ABSTRACT
OBJECTIVES: To understand SARS-Co-V-2 infection and transmission in UK nursing homes in order to develop preventive strategies for protecting the frail elderly residents. METHODS: An outbreak investigation involving 394 residents and 70 staff, was carried out in 4 nursing homes affected by COVID-19 outbreaks in central London. Two point-prevalence surveys were performed one week apart where residents underwent SARS-CoV-2 testing and had relevant symptoms documented. Asymptomatic staff from three of the four homes were also offered SARS-CoV-2 testing. RESULTS: Overall, 26% (95% CI 22-31) of residents died over the two-month period. All-cause mortality increased by 203% (95% CI 70-336) compared with previous years. Systematic testing identified 40% (95% CI 35-46) of residents as positive for SARS-CoV-2, and of these 43% (95% CI 34-52) were asymptomatic and 18% (95% CI 11-24) had only atypical symptoms; 4% (95% CI -1 to 9) of asymptomatic staff also tested positive. CONCLUSIONS: The SARS-CoV-2 outbreak in four UK nursing homes was associated with very high infection and mortality rates. Many residents developed either atypical or had no discernible symptoms. A number of asymptomatic staff members also tested positive, suggesting a role for regular screening of both residents and staff in mitigating future outbreaks.
Subject(s)
Betacoronavirus , Coronavirus Infections/pathology , Nursing Homes , Pneumonia, Viral/pathology , Aged , Aged, 80 and over , COVID-19 , COVID-19 Testing , Clinical Laboratory Techniques , Coronavirus Infections/diagnosis , Coronavirus Infections/epidemiology , Coronavirus Infections/mortality , Female , Humans , Male , Pandemics , Pneumonia, Viral/epidemiology , Pneumonia, Viral/mortality , SARS-CoV-2 , Time Factors , United Kingdom/epidemiologyABSTRACT
Biofoundries have enabled the ability to automate the construction of genetic constructs using computer-aided design. In this study, we have developed the methodology required to abstract and automate the construction of yeast-compatible designs. We demonstrate the use of our in-house software tool, AMOS, to coordinate with design software, JMP, and robotic liquid handling platforms to successfully manage the construction of a library of 88 yeast expression plasmids. In this proof-of-principle study, we used three fluorescent genes as proxy for three enzyme coding sequences. Our platform has been designed to quickly iterate around a design cycle of four protein coding sequences per plasmid, with larger numbers possible with multiplexed genome integrations in Saccharomyces cerevisiae. This work highlights how developing scalable new biotechnology applications requires a close integration between software development, liquid handling robotics, and protocol development.
Subject(s)
Automation, Laboratory/methods , Genetics, Microbial/methods , Molecular Biology/methods , Saccharomyces cerevisiae/genetics , High-Throughput Screening Assays , Robotics/methods , Saccharomyces cerevisiae/growth & development , Software , Specimen Handling/methodsABSTRACT
Parasitic diseases affect millions of people worldwide, causing debilitating illnesses and death. Rapid and cost-effective approaches to detect parasites are needed, especially in resource-limited settings. A common signature of parasitic diseases is the release of specific proteases by the parasites at multiple stages during their life cycles. To this end, we engineered several modular Escherichia coli and Bacillus subtilis whole-cell-based biosensors which incorporate an interchangeable protease recognition motif into their designs. Herein, we describe how several of our engineered biosensors have been applied to detect the presence and activity of elastase, an enzyme released by the cercarial larvae stage of Schistosoma mansoni. Collectively, S. mansoni and several other schistosomes are responsible for the infection of an estimated 200 million people worldwide. Since our biosensors are maintained in lyophilised cells, they could be applied for the detection of S. mansoni and other parasites in settings without reliable cold chain access.
