ABSTRACT
The mammalian SID-1 transmembrane family members, SIDT1 and SIDT2, are multipass transmembrane proteins that mediate the cellular uptake and intracellular trafficking of nucleic acids, playing important roles in the immune response and tumorigenesis. Previous work has suggested that human SIDT1 and SIDT2 are N-glycosylated, but the precise site-specific N-glycosylation information and its functional contribution remain unclear. In this study, we use high-resolution liquid chromatography tandem mass spectrometry to comprehensively map the N-glycosites and quantify the N-glycosylation profiles of SIDT1 and SIDT2. Further molecular mechanistic probing elucidates the essential role of N-linked glycans in regulating cell surface expression, RNA binding, protein stability, and RNA uptake of SIDT1. Our results provide crucial information about the potential functional impact of N-glycosylation in the regulation of SIDT1-mediated RNA uptake and provide insights into the molecular mechanisms of this promising nucleic acid delivery system with potential implications for therapeutic applications.
Subject(s)
Nucleotide Transport Proteins , RNA , Humans , Biological Transport , Glycosylation , Mammals/metabolism , Membrane Proteins/metabolism , Nucleotide Transport Proteins/metabolism , RNA/metabolismABSTRACT
The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a key enzyme, which extensively digests CoV replicase polyproteins essential for viral replication and transcription, making it an attractive target for antiviral drug development. However, the molecular mechanism of how Mpro of SARS-CoV-2 digests replicase polyproteins, releasing the nonstructural proteins (nsps), and its substrate specificity remain largely unknown. Here, we determine the high-resolution structures of SARS-CoV-2 Mpro in its resting state, precleavage state, and postcleavage state, constituting a full cycle of substrate cleavage. The structures show the delicate conformational changes that occur during polyprotein processing. Further, we solve the structures of the SARS-CoV-2 Mpro mutant (H41A) in complex with six native cleavage substrates from replicase polyproteins, and demonstrate that SARS-CoV-2 Mpro can recognize sequences as long as 10 residues but only have special selectivity for four subsites. These structural data provide a basis to develop potent new inhibitors against SARS-CoV-2.
Subject(s)
Coronavirus 3C Proteases , Coronavirus RNA-Dependent RNA Polymerase , SARS-CoV-2 , Antiviral Agents/chemistry , Coronavirus 3C Proteases/chemistry , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Coronavirus RNA-Dependent RNA Polymerase/genetics , Polyproteins/chemistry , Protein Conformation , Proteolysis , SARS-CoV-2/enzymology , Substrate Specificity/geneticsABSTRACT
Guanylate-binding proteins (GBPs) form a family of dynamin-related large GTPases which mediate important innate immune functions. They were proposed to form oligomers upon GTP binding/hydrolysis, but the molecular mechanisms remain elusive. Here, we present crystal structures of C-terminally truncated human GBP5 (hGBP51-486), comprising the large GTPase (LG) and middle (MD) domains, in both its nucleotide-free monomeric and nucleotide-bound dimeric states, together with nucleotide-free full-length human GBP2. Upon GTP-loading, hGBP51-486 forms a closed face-to-face dimer. The MD of hGBP5 undergoes a drastic movement relative to its LG domain and forms extensive interactions with the LG domain and MD of the pairing molecule. Disrupting the MD interface (for hGBP5) or mutating the hinge region (for hGBP2/5) impairs their ability to inhibit HIV-1. Our results point to a GTP-induced dimerization mode that is likely conserved among all GBP members and provide insights into the molecular determinants of their antiviral function.
