ABSTRACT
Microbial hydrogen (H2) cycling underpins the diversity and functionality of diverse anoxic ecosystems. Among the three evolutionarily distinct hydrogenase superfamilies responsible, [FeFe] hydrogenases were thought to be restricted to bacteria and eukaryotes. Here, we show that anaerobic archaea encode diverse, active, and ancient lineages of [FeFe] hydrogenases through combining analysis of existing and new genomes with extensive biochemical experiments. [FeFe] hydrogenases are encoded by genomes of nine archaeal phyla and expressed by H2-producing Asgard archaeon cultures. We report an ultraminimal hydrogenase in DPANN archaea that binds the catalytic H-cluster and produces H2. Moreover, we identify and characterize remarkable hybrid complexes formed through the fusion of [FeFe] and [NiFe] hydrogenases in ten other archaeal orders. Phylogenetic analysis and structural modeling suggest a deep evolutionary history of hybrid hydrogenases. These findings reveal new metabolic adaptations of archaea, streamlined H2 catalysts for biotechnological development, and a surprisingly intertwined evolutionary history between the two major H2-metabolizing enzymes.
Subject(s)
Archaea , Hydrogen , Hydrogenase , Phylogeny , Archaea/genetics , Archaea/enzymology , Archaeal Proteins/metabolism , Archaeal Proteins/chemistry , Archaeal Proteins/genetics , Genome, Archaeal , Hydrogen/metabolism , Hydrogenase/metabolism , Hydrogenase/genetics , Hydrogenase/chemistry , Iron-Sulfur Proteins/metabolism , Iron-Sulfur Proteins/genetics , Iron-Sulfur Proteins/chemistry , Models, Molecular , Protein Structure, TertiaryABSTRACT
Thermostable clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas9) enzymes could improve genome-editing efficiency and delivery due to extended protein lifetimes. However, initial experimentation demonstrated Geobacillus stearothermophilus Cas9 (GeoCas9) to be virtually inactive when used in cultured human cells. Laboratory-evolved variants of GeoCas9 overcome this natural limitation by acquiring mutations in the wedge (WED) domain that produce >100-fold-higher genome-editing levels. Cryoelectron microscopy (cryo-EM) structures of the wild-type and improved GeoCas9 (iGeoCas9) enzymes reveal extended contacts between the WED domain of iGeoCas9 and DNA substrates. Biochemical analysis shows that iGeoCas9 accelerates DNA unwinding to capture substrates under the magnesium-restricted conditions typical of mammalian but not bacterial cells. These findings enabled rational engineering of other Cas9 orthologs to enhance genome-editing levels, pointing to a general strategy for editing enzyme improvement. Together, these results uncover a new role for the Cas9 WED domain in DNA unwinding and demonstrate how accelerated target unwinding dramatically improves Cas9-induced genome-editing activity.
Subject(s)
CRISPR-Associated Protein 9 , CRISPR-Cas Systems , Cryoelectron Microscopy , DNA , Gene Editing , Humans , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/chemistry , CRISPR-Associated Protein 9/metabolism , CRISPR-Associated Protein 9/genetics , CRISPR-Cas Systems/genetics , DNA/metabolism , DNA/genetics , Gene Editing/methods , Geobacillus stearothermophilus/genetics , Geobacillus stearothermophilus/metabolism , HEK293 Cells , Protein Domains , Genome, Human , Models, Molecular , Protein Structure, Tertiary , Nucleic Acid Conformation , Biocatalysis , Magnesium/chemistry , Magnesium/metabolismABSTRACT
The cleavage of zygotes generates totipotent blastomeres. In human 8-cell blastomeres, zygotic genome activation (ZGA) occurs to initiate the ontogenesis program. However, capturing and maintaining totipotency in human cells pose significant challenges. Here, we realize culturing human totipotent blastomere-like cells (hTBLCs). We find that splicing inhibition can transiently reprogram human pluripotent stem cells into ZGA-like cells (ZLCs), which subsequently transition into stable hTBLCs after long-term passaging. Distinct from reported 8-cell-like cells (8CLCs), both ZLCs and hTBLCs widely silence pluripotent genes. Interestingly, ZLCs activate a particular group of ZGA-specific genes, and hTBLCs are enriched with pre-ZGA-specific genes. During spontaneous differentiation, hTBLCs re-enter the intermediate ZLC stage and further generate epiblast (EPI)-, primitive endoderm (PrE)-, and trophectoderm (TE)-like lineages, effectively recapitulating human pre-implantation development. Possessing both embryonic and extraembryonic developmental potency, hTBLCs can autonomously generate blastocyst-like structures in vitro without external cell signaling. In summary, our study provides key criteria and insights into human cell totipotency.