Subject(s)
Biosensing Techniques , Endopeptidases/metabolism , Larva , Schistosoma mansoni/growth & development , Animals , Freeze DryingABSTRACT
The AAA (ATPase associated with various cellular activities) ATPase, p97, is a hexameric protein of chaperone-like function, which has been reported to interact with a number of proteins of seemingly unrelated functions. For the first time, we report a classification of these proteins and aim to elucidate any common structural or functional features they may share. The interactors are grouped into those containing ubiquitin regulatory X domains, which presumably bind to p97 in the same way as the p47 adaptor, and into non-ubiquitin regulatory X domain proteins of different functional subgroups that may employ a different mode of interaction (assuming they also bind directly to p97 and are not experimental artifacts). Future studies will show whether interacting proteins direct p97 to different cellular pathways or a common one and structural elucidation of these interactions will be crucial in understanding these underlying functions.
Subject(s)
Neoplasm Proteins/physiology , Animals , Antigens, Neoplasm , Cell Cycle Proteins/chemistry , Humans , Melanoma-Specific Antigens , Models, Molecular , Molecular Chaperones/metabolism , Neoplasm Proteins/metabolism , Protein Binding , Protein Structure, Tertiary , Ubiquitin/chemistry , Ubiquitin/metabolismABSTRACT
Promyelocytic leukemia (PML) bodies are nuclear multi-protein domains. The observations that viruses transcribe their genomes adjacent to PML bodies and that nascent RNA accumulates at their periphery suggest that PML bodies function in transcription. We have used immuno-FISH in primary human fibroblasts to determine the 3D spatial organisation of gene-rich and gene-poor chromosomal regions relative to PML bodies. We find a highly non-random association of the gene-rich major histocompatibilty complex (MHC) on chromosome 6 with PML bodies. This association is specific for the centromeric end of the MHC and extends over a genomic region of at least 1.6 megabases. We also show that PML association is maintained when a subsection of this region is integrated into another chromosomal location. This is the first demonstration that PML bodies have specific chromosomal associations and supports a model for PML bodies as part of a functional nuclear compartment.
Subject(s)
Cell Nucleus/metabolism , Interphase/physiology , Major Histocompatibility Complex/genetics , Neoplasm Proteins/metabolism , Nuclear Proteins , Transcription Factors/metabolism , Cells, Cultured , Chromosomes, Human, Pair 6/genetics , Fibroblasts/cytology , Fibroblasts/physiology , Humans , In Situ Hybridization, Fluorescence/methods , Microscopy, Fluorescence/methods , Promyelocytic Leukemia Protein , Tumor Suppressor ProteinsABSTRACT
PML is a component of a multiprotein complex, termed nuclear bodies, and the PML protein was originally discovered in patients suffering from acute promyelocytic leukaemia (APL). APL is associated with a reciprocal chromosomal translocation of chromosomes 15 and 17, which results in a fusion protein comprising PML and the retinoic acid receptor alpha. The PML genomic locus is approximately 35 kb and is subdivided into nine exons. A large number of alternative spliced transcripts are synthesized from the PML gene, resulting in a variety of PML proteins ranging in molecular weight from 48-97 kDa. In this review we summarize the data on the known PML isoforms and splice variants and present a new unifying nomenclature. Although, the function/s of the PML variants are unclear, all PML isoforms contain an identical N-terminal region, suggesting that these sequences are indispensable for function, but differ in their C-terminal sequences. The N-terminal region harbours a RING-finger, two B-boxes and a predicted alpha-helical Coiled-Coil domain, that together form the RBCC/TRIM motif found in a large family of proteins. In PML this motif is essential for PML nuclear body formation in vivo and PML-homo and hetero interactions conferring growth suppressor, apoptotic and anti-viral activities. In APL oligomerization mediated by the RBCC/TRIM motif is essential for the transformation potential of the PML-RARalpha fusion protein.