Subject(s)
GTP-Binding Proteins/chemistry , Protein Multimerization , Binding Sites , GTP-Binding Proteins/genetics , GTP-Binding Proteins/metabolism , Guanosine Triphosphate/chemistry , Guanosine Triphosphate/metabolism , HEK293 Cells , Humans , Molecular Dynamics Simulation , Protein Binding , env Gene Products, Human Immunodeficiency Virus/chemistry , env Gene Products, Human Immunodeficiency Virus/metabolismABSTRACT
Wolbachia bacteria, inherited through the female germ line, infect a large fraction of arthropod species. Many Wolbachia strains manipulate host reproduction, most commonly through cytoplasmic incompatibility (CI). CI, a conditional male sterility, results when Wolbachia-infected male insects mate with uninfected females; viability is restored if the female is similarly infected (called "rescue"). CI is used to help control mosquito-borne viruses such as dengue and Zika, but its mechanisms remain unknown. The coexpressed CI factors CifA and CifB form stable complexes in vitro, but the timing and function of this interaction in the insect are unresolved. CifA expression in the female germ line is sufficient for rescue. We report high-resolution structures of a CI-factor complex, CinA-CinB, which utilizes a unique binding mode between the CinA rescue factor and the CinB nuclease; the structures were validated by biochemical and yeast growth analyses. Importantly, transgenic expression in Drosophila of a nonbinding CinA mutant, designed based on the CinA-CinB structure, suggests CinA expressed in females must bind CinB imported by sperm in order to rescue embryonic viability. Binding between cognate factors is conserved in an enzymatically distinct CI system, CidA-CidB, suggesting universal features in Wolbachia CI induction and rescue.
Subject(s)
Drosophila melanogaster/microbiology , Embryo, Nonmammalian/embryology , Infertility, Male/physiopathology , Reproduction/physiology , Wolbachia/metabolism , Animals , Animals, Genetically Modified , Drosophila melanogaster/genetics , Embryonic Development , Female , Male , Mosquito Control/methods , Multiprotein Complexes/metabolism , Protein Binding , Symbiosis , Vector Borne Diseases/prevention & control , Vector Borne Diseases/transmission , Vector Borne Diseases/virologyABSTRACT
The COVID-19 pandemic, caused by SARS-CoV-2, has led to over 750 million infections and 6.8 million deaths worldwide since late 2019. Due to the continuous evolution of SARS-CoV-2, many significant variants have emerged, creating ongoing challenges to the prevention and treatment of the pandemic. Therefore, the study of antibody responses against SARS-CoV-2 is essential for the development of vaccines and therapeutics. Here we perform single particle cryo-electron microscopy (cryo-EM) structure determination of a rabbit monoclonal antibody (RmAb) 9H1 in complex with the SARS-CoV-2 wild-type (WT) spike trimer. Our structural analysis shows that 9H1 interacts with the receptor-binding motif (RBM) region of the receptor-binding domain (RBD) on the spike protein and by directly competing with angiotensin-converting enzyme 2 (ACE2), it blocks the binding of the virus to the receptor and achieves neutralization. Our findings suggest that utilizing rabbit-derived mAbs provides valuable insights into the molecular interactions between neutralizing antibodies and spike proteins and may also facilitate the development of therapeutic antibodies and expand the antibody library.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/metabolism , Antibodies, Monoclonal , Pandemics , Cryoelectron Microscopy , Antibodies, Viral , Receptors, Virus/metabolism , Antibodies, Neutralizing , Protein Binding , Spike Glycoprotein, Coronavirus/chemistryABSTRACT
BACKGROUND: Sepsis-caused multi-organ failure remains the major cause of morbidity and mortality in intensive care units with limited therapeutics. Nicotinamide mononucleotide (NMN), a precursor of nicotinamide adenine dinucleotide (NAD+), has been recently reported to be protective in sepsis; however, its therapeutic effects remain to be determined. This study sought to investigate the therapeutic effects of NMN in septic organ failure and its underlying mechanisms. METHODS: Sepsis was induced by feces-injection-in-peritoneum in mice. NMN was given after an hour of sepsis onset. Cultured neutrophils, macrophages and endothelial cells were incubated with various agents. RESULTS: We demonstrate that administration of NMN elevated NAD+ levels and reduced serum lactate levels, oxidative stress, inflammation, and caspase-3 activity in multiple organs of septic mice, which correlated with the attenuation of heart dysfunction, pulmonary microvascular permeability, liver injury, and kidney dysfunction, leading to lower mortality. The therapeutic effects of NMN were associated with lower bacterial burden in blood, and less ROS production in septic mice. NMN improved bacterial phagocytosis and bactericidal activity of macrophages and neutrophils while reducing the lipopolysaccharides-induced inflammatory response of macrophages. In cultured endothelial cells, NMN mitigated mitochondrial dysfunction, inflammation, apoptosis, and barrier dysfunction induced by septic conditions, all of which were offset by SIRT3 inhibition. CONCLUSION: NAD+ repletion with NMN prevents mitochondrial dysfunction and restrains bacterial dissemination while limiting inflammatory damage through SIRT3 signaling in sepsis. Thus, NMN may represent a therapeutic option for sepsis.