Subject(s)
Cell Differentiation , Spliceosomes , Animals , Humans , Mice , Blastocyst/metabolism , Blastocyst/cytology , Blastomeres/metabolism , Blastomeres/cytology , Cellular Reprogramming , Embryonic Development/genetics , Germ Layers/metabolism , Germ Layers/cytology , Pluripotent Stem Cells/metabolism , Pluripotent Stem Cells/cytology , RNA Splicing , Spliceosomes/metabolism , Totipotent Stem Cells/metabolism , Totipotent Stem Cells/cytology , Zygote/metabolism , Cells, Cultured , Models, Molecular , Protein Structure, Tertiary , Genome, Human , Single-Cell Analysis , Growth Differentiation Factor 15/chemistry , Growth Differentiation Factor 15/genetics , Growth Differentiation Factor 15/metabolism , Epigenomics , Cell LineageABSTRACT
Antibodies are crucial to immune protection against SARS-CoV-2, with some in emergency use as therapeutics. Here, we identify 377 human monoclonal antibodies (mAbs) recognizing the virus spike and focus mainly on 80 that bind the receptor binding domain (RBD). We devise a competition data-driven method to map RBD binding sites. We find that although antibody binding sites are widely dispersed, neutralizing antibody binding is focused, with nearly all highly inhibitory mAbs (IC50 < 0.1 µg/mL) blocking receptor interaction, except for one that binds a unique epitope in the N-terminal domain. Many of these neutralizing mAbs use public V-genes and are close to germline. We dissect the structural basis of recognition for this large panel of antibodies through X-ray crystallography and cryoelectron microscopy of 19 Fab-antigen structures. We find novel binding modes for some potently inhibitory antibodies and demonstrate that strongly neutralizing mAbs protect, prophylactically or therapeutically, in animal models.
Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Binding Sites, Antibody , CHO Cells , Chlorocebus aethiops , Cricetulus , Epitopes , Female , HEK293 Cells , Humans , Male , Mice , Mice, Transgenic , Models, Molecular , Protein Binding , Protein Structure, Tertiary , SARS-CoV-2/immunology , Vero CellsABSTRACT
Pathogenic clostridial species secrete potent toxins that induce severe host tissue damage. Paeniclostridium sordellii lethal toxin (TcsL) causes an almost invariably lethal toxic shock syndrome associated with gynecological infections. TcsL is 87% similar to C. difficile TcdB, which enters host cells via Frizzled receptors in colon epithelium. However, P. sordellii infections target vascular endothelium, suggesting that TcsL exploits another receptor. Here, using CRISPR/Cas9 screening, we establish semaphorins SEMA6A and SEMA6B as TcsL receptors. We demonstrate that recombinant SEMA6A can protect mice from TcsL-induced edema. A 3.3 Å cryo-EM structure shows that TcsL binds SEMA6A with the same region that in TcdB binds structurally unrelated Frizzled. Remarkably, 15 mutations in this evolutionarily divergent surface are sufficient to switch binding specificity of TcsL to that of TcdB. Our findings establish semaphorins as physiologically relevant receptors for TcsL and reveal the molecular basis for the difference in tissue targeting and disease pathogenesis between highly related toxins.
Subject(s)
Bacterial Toxins/metabolism , Clostridium sordellii/metabolism , Semaphorins/metabolism , Animals , Bacterial Toxins/chemistry , Bacterial Toxins/toxicity , Binding Sites , CRISPR-Cas Systems/genetics , Cell Line , Cryoelectron Microscopy , Edema/pathology , Edema/prevention & control , Female , Humans , Lung/drug effects , Lung/pathology , Mice , Mice, Inbred C57BL , Molecular Dynamics Simulation , Mutagenesis, Site-Directed , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Recombinant Proteins/therapeutic use , Semaphorins/chemistry , Semaphorins/geneticsABSTRACT
Group II introns are a class of retroelements that invade DNA through a copy-and-paste mechanism known as retrotransposition. Their coordinated activities occur within a complex that includes a maturase protein, which promotes splicing through an unknown mechanism. The mechanism of splice site exchange within the RNA active site during catalysis also remains unclear. We determined two cryo-EM structures at 3.6-Å resolution of a group II intron reverse splicing into DNA. These structures reveal that the branch-site domain VI helix swings 90°, enabling substrate exchange during DNA integration. The maturase assists catalysis through a transient RNA-protein contact with domain VI that positions the branch-site adenosine for lariat formation during forward splicing. These findings provide the first direct evidence of the role the maturase plays during group II intron catalysis. The domain VI dynamics closely parallel spliceosomal branch-site helix movement and provide strong evidence for a retroelement origin of the spliceosome.