Subject(s)
Microtubule Proteins , Neoplasm Proteins/classification , Neoplasm Proteins/physiology , Transcription Factors/classification , Transcription Factors/physiology , Alternative Splicing , Amino Acid Motifs/genetics , Animals , Humans , Leukemia, Promyelocytic, Acute/genetics , Ligases/genetics , Nuclear Proteins/genetics , Oncogene Proteins, Fusion/genetics , Promyelocytic Leukemia Protein , Protein Binding , Protein Isoforms/classification , Protein Isoforms/physiology , Protein Structure, Tertiary/genetics , Structure-Activity Relationship , Terminology as Topic , Transcription Factors/genetics , Tripartite Motif Proteins , Tumor Suppressor Proteins , Ubiquitin-Protein LigasesABSTRACT
p47 is the major protein identified in complex with the cytosolic AAA ATPase p97. It functions as an essential cofactor of p97-regulated membrane fusion, which has been suggested to disassemble t-t-SNARE complexes and prepare them for further rounds of membrane fusion. Here, we report the high-resolution NMR structure of the C-terminal domain from p47. It comprises a UBX domain and a 13 residue long structured N-terminal extension. The UBX domain adopts a characteristic ubiquitin fold with a betabetaalphabetabetaalphabeta secondary structure arrangement. Three hydrophobic residues from the N-terminal extension pack closely against a cleft in the UBX domain. We also identify, for the first time, the p97 interaction surface using NMR chemical shift perturbation studies.
Subject(s)
Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/metabolism , Membrane Proteins/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Vesicular Transport Proteins , Amino Acid Sequence , Animals , Binding Sites , DEAD-box RNA Helicases , Membrane Proteins/chemistry , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Protein Structure, Secondary , Protein Structure, Tertiary , Rats , SNARE Proteins , Sequence Alignment , Solutions , Ubiquitins/chemistryABSTRACT
beta-Glucosyltransferase (BGT) is a DNA-modifying enzyme encoded by bacteriophage T4 that transfers glucose from uridine diphosphoglucose to 5-hydroxymethyl cytosine bases of phage T4 DNA. We report six X-ray structures of the substrate-free and the UDP-bound enzyme. Four also contain metal ions which activate the enzyme, including Mg(2+) in forms 1 and 2 and Mn(2+) or Ca(2+). The substrate-free BGT structure differs by a domain movement from one previously determined in another space group. Further domain movements are seen in the complex with UDP and the four UDP-metal complexes. Mg(2+), Mn(2+) and Ca(2+) bind near the beta-phosphate of the nucleotide, but they occupy slightly different positions and have different ligands depending on the metal and the crystal form. Whilst the metal site observed in these complexes with the product UDP is not compatible with a role in activating glucose transfer, it approximates the position of the positive charge in the oxocarbonium ion thought to form on the glucose moiety of the substrate during catalysis.
Subject(s)
Bacteriophage T4/enzymology , Glucosyltransferases/chemistry , Glucosyltransferases/metabolism , Metals/metabolism , Uridine Diphosphate/metabolism , Allosteric Site , Calcium/metabolism , Crystallography, X-Ray , Enzyme Activation , Ligands , Magnesium/chemistry , Magnesium/metabolism , Manganese/metabolism , Metals/chemistry , Models, Molecular , Movement , Protein Binding , Protein Structure, TertiaryABSTRACT
Promyelocytic leukemia (PML) is the organizer of nuclear matrix domains, PML nuclear bodies (NBs), with a proposed role in apoptosis control. In acute promyelocytic leukemia, PML/retinoic acid receptor (RAR) alpha expression disrupts NBs, but therapies such as retinoic acid or arsenic trioxide (As2O3) restore them. PML is conjugated by the ubiquitin-related peptide SUMO-1, a process enhanced by As2O3 and proposed to target PML to the nuclear matrix. We demonstrate that As2O3 triggers the proteasome-dependent degradation of PML and PML/RARalpha and that this process requires a specific sumolation site in PML, K160. PML sumolation is dispensable for its As2O3-induced matrix targeting and formation of primary nuclear aggregates, but is required for the formation of secondary shell-like NBs. Interestingly, only these mature NBs harbor 11S proteasome components, which are further recruited upon As2O3 exposure. Proteasome recruitment by sumolated PML only likely accounts for the failure of PML-K160R to be degraded. Therefore, studying the basis of As2O3-induced PML/RARalpha degradation we show that PML sumolation directly or indirectly promotes its catabolism, suggesting that mature NBs could be sites of intranuclear proteolysis and opening new insights into NB alterations found in viral infections or transformation.