Subject(s)
Mitochondrial Diseases , Sepsis , Sirtuin 3 , Mice , Animals , NAD , Nicotinamide Mononucleotide/pharmacology , Nicotinamide Mononucleotide/therapeutic use , Endothelial Cells , Inflammation/complications , Inflammation/drug therapy , Sepsis/complications , Sepsis/drug therapyABSTRACT
RNA 2'-O-methylation is widely distributed and plays important roles in various cellular processes. Mycoplasma genitalium RNase R (MgR), a prokaryotic member of the RNase II/RNB family, is a 3'-5' exoribonuclease and is particularly sensitive to RNA 2'-O-methylation. However, how RNase R interacts with various RNA species and exhibits remarkable sensitivity to substrate 2'-O-methyl modifications remains elusive. Here we report high-resolution crystal structures of MgR in apo form and in complex with various RNA substrates. The structural data together with extensive biochemical analysis quantitively illustrate MgR's ribonuclease activity and significant sensitivity to RNA 2'-O-methylation. Comparison to its related homologs reveals an exquisite mechanism for the recognition and degradation of RNA substrates. Through structural and mutagenesis studies, we identified proline 277 to be responsible for the significant sensitivity of MgR to RNA 2'-O-methylation within the RNase II/RNB family. We also generated several MgR variants with modulated activities. Our work provides a mechanistic understanding of MgR activity that can be harnessed as a powerful RNA analytical tool that will open up a new venue for RNA 2'-O-methylations research in biological and clinical samples.
Subject(s)
Exoribonucleases/chemistry , Mycoplasma genitalium/chemistry , RNA/chemistry , Ribose/metabolism , Catalysis , Catalytic Domain/genetics , Chromatography, Liquid , Crystallography, X-Ray , Escherichia coli/metabolism , Exoribonucleases/metabolism , Methylation , Mutagenesis , Mutation , Mycoplasma genitalium/enzymology , Protein Binding , Protein Domains , RNA/metabolism , RNA Stability , Recombinant Proteins , Substrate Specificity , Tandem Mass SpectrometryABSTRACT
BACKGROUND: Cardiomyocyte death contributes to cardiac pathology of diabetes. Studies have shown that the RIPK3/MLKL necroptosis signaling is activated in diabetic hearts. Deletion of RIPK3 was reported to attenuate myocardial injury and heart dysfunction in streptozocin (STZ)-induced diabetic mice, suggesting a potential role of necroptosis in diabetic cardiomyopathy. This study characterized cardiomyocyte necroptosis in diabetic hearts and investigated whether MLKL-mediated necroptosis is a target for cardiac protection in diabetes. METHODS: Type 1 diabetes was induced in RIPK3 knockout, MLKL knockout and wild-type mice. Akita Type-1 diabetic mice were injected with shRNA for MLKL. Myocardial function was assessed by echocardiography. Immuno-histological analyses determined cardiomyocyte death and fibrosis in the heart. Cultured adult mouse cardiomyocytes were incubated with high glucose in the presence of various drugs. Cell death and phosphorylation of RIPK3 and MLKL were analysed. RESULTS: We showed that the levels of phosphorylated RIPK3 and MLKL were higher in high glucose-stimulated cardiomyocytes and hearts of STZ-induced type-1 diabetic mice, akita mice and type-1 diabetic monkeys when compared to non-diabetic controls. Inhibition of RIPK3 by its pharmacological inhibitor or gene deletion, or MLKL deletion prevented high glucose-induced MLKL phosphorylation and attenuated necroptosis in cardiomyocytes. In STZ-induced type-1 diabetic mice, cardiomyocyte necroptosis was present along with elevated cardiac troponin I in serum and MLKL oligomerization, and co-localized with phosphorylated MLKL. Deletion of RIPK3 or MLKL prevented MLKL phosphorylation and cardiac necroptosis, attenuated serum cardiac troponin I levels, reduced myocardial collagen deposition and improved myocardial function in STZ-injected mice. Additionally, shRNA-mediated down-regulation of MLKL reduced cardiomyocyte necroptosis in akita mice. Interestingly, incubation with anti-diabetic drugs (empagliflozin and metformin) prevented phosphorylation of RIPK3 and MLKL, and reduced cell death in high glucose-induced cardiomyocytes. CONCLUSIONS: We have provided evidence that cardiomyocyte necroptosis is present in diabetic hearts and that MLKL-mediated cardiomyocyte necroptosis contributes to diabetic cardiomyopathy. These findings highlight MLKL-mediated necroptosis as a target for cardiac protection in diabetes.
