. A core component of these complexes is the WD40 protein MEP50/WDR77/p44, which mediates interactions with binding partners and substrates. We have determined the crystal structure of human PRMT5 in complex with MEP50 (methylosome protein 50), bound to an S-adenosylmethionine analog and a peptide substrate derived from histone H4. The structure of the surprising hetero-octameric complex reveals the close interaction between the seven-bladed β-propeller MEP50 and the N-terminal domain of PRMT5, and delineates the structural elements of substrate recognition.
Cobalamin (vitamin B12) is a complex metabolite and essential cofactor required by many branches of life, including most eukaryotic phytoplankton. Algae and other cobalamin auxotrophs rely on ...environmental cobalamin supplied from a relatively small set of cobalamin-producing prokaryotic taxa. Although several Bacteria have been implicated in cobalamin biosynthesis and associated with algal symbiosis, the involvement of Archaea in cobalamin production is poorly understood, especially with respect to the Thaumarchaeota. Based on the detection of cobalamin synthesis genes in available thaumarchaeotal genomes, we hypothesized that Thaumarchaeota, which are ubiquitous and abundant in aquatic environments, have an important role in cobalamin biosynthesis within global aquatic ecosystems. To test this hypothesis, we examined cobalamin synthesis genes across sequenced thaumarchaeotal genomes and 430 metagenomes from a diverse range of marine, freshwater and hypersaline environments. Our analysis demonstrates that all available thaumarchaeotal genomes possess cobalamin synthesis genes, predominantly from the anaerobic pathway, suggesting widespread genetic capacity for cobalamin synthesis. Furthermore, although bacterial cobalamin genes dominated most surface marine metagenomes, thaumarchaeotal cobalamin genes dominated metagenomes from polar marine environments, increased with depth in marine water columns, and displayed seasonality, with increased winter abundance observed in time-series datasets (e.g., L4 surface water in the English Channel). Our results also suggest niche partitioning between thaumarchaeotal and cyanobacterial ribosomal and cobalamin synthesis genes across all metagenomic datasets analyzed. These results provide strong evidence for specific biogeographical distributions of thaumarchaeotal cobalamin genes, expanding our understanding of the global biogeochemical roles played by Thaumarchaeota in aquatic environments.
Lipoprotein(a) (Lp(a)), an independent, causal cardiovascular risk factor, is a lipoprotein particle that is formed by the interaction of a low-density lipoprotein (LDL) particle and ...apolipoprotein(a) (apo(a))
. Apo(a) first binds to lysine residues of apolipoprotein B-100 (apoB-100) on LDL through the Kringle IV (K
) 7 and 8 domains, before a disulfide bond forms between apo(a) and apoB-100 to create Lp(a) (refs.
). Here we show that the first step of Lp(a) formation can be inhibited through small-molecule interactions with apo(a) K
7-8. We identify compounds that bind to apo(a) K
7-8, and, through chemical optimization and further application of multivalency, we create compounds with subnanomolar potency that inhibit the formation of Lp(a). Oral doses of prototype compounds and a potent, multivalent disruptor, LY3473329 (muvalaplin), reduced the levels of Lp(a) in transgenic mice and in cynomolgus monkeys. Although multivalent molecules bind to the Kringle domains of rat plasminogen and reduce plasmin activity, species-selective differences in plasminogen sequences suggest that inhibitor molecules will reduce the levels of Lp(a), but not those of plasminogen, in humans. These data support the clinical development of LY3473329-which is already in phase 2 studies-as a potent and specific orally administered agent for reducing the levels of Lp(a).
The nuclear pore complex (NPC) is the sole passageway for the transport of macromolecules across the nuclear envelope. Nup133, a major component in the essential Y-shaped Nup84 complex, is a large ...scaffold protein of the NPC's outer ring structure. Here, we describe an integrative modeling approach that produces atomic models for multiple states of Saccharomyces cerevisiae (Sc) Nup133, based on the crystal structures of the sequence segments and their homologs, including the related Vanderwaltozyma polyspora (Vp) Nup133 residues 55 to 502 (VpNup13355–502) determined in this study, small angle X-ray scattering profiles for 18 constructs of ScNup133 and one construct of VpNup133, and 23 negative-stain electron microscopy class averages of ScNup1332–1157. Using our integrative approach, we then computed a multi-state structural model of the full-length ScNup133 and validated it with mutational studies and 45 chemical cross-links determined via mass spectrometry. Finally, the model of ScNup133 allowed us to annotate a potential ArfGAP1 lipid packing sensor (ALPS) motif in Sc and VpNup133 and discuss its potential significance in the context of the whole NPC; we suggest that ALPS motifs are scattered throughout the NPC's scaffold in all eukaryotes and play a major role in the assembly and membrane anchoring of the NPC in the nuclear envelope. Our results are consistent with a common evolutionary origin of Nup133 with membrane coating complexes (the protocoatomer hypothesis); the presence of the ALPS motifs in coatomer-like nucleoporins suggests an ancestral mechanism for membrane recognition present in early membrane coating complexes.