Subject(s)
Adenosine Triphosphatases/metabolism , Arsenicals/pharmacology , Endopeptidases , Neoplasm Proteins/metabolism , Nuclear Matrix/metabolism , Nuclear Proteins , Oxides/pharmacology , Receptors, Retinoic Acid/metabolism , Transcription Factors/metabolism , Ubiquitins/metabolism , Amino Acid Motifs , Animals , Arsenic Trioxide , CHO Cells , Cell Line , Cell Nucleus/metabolism , Cells, Cultured , Cricetinae , Mice , Models, Biological , Mutation , Neoplasm Proteins/chemistry , Neoplasm Proteins/genetics , Promyelocytic Leukemia Protein , Proteasome Endopeptidase Complex , Protein Isoforms/chemistry , Protein Transport , Retinoic Acid Receptor alpha , SUMO-1 Protein , Transcription Factors/chemistry , Transcription Factors/genetics , Tumor Suppressor ProteinsABSTRACT
Proteins involved in DNA repair, or its coordination with DNA replication and mitosis through cell cycle checkpoints, are vital in the concerted cellular response to DNA damage that maintains the integrity of the genome. The "BRCT" domain (BRCA1 carboxy terminal) was noted as a putative protein-protein interaction motif in the breast cancer suppressor gene, BRCA1, and subsequently identified in over 50 proteins involved in DNA repair, recombination, or cell cycle control. The heterodimer of the DNA repair proteins, XRCC1 and DNA ligase III, was the first example of a functional interaction via BRCT modules. The only three-dimensional crystal structure of a BRCT domain was solved for this region of XRCC1. Key amino acid residues mediating the interaction with DNA ligase III were identified here by targeted mutagenesis of the XRCC1 BRCT domain. The consequences of these mutations on protein folding were assessed. A structural model of the DNA ligase III BRCT domain was constructed and similarly tested by mutation of corresponding residues required for the interaction with XRCC1. These data identify the XRCC1-DNA ligase III heterodimer interface and provide the first demonstration of the surface contacts coordinating a functional BRCT-BRCT protein interaction.
Subject(s)
BRCA1 Protein/metabolism , DNA Ligases/metabolism , DNA Repair , DNA-Binding Proteins/metabolism , Amino Acid Substitution/genetics , BRCA1 Protein/chemistry , BRCA1 Protein/genetics , Circular Dichroism , DNA Ligase ATP , DNA Ligases/chemistry , DNA-Binding Proteins/chemistry , Dimerization , Humans , Models, Molecular , Mutagenesis, Site-Directed , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Poly-ADP-Ribose Binding Proteins , Protein Binding/genetics , Protein Structure, Secondary/genetics , Protein Structure, Tertiary/genetics , Thermodynamics , Tryptophan/genetics , X-ray Repair Cross Complementing Protein 1 , Xenopus ProteinsABSTRACT
The BRCA1 C-terminal region contains a duplicated globular domain termed BRCT that is found within many DNA damage repair and cell cycle checkpoint proteins. The unique diversity of this domain superfamily allows BRCT modules to interact forming homo/hetero BRCT multimers, BRCT-non-BRCT interactions, and interactions with DNA strand breaks. The sequence and functional diversity of the BRCT superfamily suggests that BRCT domains are evolutionarily convenient interaction modules.