Subject(s)
Diabetes Mellitus, Experimental , Diabetes Mellitus, Type 1 , Diabetic Cardiomyopathies , Necroptosis , Protein Kinases , Animals , Diabetes Mellitus, Experimental/pathology , Diabetes Mellitus, Type 1/pathology , Diabetic Cardiomyopathies/pathology , Disease Models, Animal , Glucose , Mice , Protein Kinases/metabolism , RNA, Small Interfering , Troponin IABSTRACT
Calpains have been implicated in heart diseases. While calpain-1 has been detrimental to the heart, the role of calpain-2 in cardiac pathology remains controversial. In this study we investigated whether sustained over-expression of calpain-2 had any adverse effects on the heart and the underlying mechanisms. Double transgenic mice (Tg-Capn2/tTA) were generated, which express human CAPN2 restricted to cardiomyocytes. The mice were subjected to echocardiography at age 3, 6, 8 and 12 months, and their heart tissues and sera were collected for analyses. We showed that transgenic mice over-expressing calpain-2 restricted to cardiomyocytes had normal heart function with no evidence of cardiac pathological remodeling at age 3 months. However, they exhibited features of dilated cardiomyopathy including increased heart size, enlarged heart chambers and heart dysfunction from age 8 months; histological analysis revealed loss of cardiomyocytes replaced by myocardial fibrosis and cardiomyocyte hypertrophy in transgenic mice from age 8 months. These cardiac alterations closely correlated with aberrant autophagy evidenced by significantly increased LC3BII and p62 protein levels and accumulation of autophagosomes in the hearts of transgenic mice. Notably, injection of 3-methyladenine, a well-established inhibitor of autophagy (30 mg/kg, i.p. once every 3 days starting from age 6 months for 2 months) prevented aberrant autophagy, attenuated myocardial injury and improved heart function in the transgenic mice. In cultured cardiomyocytes, over-expression of calpain-2 blocked autophagic flux by impairing lysosomal function. Furthermore, over-expression of calpain-2 resulted in lower levels of junctophilin-2 protein in the heart of transgenic mice and in cultured cardiomyocytes, which was attenuated by 3-methyladenine. In addition, blockade of autophagic flux by bafilomycin A (100 nM) induced a reduction of junctophilin-2 protein in cardiomyocytes. In summary, transgenic over-expression of calpain-2 induces age-dependent dilated cardiomyopathy in mice, which may be mediated through aberrant autophagy and a reduction of junctophilin-2. Thus, a sustained increase in calpain-2 may be detrimental to the heart.