Extracellular domain (ECD) antigens are crucial components for antibody discovery, in vitro assays, and epitope mapping during therapeutical antibody development. Oftentimes, those antigens are ...difficult to produce while retaining the biologic function/activity upon extracellular secretion in commonly used expression systems. We have developed an effective method to cope with the challenge of generating quality antigen ECDs. In this method, a monoclonal antibody (Mab) or antibody fragment antigen-binding (Fab) region acts as a "chaperone" to stabilize the antigen ECD through forming an antibody:antigen complex. This methodology includes transient co-expression of the complex in Chinese hamster ovary cells and then dissociation of the purified complex into individual components by low pH treatment in the presence of arginine. The antigen is then separated from the chaperone on a preparative size exclusion chromatography (pSEC) followed by an optional affinity chromatography process to remove residual Mab or Fab. We demonstrate this co-expression/disassociation methodology on two difficult-to-express antigen ECDs from cluster-of-differentiation/cytokine family and were successful in producing stable, biologically active antigens when the common methods using Histidine-tagged and/or Fc-fused protein failed. This can be applied as a general approach for antigen production if a Mab or binding partner is available.
The nuclear pore complex, composed of proteins termed nucleoporins (Nups), is responsible for nucleocytoplasmic transport in eukaryotes. Nuclear pore complexes (NPCs) form an annular structure ...composed of the nuclear ring, cytoplasmic ring, a membrane ring, and two inner rings. Nup192 is a major component of the NPC’s inner ring. We report the crystal structure of Saccharomyces cerevisiae Nup192 residues 2–960 ScNup192(2–960), which adopts an α-helical fold with three domains (i.e., D1, D2, and D3). Small angle X-ray scattering and electron microscopy (EM) studies reveal that ScNup192(2–960) could undergo long-range transition between “open” and “closed” conformations. We obtained a structural model of full-length ScNup192 based on EM, the structure of ScNup192(2–960), and homology modeling. Evolutionary analyses using the ScNup192(2–960) structure suggest that NPCs and vesicle-coating complexes are descended from a common membrane-coating ancestral complex. We show that suppression of Nup192 expression leads to compromised nuclear transport and hypothesize a role for Nup192 in modulating the permeability of the NPC central channel.
► Structure of the N-terminal half of the yeast Nup192, a major component in the NPC ► Structural insights in to the conformational dynamics of ScNup192 by SAXS and EM ► Nup192 appears structurally related to karyopherins and vesicle coating proteins ► Functional data suggests that the Nup192 is necessary for efficient nuclear transport
Sampathkumar et al. describe the crystal structure of the N-terminal half of S. cerevisiae Nup192, a major component of the nuclear pore complex (NPC), and an electron microscopy model of the full-length protein. Nup192 is flexible, structurally related to karyopherins, and may help modulate the NPC permeability.
Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP‐binding cassette (ABC) transporter that functions as a chloride channel. Nucleotide‐binding domain 1 (NBD1), one of two ABC ...domains in CFTR, also contains sites for the predominant CF‐causing mutation and, potentially, for regulatory phosphorylation. We have determined crystal structures for mouse NBD1 in unliganded, ADP‐ and ATP‐bound states, with and without phosphorylation. This NBD1 differs from typical ABC domains in having added regulatory segments, a foreshortened subdomain interconnection, and an unusual nucleotide conformation. Moreover, isolated NBD1 has undetectable ATPase activity and its structure is essentially the same independent of ligand state. Phe508, which is commonly deleted in CF, is exposed at a putative NBD1‐transmembrane interface. Our results are consistent with a CFTR mechanism, whereby channel gating occurs through ATP binding in an NBD1–NBD2 nucleotide sandwich that forms upon displacement of NBD1 regulatory segments.
The MET receptor tyrosine kinase has emerged as an important target for the development of novel cancer therapeutics. Activation
of MET by mutation or gene amplification has been linked to kidney, ...gastric, and lung cancers. In other cancers, such as glioblastoma,
autocrine activation of MET has been demonstrated. Several classes of ATP-competitive inhibitor have been described, which
inhibit MET but also other kinases. Here, we describe SGX523, a novel, ATP-competitive kinase inhibitor remarkable for its
exquisite selectivity for MET. SGX523 potently inhibited MET with an IC 50 of 4 nmol/L and is >1,000-fold selective versus the >200-fold selectivity of other protein kinases tested in biochemical
assays. Crystallographic study revealed that SGX523 stabilizes MET in a unique inactive conformation that is inaccessible
to other protein kinases, suggesting an explanation for the selectivity. SGX523 inhibited MET-mediated signaling, cell proliferation,
and cell migration at nanomolar concentrations but had no effect on signaling dependent on other protein kinases, including
the closely related RON, even at micromolar concentrations. SGX523 inhibition of MET in vivo was associated with the dose-dependent inhibition of growth of tumor xenografts derived from human glioblastoma and lung
and gastric cancers, confirming the dependence of these tumors on MET catalytic activity. Our results show that SGX523 is
the most selective inhibitor of MET catalytic activity described to date and is thus a useful tool to investigate the role
of MET kinase in cancer without the confounding effects of promiscuous protein kinase inhibition. Mol Cancer Ther 2009;8(12):3181–90
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