Subject(s)
RNA Splicing , RNA-Directed DNA Polymerase/chemistry , RNA/chemistry , Catalytic Domain , Cryoelectron Microscopy , Escherichia coli/genetics , Escherichia coli/metabolism , Nucleic Acid Conformation , Protein Structure, Tertiary , RNA/metabolism , RNA-Directed DNA Polymerase/metabolism , Retroelements , Spliceosomes/chemistryABSTRACT
The CC chemokine receptor 7 (CCR7) balances immunity and tolerance by homeostatic trafficking of immune cells. In cancer, CCR7-mediated trafficking leads to lymph node metastasis, suggesting the receptor as a promising therapeutic target. Here, we present the crystal structure of human CCR7 fused to the protein Sialidase NanA by using data up to 2.1 Å resolution. The structure shows the ligand Cmp2105 bound to an intracellular allosteric binding pocket. A sulfonamide group, characteristic for various chemokine receptor ligands, binds to a patch of conserved residues in the Gi protein binding region between transmembrane helix 7 and helix 8. We demonstrate how structural data can be used in combination with a compound repository and automated thermal stability screening to identify and modulate allosteric chemokine receptor antagonists. We detect both novel (CS-1 and CS-2) and clinically relevant (CXCR1-CXCR2 phase-II antagonist Navarixin) CCR7 modulators with implications for multi-target strategies against cancer.
Subject(s)
Ligands , Receptors, CCR7/metabolism , Allosteric Regulation , Binding Sites , Crystallography, X-Ray , Humans , Molecular Dynamics Simulation , Neuraminidase/genetics , Neuraminidase/metabolism , Protein Binding , Protein Structure, Tertiary , Receptors, CCR2/chemistry , Receptors, CCR2/metabolism , Receptors, CCR7/antagonists & inhibitors , Receptors, CCR7/genetics , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purificationABSTRACT
The vaccine-mediated elicitation of antibodies (Abs) capable of neutralizing diverse HIV-1 strains has been a long-standing goal. To understand how broadly neutralizing antibodies (bNAbs) can be elicited, we identified, characterized, and tracked five neutralizing Ab lineages targeting the HIV-1-fusion peptide (FP) in vaccinated macaques over time. Genetic and structural analyses revealed two of these lineages to belong to a reproducible class capable of neutralizing up to 59% of 208 diverse viral strains. B cell analysis indicated each of the five lineages to have been initiated and expanded by FP-carrier priming, with envelope (Env)-trimer boosts inducing cross-reactive neutralization. These Abs had binding-energy hotspots focused on FP, whereas several FP-directed Abs induced by immunization with Env trimer-only were less FP-focused and less broadly neutralizing. Priming with a conserved subregion, such as FP, can thus induce Abs with binding-energy hotspots coincident with the target subregion and capable of broad neutralization.