Subject(s)
Cardiomyopathy, Dilated , Mice , Animals , Humans , Infant , Cardiomyopathy, Dilated/metabolism , Cardiomyopathy, Dilated/pathology , Calpain , Myocytes, Cardiac , Autophagy , Mice, TransgenicABSTRACT
Lipid overload contributes to cardiac complications of diabetes and obesity. However, the underlying mechanisms remain obscure. This study investigates the role of gamma-aminobutyrate transaminase (ABAT), the key enzyme involved in the catabolism of γ-aminobutyric acid (GABA), in lipid overload-induced cardiac injury. Microarray revealed a down-regulation of ABAT mRNA expression in high fat diet (HFD)-fed mouse hearts, which correlated with a reduction in ABAT protein level and its GABA catabolic activity. Transgenic mice with cardiomyocyte-specific ABAT over-expression (Tg-ABAT/tTA) were generated to determine the role of ABAT in lipid overload-induced cardiac injury. Feeding with a HFD to control mice for 4 months reduced ATP production and the mitochondrial DNA copy number, and induced myocardial oxidative stress, hypertrophy, fibrosis and dysfunction. Such pathological effects of HFD were mitigated by ABAT over-expression in Tg-ABAT/tTA mice. In cultured cardiomyocytes, palmitate increased mitochondrial ROS production, depleted ATP production and promoted apoptosis, all of which were attenuated by ABAT over-expression. With the inhibition of ABAT's GABA catabolic activity, the protective effects of ABAT remained unchanged in palmitate-induced cardiomyocytes. Thus, ABAT protects the mitochondrial function in defending the heart against lipid overload-induced injury through mechanisms independent of its GABA catabolic activity, and may represent a new therapeutic target for lipid overload-induced cardiac injury.
Subject(s)
4-Aminobutyrate Transaminase , Heart Injuries , 4-Aminobutyrate Transaminase/genetics , 4-Aminobutyrate Transaminase/metabolism , Adenosine Triphosphate/metabolism , Animals , Diet, High-Fat/adverse effects , Heart Injuries/etiology , Mice , Mice, Inbred C57BL , Myocytes, Cardiac/metabolism , Palmitates/metabolism , gamma-Aminobutyric Acid/metabolismABSTRACT
SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase (dNTPase) that depletes cellular dNTPs in noncycling cells to promote genome stability and to inhibit retroviral and herpes viral replication. In addition to being substrates, cellular nucleotides also allosterically regulate SAMHD1 activity. Recently, it was shown that high expression levels of SAMHD1 are also correlated with significantly worse patient responses to nucleotide analog drugs important for treating a variety of cancers, including acute myeloid leukemia (AML). In this study, we used biochemical, structural, and cellular methods to examine the interactions of various cancer drugs with SAMHD1. We found that both the catalytic and the allosteric sites of SAMHD1 are sensitive to sugar modifications of the nucleotide analogs, with the allosteric site being significantly more restrictive. We crystallized cladribine-TP, clofarabine-TP, fludarabine-TP, vidarabine-TP, cytarabine-TP, and gemcitabine-TP in the catalytic pocket of SAMHD1. We found that all of these drugs are substrates of SAMHD1 and that the efficacy of most of these drugs is affected by SAMHD1 activity. Of the nucleotide analogs tested, only cladribine-TP with a deoxyribose sugar efficiently induced the catalytically active SAMHD1 tetramer. Together, these results establish a detailed framework for understanding the substrate specificity and allosteric activation of SAMHD1 with regard to nucleotide analogs, which can be used to improve current cancer and antiviral therapies.
Subject(s)
Allosteric Site/drug effects , Catalytic Domain/drug effects , Drug Interactions/physiology , Leukemia, Myeloid, Acute/metabolism , SAM Domain and HD Domain-Containing Protein 1/metabolism , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Crystallography, X-Ray/methods , HL-60 Cells , Humans , Leukemia, Myeloid, Acute/drug therapy , Nucleotides/pharmacology , Substrate SpecificityABSTRACT
Asthma is a disease of the respiratory system that is commonly considered a T-helper 2 (Th2) cell-associated inflammatory disease. Group 2 innate lymphoid cells (ILC2s) promote the inflammatory responses in asthma by secreting type 2 cytokines. Interleukin (IL)-9 also serves as a promoting factor in asthma and it is well known that ILC2s have an autocrine effect of IL-9 to sustain their survival and proliferation. However, the specific role of ILC2-derived IL-9 in asthma remains unclear. HMGB1 (High-Mobility Group Box-1) is a nuclear protein, and Previous studies have shown that HMGB1 can regulate the differentiation of T-helper cells and participate in the development of asthma. But whether HMGB1 can regulate the innate lymphocytes in the pathological process of asthma is unknown. In this study we have shown increased presence of HMGB1 protein in the lung of mice with asthma, which was associated with increased secretion of IL-9 by ILC2s. This led to the activation of dendritic cells (DCs) that can accelerate the differentiation of Th2 cells and worsen the severity of asthma. Taken together, our study provides a complementary understanding of the asthma development and highlights a novel inflammatory pathway in the pathogenesis of asthma.