Subject(s)
BRCA1 Protein/chemistry , Tumor Suppressor Proteins , Ubiquitin-Protein Ligases , Amino Acid Sequence , BRCA1 Protein/physiology , Bacterial Proteins/chemistry , Biopolymers , Breast Neoplasms/genetics , Carrier Proteins/chemistry , Carrier Proteins/physiology , Cell Cycle Proteins/chemistry , DNA Damage , DNA Ligases/chemistry , DNA Nucleotidylexotransferase/chemistry , DNA Nucleotidylexotransferase/physiology , DNA Repair , DNA-Binding Proteins/chemistry , Evolution, Molecular , Female , Fungal Proteins/chemistry , Genes, BRCA1 , Humans , Models, Molecular , Molecular Sequence Data , Mutation, Missense , Neoplastic Syndromes, Hereditary/genetics , Nuclear Proteins , Poly(ADP-ribose) Polymerases/chemistry , Protein Conformation , Protein Structure, Tertiary , Sequence Alignment , Sequence Homology, Amino Acid , Structure-Activity Relationship , X-ray Repair Cross Complementing Protein 1ABSTRACT
Leukaemogenesis correlates with alterations in chromatin structure brought about by the gain or loss of interactive domains from regulatory factors that are disrupted by chromosomal translocations. The gene MLL, a target of such translocation events, forms chimaeric fusion products with a variety of partner genes. While MLL appears to be involved in chromatin-mediated gene regulation, the functions of its partner genes are largely speculative. We report the biochemical analysis of the MLL partner gene AF10 and its possible role in leukaemogenesis. AF10 has been reported to be re-arranged with genes other than MLL leading to the same phenotype, a myeloid leukaemia. We have identified a novel protein-protein interaction motif in the AF10 protein comprising the extended LAP/PHD-finger. This domain mediates homo-oligomerisation of recombinant AF10 and is conserved in several proteins, including MLL itself. AF10 binds cruciform DNA via a specific interaction with an AT-hook motif and is localised to the nucleus by a defined bipartite nuclear localisation signal in the N-terminal region.
Subject(s)
Transcription Factors/chemistry , Transcription Factors/metabolism , Amino Acid Motifs , Amino Acid Sequence , Biological Transport , Cell Nucleus/metabolism , Chromatography, Gel , Conserved Sequence , Cross-Linking Reagents/metabolism , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/genetics , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/ultrastructure , HeLa Cells , Humans , Leukemia, Myeloid/genetics , Microscopy, Electron , Molecular Sequence Data , Molecular Weight , Mutation/genetics , Nuclear Localization Signals/genetics , Nuclear Localization Signals/physiology , Nucleic Acid Conformation , Protein Binding , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/ultrastructure , Sequence Alignment , Transcription Factors/genetics , Transcription Factors/ultrastructureABSTRACT
HAP1, also known as APE/Ref-1, is the major apurinic/apyrimidinic (AP) endonuclease in human cells. Previous structural studies have suggested a possible role for the Asp-210 residue of HAP1 in the enzymatic function of this enzyme. Here, we demonstrate that substitution of Asp-210 by Asn or Ala eliminates the AP endonuclease activity of HAP1, while substitution by Glu reduces specific activity approximately 500-fold. Nevertheless, these mutant proteins still bind efficiently to oligonucleotides containing either AP sites or the chemically unrelated bulky p-benzoquinone (pBQ) derivatives of dC, dA and dG, all of which are substrates for HAP1. These results indicate that Asp-210 is required for catalysis, but not substrate recognition, consistent with enzyme kinetic data indicating that the HAP1-D210E protein has a 3000-fold reduced K(cat )for AP site cleavage, but an unchanged K(m). Through analysis of the binding of Asp-210 substitution mutants to oligonucleotides containing either an AP site or a pBQ adduct, we conclude that the absence of Asp-210 allows the formation of a stable HAP1-substrate complex that exists only transiently during the catalytic cycle of wild-type HAP1 protein. We interpret these data in the context of the structure of the HAP1 active site and the recently determined co-crystal structure of HAP1 bound to DNA substrates.