Subject(s)
AIDS Vaccines/immunology , Antibodies, Neutralizing/immunology , HIV Antibodies/immunology , Peptides/immunology , Amino Acid Sequence , Animals , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/classification , B-Lymphocytes/cytology , B-Lymphocytes/metabolism , Crystallography, X-Ray , Female , HEK293 Cells , HIV Antibodies/chemistry , HIV Antibodies/classification , HIV-1/metabolism , Humans , Macaca mulatta , Male , Peptides/chemistry , Protein Structure, Tertiary , env Gene Products, Human Immunodeficiency Virus/chemistry , env Gene Products, Human Immunodeficiency Virus/immunology , env Gene Products, Human Immunodeficiency Virus/metabolismABSTRACT
During autophagy, vesicle dynamics and cargo recruitment are driven by numerous adaptors and receptors that become tethered to the phagophore through interactions with lipidated ATG8/LC3 decorating the expanding membrane. Most currently described ATG8-binding proteins exploit a well-defined ATG8-interacting motif (AIM, or LC3-interacting region [LIR]) that contacts a hydrophobic patch on ATG8 known as the LIR/AIM docking site (LDS). Here we describe a new class of ATG8 interactors that exploit ubiquitin-interacting motif (UIM)-like sequences for high-affinity binding to an alternative ATG8 interaction site. Assays with candidate UIM-containing proteins together with unbiased screens identified a large collection of UIM-based ATG8 interactors in plants, yeast, and humans. Analysis of a subset also harboring ubiquitin regulatory X (UBX) domains revealed a role for UIM-directed autophagy in clearing non-functional CDC48/p97 complexes, including some impaired in human disease. With this new class of adaptors and receptors, we greatly extend the reach of selective autophagy and identify new factors regulating autophagic vesicle dynamics.
Subject(s)
Autophagy-Related Protein 8 Family/metabolism , Autophagy , Microtubule-Associated Proteins/metabolism , Amino Acid Motifs , Arabidopsis/metabolism , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/metabolism , Autophagy-Related Protein 8 Family/chemistry , Binding Sites , Humans , Microtubule-Associated Proteins/chemistry , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Tertiary , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Sequence AlignmentABSTRACT
How the central innate immune protein, STING, is activated by its ligands remains unknown. Here, using structural biology and biochemistry, we report that the metazoan second messenger 2'3'-cGAMP induces closing of the human STING homodimer and release of the STING C-terminal tail, which exposes a polymerization interface on the STING dimer and leads to the formation of disulfide-linked polymers via cysteine residue 148. Disease-causing hyperactive STING mutations either flank C148 and depend on disulfide formation or reside in the C-terminal tail binding site and cause constitutive C-terminal tail release and polymerization. Finally, bacterial cyclic-di-GMP induces an alternative active STING conformation, activates STING in a cooperative manner, and acts as a partial antagonist of 2'3'-cGAMP signaling. Our insights explain the tight control of STING signaling given varying background activation signals and provide a therapeutic hypothesis for autoimmune syndrome treatment.
Subject(s)
Membrane Proteins/metabolism , Binding Sites , Cyclic GMP/analogs & derivatives , Cyclic GMP/metabolism , Dimerization , Endoplasmic Reticulum/metabolism , HEK293 Cells , Humans , Ligands , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Models, Molecular , Mutagenesis, Site-Directed , Nucleotides, Cyclic/metabolism , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Signal TransductionABSTRACT
Ribonuclease (RNase) P is a ubiquitous ribozyme that cleaves the 5' leader from precursor tRNAs. Here, we report cryo-electron microscopy structures of the human nuclear RNase P alone and in complex with tRNAVal. Human RNase P is a large ribonucleoprotein complex that contains 10 protein components and one catalytic RNA. The protein components form an interlocked clamp that stabilizes the RNA in a conformation optimal for substrate binding. Human RNase P recognizes the tRNA using a double-anchor mechanism through both protein-RNA and RNA-RNA interactions. Structural comparison of the apo and tRNA-bound human RNase P reveals that binding of tRNA induces a local conformational change in the catalytic center, transforming the ribozyme into an active state. Our results also provide an evolutionary model depicting how auxiliary RNA elements in bacterial RNase P, essential for substrate binding, and catalysis, were replaced by the much more complex and multifunctional protein components in higher organisms.
Subject(s)
Cryoelectron Microscopy , RNA, Transfer/chemistry , Ribonuclease P/chemistry , Binding Sites , Evolution, Molecular , HEK293 Cells , Holoenzymes/chemistry , Humans , Molecular Dynamics Simulation , Nucleic Acid Conformation , Protein Domains , Protein Structure, Tertiary , RNA, Transfer/metabolism , Ribonuclease P/isolation & purification , Ribonuclease P/metabolismABSTRACT
The hetero-oligomeric chaperonin of eukarya, TRiC, is required to fold the cytoskeletal protein actin. The simpler bacterial chaperonin system, GroEL/GroES, is unable to mediate actin folding. Here, we use spectroscopic and structural techniques to determine how TRiC promotes the conformational progression of actin to the native state. We find that actin fails to fold spontaneously even in the absence of aggregation but populates a kinetically trapped, conformationally dynamic state. Binding of this frustrated intermediate to TRiC specifies an extended topology of actin with native-like secondary structure. In contrast, GroEL stabilizes bound actin in an unfolded state. ATP binding to TRiC effects an asymmetric conformational change in the chaperonin ring. This step induces the partial release of actin, priming it for folding upon complete release into the chaperonin cavity, mediated by ATP hydrolysis. Our results reveal how the unique features of TRiC direct the folding pathway of an obligate eukaryotic substrate.