Subject(s)
Asthma/metabolism , HMGB1 Protein/metabolism , Lymphocytes/immunology , Animals , Asthma/physiopathology , Cell Differentiation/drug effects , Cytokines/metabolism , Dendritic Cells/metabolism , Disease Models, Animal , Female , HMGB Proteins/metabolism , HMGB Proteins/physiology , HMGB1 Protein/physiology , Immunity, Innate/drug effects , Interleukin-9/immunology , Interleukin-9/metabolism , Lung/immunology , Male , Mice , Mice, Inbred BALB C , Th2 Cells/immunologyABSTRACT
Interleukin (IL)-9 belongs to the IL-2Rγc chain family and is a multifunctional cytokine that can regulate the function of many kinds of cells. It was originally identified as a growth factor of T cells and mast cells. In previous studies, IL-9 was mainly involved in the development of allergic diseases, autoimmune diseases and parasite infections. Recently, IL-9, as a double-edged sword in the development of cancers, has attracted extensive attention. Since T-helper 9 (Th9) cell-derived IL-9 was verified to play a powerful antitumor role in solid tumors, an increasing number of researchers have started to pay attention to the role of IL-9-skewed CD8+ T (Tc9) cells, mast cells and Vδ2 T cell-derived IL-9 in tumor immunity. Here, we review recent studies on IL-9 and several kinds of IL-9-producing cells in tumor immunity to provide useful insight into tumorigenesis and treatment. Video Abstract.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Interleukin-9/immunology , Mast Cells/immunology , Neoplasms/immunology , T-Lymphocytes, Helper-Inducer/immunology , Animals , CD8-Positive T-Lymphocytes/cytology , Humans , Mast Cells/cytology , T-Lymphocytes, Helper-Inducer/cytologyABSTRACT
During its life cycle, Zika virus (ZIKV), an arthropod-borne flavivirus that is associated with Guillain-Barré syndrome and causes microencephaly in fetuses and newborn children, encodes a critical and indispensable helicase domain that has 5'-triphosphatase activity and performs ATP hydrolysis to generate energy and thus, sustains unwinding of double-stranded RNA during ZIKV genome replication. Of these processes, ATP hydrolysis represents the most basic event; however, its dynamic mechanisms remain largely unknown, impeding the further understanding of the function of ZIKV helicase and the ongoing anti-ZIKV drug design. In this work, we determined the crystal structure of ZIKV helicase in complex with ADP-AlF3-Mn2+ and ADP-Mn2+ separately. The structural analysis indicates that these structures represent the intermediate state and posthydrolysis state, respectively, of the ATP hydrolysis process of ZIKV helicase. These findings, together with our earlier work, which identified the prehydrolysis state of ZIKV helicase, lead to a proposal of the ATP hydrolysis cycle for ZIKV helicase. On this basis, we used site-directed mutagenesis combined with an enzymatic study to identify successfully residues that are critical for the ATPase activity of ZIKV helicase; this will provide new ideas to understand the function for the key enzyme of ZIKV.-Yang, X., Chen, C., Tian, H., Chi, H., Mu, Z., Zhang, T., Yang, K., Zhao, Q., Liu, X., Wang, Z., Ji, X., Yang, H. Mechanism of ATP hydrolysis by the Zika virus helicase.