Subject(s)
Carbon-Oxygen Lyases/genetics , DNA-(Apurinic or Apyrimidinic Site) Lyase , Benzoquinones/metabolism , Binding Sites , Carbon-Oxygen Lyases/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Escherichia coli , Humans , Hydrogen Bonding , Kinetics , Models, Molecular , Mutation , Oligodeoxyribonucleotides/metabolism , Protein Structure, Secondary , Substrate SpecificityABSTRACT
Polycomb group (PcG) proteins were first described in Drosophila as factors responsible for maintaining the transcriptionally repressed state of Hox/homeotic genes in a stable and heritable manner throughout development. A growing number of vertebrate genes related to the Drosophila PcG proteins have recently been identified, including two Polycomb orthologues, Pc2 and M33. PcG proteins form multiprotein complexes, termed PcG bodies, that are thought to repress transcription by altering chromatin structure. Here we report the identification and characterization of HPC3 (human Polycomb 3), a novel PcG protein isolated in a yeast two-hybrid screen using human RING1 as bait. HPC3 shows strong sequence similarity to Drosophila Pc and also to vertebrate Pc2 and M33, particularly within the chromodomain and C-box. Previous studies indicate that M33 and human Pc2 (HPC2) can interact with RING1, and we show here that HPC3 also binds to RING1. This interaction is dependent upon the HPC3 C-box but, only partially on the RING finger of RING1. In contrast to HPC2, HPC3 interactions with RING1 are only observed in vivo with covalently modified forms of RING1. HPC3 also colocalizes with other PcG proteins in human PcG bodies. Consistent with its role as a PcG member, HPC3 is able to act as a long range transcriptional silencer when targeted to a reporter gene by a heterologous DNA-binding domain. Taken together, these data suggest that HPC3 is part of a large multiprotein complex that also contains other PcG proteins and is involved in repression of transcriptional activity.
Subject(s)
DNA-Binding Proteins/chemistry , Drosophila Proteins , Nuclear Proteins/chemistry , Proto-Oncogene Proteins/chemistry , Repressor Proteins/chemistry , Amino Acid Sequence , Binding Sites , DNA, Complementary/isolation & purification , Humans , Molecular Sequence Data , Polycomb Repressive Complex 1 , Repressor Proteins/genetics , Repressor Proteins/physiology , Sequence HomologyABSTRACT
The tumor-suppressive promyelocytic leukemia (PML) protein of acute promyelocytic leukemia (APL) has served as one of the defining components of a class of distinctive nuclear bodies (NBs). PML is delocalized from NBs in APL cells and is degraded in cells infected by several viruses. In these cells, NBs are disrupted, leading to the aberrant localization of NB proteins. These results have suggested a critical role for the NB in immune response and tumor suppression and raised the question of whether PML is crucial for the formation or stability of NB. In addition, PML is, among other proteins, covalently modified by SUMO-1. However, the functional relevance of this modification is unclear. Here, we show in primary PML(-/-) cells of various histologic origins, that in the absence of PML, several NB proteins such as Sp100, CBP, ISG20, Daxx, and SUMO-1 fail to accumulate in the NB and acquire aberrant localization patterns. Transfection of PML in PML(-/-) cells causes the relocalization of NB proteins. By contrast, a PML mutant that can no longer be modified by SUMO-1 fails to do so and displays an aberrant nuclear localization pattern. Therefore, PML is required for the proper formation of the NB. Conjugation to SUMO-1 is a prerequisite for PML to exert this function. These data shed new light on both the mechanisms underlying the formation of the NBs and the pathogenesis of APL. (Blood. 2000;95:2748-2752)
Subject(s)
Antigens, Nuclear , Cell Nucleus/metabolism , Exonucleases , Intracellular Signaling Peptides and Proteins , Keratinocytes/physiology , Lymphocytes/physiology , Neoplasm Proteins/metabolism , Transcription Factors/metabolism , Ubiquitins/metabolism , Animals , Apoptosis , Autoantigens/metabolism , Carrier Proteins/metabolism , Cells, Cultured , Co-Repressor Proteins , Exoribonucleases , Fibroblasts/cytology , Fibroblasts/physiology , Keratinocytes/cytology , Lymphocytes/cytology , Mice , Molecular Chaperones , Mutagenesis, Site-Directed , Neoplasm Proteins/genetics , Nuclear Proteins/metabolism , Promyelocytic Leukemia Protein , Recombinant Proteins/metabolism , SUMO-1 Protein , Transcription Factors/genetics , Tumor Suppressor ProteinsABSTRACT
Ubiquitination targets proteins for degradation and is a potent regulator of cellular protein function. Recent results implicate the RING finger domain in specific ubiquitination events; it is possible that all RING proteins act as E3 ubiquitin protein ligases, with implications for a variety of biological areas.