Subject(s)
Actins/metabolism , Chaperonin 10/metabolism , Chaperonin 60/metabolism , Actins/chemistry , Adenosine Triphosphate/metabolism , Animals , Cattle , Chaperonin 10/chemistry , Chaperonin 60/chemistry , Cryoelectron Microscopy , Deoxyribonuclease I/chemistry , Deoxyribonuclease I/metabolism , Deuterium Exchange Measurement , Humans , Protein Binding , Protein Folding , Protein Structure, TertiaryABSTRACT
The single most frequent cancer-causing mutation across all heterotrimeric G proteins is R201C in Gαs. The current model explaining the gain-of-function activity of the R201 mutations is through the loss of GTPase activity and resulting inability to switch off to the GDP state. Here, we find that the R201C mutation can bypass the need for GTP binding by directly activating GDP-bound Gαs through stabilization of an intramolecular hydrogen bond network. Having found that a gain-of-function mutation can convert GDP into an activator, we postulated that a reciprocal mutation might disrupt the normal role of GTP. Indeed, we found R228C, a loss-of-function mutation in Gαs that causes pseudohypoparathyroidism type 1a (PHP-Ia), compromised the adenylyl cyclase-activating activity of Gαs bound to a non-hydrolyzable GTP analog. These findings show that disease-causing mutations in Gαs can subvert the canonical roles of GDP and GTP, providing new insights into the regulation mechanism of G proteins.
Subject(s)
GTP-Binding Protein alpha Subunits, Gs/metabolism , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/metabolism , Adenylyl Cyclases/chemistry , Adenylyl Cyclases/metabolism , Crystallography, X-Ray , GTP-Binding Protein alpha Subunits, Gs/chemistry , GTP-Binding Protein alpha Subunits, Gs/genetics , Humans , Hydrogen Bonding , Mutagenesis, Site-Directed , Protein Binding , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Structure, Tertiary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purificationABSTRACT
The seven-transmembrane-spanning protein Smoothened is the central transducer in Hedgehog signaling, a pathway fundamental in development and in cancer. Smoothened is activated by cholesterol binding to its extracellular cysteine-rich domain (CRD). How this interaction leads to changes in the transmembrane domain and Smoothened activation is unknown. Here, we report crystal structures of sterol-activated Smoothened. The CRD undergoes a dramatic reorientation, allosterically causing the transmembrane domain to adopt a conformation similar to active G-protein-coupled receptors. We show that Smoothened contains a unique inhibitory π-cation lock, which is broken on activation and is disrupted in constitutively active oncogenic mutants. Smoothened activation opens a hydrophobic tunnel, suggesting a pathway for cholesterol movement from the inner membrane leaflet to the CRD. All Smoothened antagonists bind the transmembrane domain and block tunnel opening, but cyclopamine also binds the CRD, inducing the active transmembrane conformation. Together, these results define the mechanisms of Smoothened activation and inhibition.