Subject(s)
Adenosine Triphosphate/chemistry , RNA Helicases/chemistry , Viral Proteins/chemistry , Zika Virus/enzymology , Adenosine Triphosphate/metabolism , Crystallography, X-Ray , Hydrolysis , RNA Helicases/genetics , RNA Helicases/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism , Zika Virus/geneticsABSTRACT
The sterile alpha motif and HD domain-containing protein 1 (SAMHD1), a dNTPase, prevents the infection of nondividing cells by retroviruses, including HIV, by depleting the cellular dNTP pool available for viral reverse transcription. SAMHD1 is a major regulator of cellular dNTP levels in mammalian cells. Mutations in SAMHD1 are associated with chronic lymphocytic leukemia (CLL) and the autoimmune condition Aicardi Goutières syndrome (AGS). The dNTPase activity of SAMHD1 can be regulated by dGTP, with which SAMHD1 assembles into catalytically active tetramers. Here we present extensive biochemical and structural data that reveal an exquisite activation mechanism of SAMHD1 via combined action of both GTP and dNTPs. We obtained 26 crystal structures of SAMHD1 in complex with different combinations of GTP and dNTP mixtures, which depict the full spectrum of GTP/dNTP binding at the eight allosteric and four catalytic sites of the SAMHD1 tetramer. Our data demonstrate how SAMHD1 is activated by binding of GTP or dGTP at allosteric site 1 and a dNTP of any type at allosteric site 2. Our enzymatic assays further reveal a robust regulatory mechanism of SAMHD1 activity, which bares resemblance to that of the ribonuclease reductase responsible for cellular dNTP production. These results establish a complete framework for a mechanistic understanding of the important functions of SAMHD1 in the regulation of cellular dNTP levels, as well as in HIV restriction and the pathogenesis of CLL and AGS.
Subject(s)
Monomeric GTP-Binding Proteins/chemistry , Monomeric GTP-Binding Proteins/metabolism , Nucleotides/chemistry , Nucleotides/metabolism , Allosteric Site , Catalytic Domain , Crystallography, X-Ray , Guanosine Triphosphate/chemistry , Guanosine Triphosphate/metabolism , Humans , Hydrolysis , Kinetics , Ligands , Models, Molecular , Protein Multimerization , Ribonucleotide Reductases/metabolism , SAM Domain and HD Domain-Containing Protein 1 , Substrate SpecificityABSTRACT
SAMHD1 is a cellular protein that plays key roles in HIV-1 restriction and regulation of cellular dNTP levels. Mutations in SAMHD1 are also implicated in the pathogenesis of chronic lymphocytic leukemia and Aicardi-Goutières syndrome. The anti-HIV-1 activity of SAMHD1 is negatively modulated by phosphorylation at residue Thr-592. The mechanism underlying the effect of phosphorylation on anti-HIV-1 activity remains unclear. SAMHD1 forms tetramers that possess deoxyribonucleotide triphosphate triphosphohydrolase (dNTPase) activity, which is allosterically controlled by the combined action of GTP and all four dNTPs. Here we demonstrate that the phosphomimetic mutation T592E reduces the stability of the SAMHD1 tetramer and the dNTPase activity of the enzyme. To better understand the underlying mechanisms, we determined the crystal structures of SAMHD1 variants T592E and T592V. Although the neutral substitution T592V does not perturb the structure, the charged T592E induces large conformational changes, likely triggered by electrostatic repulsion from a distinct negatively charged environment surrounding Thr-592. The phosphomimetic mutation results in a significant decrease in the population of active SAMHD1 tetramers, and hence the dNTPase activity is substantially decreased. These results provide a mechanistic understanding of how SAMHD1 phosphorylation at residue Thr-592 may modulate its cellular and antiviral functions.