Subject(s)
Amino Acid Motifs , Proteins/metabolism , Ubiquitins/metabolism , Amino Acid Sequence , Proteins/chemistryABSTRACT
p97, an abundant hexameric ATPase of the AAA family, is involved in homotypic membrane fusion. It is thought to disassemble SNARE complexes formed during the process of membrane fusion. Here, we report two structures: a crystal structure of the N-terminal and D1 ATPase domains of murine p97 at 2.9 A resolution, and a cryoelectron microscopy structure of full-length rat p97 at 18 A resolution. Together, these structures show that the D1 and D2 hexamers pack in a tail-to-tail arrangement, and that the N domain is flexible. A comparison with NSF D2 (ATP complex) reveals possible conformational changes induced by ATP hydrolysis. Given the D1 and D2 packing arrangement, we propose a ratchet mechanism for p97 during its ATP hydrolysis cycle.
Subject(s)
Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/ultrastructure , Cryoelectron Microscopy , Nuclear Proteins/chemistry , Nuclear Proteins/ultrastructure , Saccharomyces cerevisiae Proteins , Vesicular Transport Proteins , Adenosine Diphosphate/metabolism , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Adenylyl Imidodiphosphate/metabolism , Amino Acid Sequence , Animals , Archaeal Proteins , Binding Sites , Carrier Proteins/chemistry , Crystallography, X-Ray , Fungal Proteins/chemistry , Membrane Fusion , Mice , Models, Molecular , Molecular Sequence Data , N-Ethylmaleimide-Sensitive Proteins , Nuclear Proteins/metabolism , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Peptide Fragments/ultrastructure , Pliability , Protein Structure, Secondary , Protein Structure, Tertiary , Rats , Sequence Alignment , Valosin Containing ProteinABSTRACT
The precise biochemical role of N-ethylmaleimide-sensitive factor (NSF) in membrane fusion mediated by SNARE proteins is unclear. To provide further insight into the function of NSF, we have introduced a mutation into mammalian NSF that, in Drosophila dNSF-1, leads to temperature-sensitive neuroparalysis. This mutation is like the comatose mutation and renders the mammalian NSF temperature sensitive for fusion of postmitotic Golgi vesicles and tubules into intact cisternae. Unexpectedly, at the temperature that is permissive for membrane fusion, this mutant NSF binds to, but cannot disassemble, SNARE complexes and exhibits almost no ATPase activity. A well-charaterized NSF mutant containing an inactivating point mutation in the catalytic site of its ATPase domain is equally active in the Golgi-reassembly assay. These data indicate that the need for NSF during postmitotic Golgi membrane fusion may be distinct from its ATPase-dependent ability to break up SNARE pairs.