Subject(s)
Hedgehog Proteins/metabolism , Smoothened Receptor/chemistry , Xenopus Proteins/chemistry , Allosteric Regulation , Animals , Binding Sites , Cell Line , Cholesterol/chemistry , Cholesterol/metabolism , Crystallography, X-Ray , Flow Cytometry , Hedgehog Proteins/genetics , Humans , Mice , Molecular Dynamics Simulation , Protein Binding , Protein Domains , Protein Structure, Tertiary , Signal Transduction , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/chemistry , Small Molecule Libraries/metabolism , Smoothened Receptor/antagonists & inhibitors , Smoothened Receptor/metabolism , Veratrum Alkaloids/chemistry , Veratrum Alkaloids/metabolism , Xenopus Proteins/antagonists & inhibitors , Xenopus Proteins/metabolism , Xenopus laevis/metabolismABSTRACT
Anti-HIV-1 envelope broadly neutralizing monoclonal antibodies (bNAbs) isolated from memory B cells may not fully represent HIV-1-neutralizing profiles measured in plasma. Accordingly, we characterized near-pan-neutralizing antibodies extracted directly from the plasma of two "elite neutralizers." Circulating anti-gp120 polyclonal antibodies were deconvoluted using proteomics to guide lineage analysis of bone marrow plasma cells. In both subjects, a single lineage of anti-CD4-binding site (CD4bs) antibodies explained the plasma-neutralizing activity. Importantly, members of these lineages potently neutralized 89%-100% of a multi-tier 117 pseudovirus panel, closely matching the specificity and breadth of the circulating antibodies. X-ray crystallographic analysis of one monoclonal, N49P7, suggested a unique ability to bypass the CD4bs Phe43 cavity, while reaching deep into highly conserved residues of Layer 3 of the gp120 inner domain, likely explaining its extreme potency and breadth. Further direct analyses of plasma anti-HIV-1 bNAbs should provide new insights for developing antibody-based antiviral agents and vaccines.
Subject(s)
Antibodies, Neutralizing/immunology , HIV Envelope Protein gp120/immunology , HIV-1/metabolism , Amino Acid Sequence , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/chemistry , Binding Sites , CD4 Antigens/chemistry , CD4 Antigens/metabolism , Crystallography, X-Ray , HIV Antibodies/blood , HIV Antibodies/immunology , HIV Envelope Protein gp120/chemistry , HIV Envelope Protein gp120/metabolism , HIV-1/genetics , Humans , Molecular Dynamics Simulation , Protein Binding , Protein Structure, Tertiary , RNA, Viral/blood , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/immunologyABSTRACT
Conjugative transposition drives the emergence of multidrug resistance in diverse bacterial pathogens, yet the mechanisms are poorly characterized. The Tn1549 conjugative transposon propagates resistance to the antibiotic vancomycin used for severe drug-resistant infections. Here, we present four high-resolution structures of the conserved Y-transposase of Tn1549 complexed with circular transposon DNA intermediates. The structures reveal individual transposition steps and explain how specific DNA distortion and cleavage mechanisms enable DNA strand exchange with an absolute minimum homology requirement. This appears to uniquely allow Tn916-like conjugative transposons to bypass DNA homology and insert into diverse genomic sites, expanding gene transfer. We further uncover a structural regulatory mechanism that prevents premature cleavage of the transposon DNA before a suitable target DNA is found and generate a peptide antagonist that interferes with the transposase-DNA structure to block transposition. Our results reveal mechanistic principles of conjugative transposition that could help control the spread of antibiotic resistance genes.
Subject(s)
DNA, Bacterial/metabolism , Transposases/metabolism , Amino Acid Sequence , Base Sequence , Binding Sites , Catalytic Domain , Crystallography, X-Ray , DNA Cleavage , DNA Transposable Elements/genetics , DNA, Bacterial/chemistry , Drug Resistance, Bacterial , Enterococcus faecalis/genetics , Models, Molecular , Molecular Dynamics Simulation , Mutagenesis, Site-Directed , Nucleic Acid Conformation , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Sequence Alignment , Transposases/antagonists & inhibitors , Transposases/chemistry , Transposases/geneticsABSTRACT
Cyclic GMP-AMP synthase (cGAS) recognition of cytosolic DNA is critical for immune responses to pathogen replication, cellular stress, and cancer. Existing structures of the mouse cGAS-DNA complex provide a model for enzyme activation but do not explain why human cGAS exhibits severely reduced levels of cyclic GMP-AMP (cGAMP) synthesis compared to other mammals. Here, we discover that enhanced DNA-length specificity restrains human cGAS activation. Using reconstitution of cGAMP signaling in bacteria, we mapped the determinant of human cGAS regulation to two amino acid substitutions in the DNA-binding surface. Human-specific substitutions are necessary and sufficient to direct preferential detection of long DNA. Crystal structures reveal why removal of human substitutions relaxes DNA-length specificity and explain how human-specific DNA interactions favor cGAS oligomerization. These results define how DNA-sensing in humans adapted for enhanced specificity and provide a model of the active human cGAS-DNA complex to enable structure-guided design of cGAS therapeutics.