Subject(s)
Amino Acid Substitution , Monomeric GTP-Binding Proteins/chemistry , Mutation, Missense , Enzyme Stability/genetics , Humans , SAM Domain and HD Domain-Containing Protein 1 , Substrate Specificity/geneticsABSTRACT
Polyhedra represent an ancient system used by a number of insect viruses to protect virions during long periods of environmental exposure. We present high resolution crystal structures of polyhedra for seven previously uncharacterised types of cypoviruses, four using ab initio selenomethionine phasing (two of these required over 100 selenomethionine crystals each). Approximately 80% of residues are structurally equivalent between all polyhedrins (pairwise rmsd ⩽ 1.5 Å), whilst pairwise sequence identities, based on structural alignment, are as little as 12%. These structures illustrate the effect of 400 million years of evolution on a system where the crystal lattice is the functionally conserved feature in the face of massive sequence variability. The conservation of crystal contacts is maintained across most of the molecular surface, except for a dispensable virus recognition domain. By spreading the contacts over so much of the protein surface the lattice remains robust in the face of many individual changes. Overall these unusual structural constraints seem to have skewed the molecule's evolution so that surface residues are almost as conserved as the internal residues.
Subject(s)
Insect Viruses/ultrastructure , Viral Structural Proteins/chemistry , Amino Acid Sequence , Binding Sites , Conserved Sequence , Cytidine Triphosphate/chemistry , Evolution, Molecular , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Quaternary , Protein Structure, Secondary , Viral Structural Proteins/ultrastructureABSTRACT
Tripartite motif protein isoform 5 alpha (TRIM5α) is a potent antiviral protein that restricts infection by HIV-1 and other retroviruses. TRIM5α recognizes the lattice of the retrovirus capsid through its B30.2 (PRY/SPRY) domain in a species-specific manner. Upon binding, TRIM5α induces premature disassembly of the viral capsid and activates the downstream innate immune response. We have determined the crystal structure of the rhesus TRIM5α PRY/SPRY domain that reveals essential features for capsid binding. Combined cryo-electron microscopy and biochemical data show that the monomeric rhesus TRIM5α PRY/SPRY, but not the human TRIM5α PRY/SPRY, can bind to HIV-1 capsid protein assemblies without causing disruption of the capsid. This suggests that the PRY/SPRY domain alone constitutes an important pattern-sensing component of TRIM5α that is capable of interacting with viral capsids of different curvatures. Our results provide molecular insights into the mechanisms of TRIM5α-mediated retroviral restriction.
Subject(s)
Capsid/chemistry , Capsid/metabolism , Carrier Proteins/chemistry , Carrier Proteins/metabolism , HIV-1/chemistry , Macaca mulatta/metabolism , Amino Acid Sequence , Animals , Conserved Sequence , Crystallography, X-Ray , Evolution, Molecular , Humans , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Multimerization , Protein Structure, Tertiary , SolutionsABSTRACT
This work demonstrates that with the use of a microfocus synchrotron beam the structure of a novel viral polyhedrin could be successfully determined from microcrystals within cells, removing the preparatory step of sample isolation and maintaining a favourable biological environment. The data obtained are of high quality, comparable to that obtained from isolated crystals, and enabled a facile structure determination. A small but significant difference is observed between the unit-cell parameters and the mosaic spread of in cellulo and isolated crystals, suggesting that even these robust crystals are adversely affected by removal from the cell.
Subject(s)
Crystallography, X-Ray/methods , Reoviridae/chemistry , Viral Proteins/chemistry , Crystallography, X-Ray/instrumentation , Models, Molecular , Protein Conformation , SynchrotronsABSTRACT
Natural protein crystals (polyhedra) armour certain viruses, allowing them to survive for years under hostile conditions. We have determined the structure of polyhedra of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), revealing a highly symmetrical covalently cross-braced robust lattice, the subunits of which possess a flexible adaptor enabling this supra-molecular assembly to specifically entrap massive baculoviruses. Inter-subunit chemical switches modulate the controlled release of virus particles in the unusual high pH environment of the target insect's gut. Surprisingly, the polyhedrin subunits are more similar to picornavirus coat proteins than to the polyhedrin of cytoplasmic polyhedrosis virus (CPV). It is, therefore, remarkable that both AcMNPV and CPV polyhedra possess identical crystal lattices and crystal symmetry. This crystalline arrangement must be particularly well suited to the functional requirements of the polyhedra and has been either preserved or re-selected during evolution. The use of flexible adaptors to generate a powerful system for packaging irregular particles is characteristic of the AcMNPV polyhedrin and may provide a vehicle to sequester a wide range of objects such as biological nano-particles.