Subject(s)
Adenosine Triphosphatases/physiology , Carrier Proteins/physiology , Ethylmaleimide/metabolism , Golgi Apparatus/physiology , Intracellular Membranes/physiology , Membrane Fusion/physiology , Membrane Proteins/metabolism , Vesicular Transport Proteins , Adenosine Triphosphatases/metabolism , Animals , CHO Cells , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cricetinae , Drosophila , Ethylmaleimide/pharmacology , Golgi Apparatus/metabolism , Mitosis , Mutagenesis, Site-Directed , N-Ethylmaleimide-Sensitive Proteins , Nucleotides , Protein Conformation , SNARE Proteins , TemperatureABSTRACT
beta-Glucosyltransferase (BGT) is a DNA-modifying enzyme encoded by bacteriophage T4 which catalyses the transfer of glucose (Glc) from uridine diphosphoglucose (UDP-Glc) to 5-hydroxymethylcytosine (5-HMC) in double-stranded DNA. The glucosylation of T4 phage DNA is part of a phage DNA protection system aimed at host nucleases. We previously reported the first three-dimensional structure of BGT determined from crystals grown in ammonium sulphate containing UDP-Glc. In this previous structure, we did not observe electron density for the Glc moiety of UDP-Glc nor for two large surface loop regions (residues 68-76 and 109-122). Here we report two further BGT co-crystal structures, in the presence of UDP product (form I) and donor substrate UDP-Glc (form II), respectively. Form I crystals are grown in ammonium sulphate and the structure has been determined to 1.88 A resolution (R -factor 19.1 %). Form II crystals are grown in polyethyleneglycol 4000 and the structure has been solved to 2.3 A resolution (R -factor 19.8 %). The form I structure is isomorphous to our previous BGT UDP-Glc structure. The form II structure, however, has allowed us to model the two missing surface loop regions and thus provides the first complete structural description of BGT. In this low-salt crystal form, we see no electron density for the Glc moiety from UDP-Glc similar to previous observations. Biochemical data however, shows that BGT can cleave UDP-Glc in the absence of DNA acceptor, which probably accounts for the absence of Glc in our UDP-Glc substrate structures. The complete BGT structure now provides a basis for detailed modelling of a BGT HMC-DNA ternary complex. By using the structural similarity between the catalytic core of glycogen phosphorylase (GP) and BGT, we have modelled the position of the Glc moiety in UDP-Glc. From these two models, we propose a catalytic mechanism for BGT and identify residues involved in both DNA binding and in stabilizing a "flipped-out" 5-HMC nucleotide.
Subject(s)
Bacteriophage T4/enzymology , Glucosyltransferases/chemistry , Binding Sites , DNA/chemistry , DNA, Viral/metabolism , DNA-Binding Proteins/chemistry , Glycosylation , Hydrogen Bonding , Models, Molecular , Molecular Conformation , Protein Binding , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Uridine Diphosphate/chemistry , Uridine Diphosphate Glucose/chemistryABSTRACT
The structure and fold of the enzyme responsible for the biosynthesis of the xenotransplantation antigen, namely pig alpha3 galactosyltransferase, has been studied by means of computational methods. Secondary structure predictions indicated that alpha3-galactosyltransferase and related protein family members, including blood group A and B transferases and Forssman synthase, are likely to consist of alternating alpha-helices and beta-strands. Fold recognition studies predicted that alpha3-galactosyltransferase shares the same fold as the T4 phage DNA-modifying enzyme beta-glucosyltransferase. This latter enzyme displays a strong structural resemblance with the core of glycogen phosphorylase b. By using the three-dimensional structure of beta-glucosyltransferase and of several glycogen phosphorylases, the nucleotide binding domain of pig alpha3-galactosyltransferase was built by knowledge-based methods. Both the UDP-galactose ligand and a divalent cation were included in the model during the refinement procedure. The final three-dimensional model is in agreement with our present knowledge of the biochemistry and mechanism of alpha3-galactosyltransferases.