Subject(s)
DNA/metabolism , Immunologic Surveillance/physiology , Nucleotidyltransferases/metabolism , Animals , Benzofurans/chemistry , Benzofurans/metabolism , Binding Sites , Catalytic Domain , Chemotaxis/drug effects , DNA/chemistry , Humans , Mice , Molecular Docking Simulation , Mutagenesis, Site-Directed , Nucleotides, Cyclic/metabolism , Nucleotides, Cyclic/pharmacology , Nucleotidyltransferases/antagonists & inhibitors , Nucleotidyltransferases/genetics , Protein Multimerization , Protein Structure, Tertiary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Species Specificity , Vibrio cholerae/metabolism , Vibrio cholerae/physiologyABSTRACT
Hedgehog protein signals mediate tissue patterning and maintenance by binding to and inactivating their common receptor Patched, a 12-transmembrane protein that otherwise would suppress the activity of the 7-transmembrane protein Smoothened. Loss of Patched function, the most common cause of basal cell carcinoma, permits unregulated activation of Smoothened and of the Hedgehog pathway. A cryo-EM structure of the Patched protein reveals striking transmembrane domain similarities to prokaryotic RND transporters. A central hydrophobic conduit with cholesterol-like contents courses through the extracellular domain and resembles that used by other RND proteins to transport substrates, suggesting Patched activity in cholesterol transport. Cholesterol activity in the inner leaflet of the plasma membrane is reduced by PTCH1 expression but rapidly restored by Hedgehog stimulation, suggesting that PTCH1 regulates Smoothened by controlling cholesterol availability.
Subject(s)
Cholesterol/metabolism , Hedgehog Proteins/metabolism , Patched-1 Receptor/metabolism , Amino Acid Sequence , Animals , Cell Line , Cryoelectron Microscopy , Dimerization , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Evolution, Molecular , HEK293 Cells , Hedgehog Proteins/chemistry , Hedgehog Proteins/genetics , Humans , Mice , Multidrug Resistance-Associated Proteins/chemistry , Multidrug Resistance-Associated Proteins/metabolism , Patched-1 Receptor/chemistry , Patched-1 Receptor/genetics , Protein Structure, Tertiary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Sequence Alignment , Signal TransductionABSTRACT
Aggregates of human islet amyloid polypeptide (IAPP) in the pancreas of patients with type 2 diabetes (T2D) are thought to contribute to ß cell dysfunction and death. To understand how IAPP harms cells and how this might be overcome, we created a yeast model of IAPP toxicity. Ste24, an evolutionarily conserved protease that was recently reported to degrade peptides stuck within the translocon between the cytoplasm and the endoplasmic reticulum, was the strongest suppressor of IAPP toxicity. By testing variants of the human homolog, ZMPSTE24, with varying activity levels, the rescue of IAPP toxicity proved to be directly proportional to the declogging efficiency. Clinically relevant ZMPSTE24 variants identified in the largest database of exomes sequences derived from T2D patients were characterized using the yeast model, revealing 14 partial loss-of-function variants, which were enriched among diabetes patients over 2-fold. Thus, clogging of the translocon by IAPP oligomers may contribute to ß cell failure.
Subject(s)
Islet Amyloid Polypeptide/metabolism , Membrane Proteins/metabolism , Metalloendopeptidases/metabolism , Diabetes Mellitus, Type 2/metabolism , Diabetes Mellitus, Type 2/pathology , Endoplasmic Reticulum Stress/drug effects , Humans , Islet Amyloid Polypeptide/chemistry , Islet Amyloid Polypeptide/toxicity , Membrane Proteins/chemistry , Membrane Proteins/genetics , Metalloendopeptidases/chemistry , Metalloendopeptidases/genetics , Models, Biological , Mutagenesis , Protein Aggregates/physiology , Protein Structure, Tertiary , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Unfolded Protein Response/drug effectsABSTRACT
Tandem zinc finger (ZF) proteins are the largest and most rapidly diverging family of DNA-binding transcription regulators in mammals. ZFP568 represses a transcript of placental-specific insulin like growth factor 2 (Igf2-P0) in mice. ZFP568 binds a 24-base pair sequence-specific element upstream of Igf2-P0 via the eleven-ZF array. Both DNA and protein conformations deviate from the conventional one finger-three bases recognition, with individual ZFs contacting 2, 3, or 4 bases and recognizing thymine on the opposite strand. These interactions arise from a shortened minor groove caused by an AT-rich stretch, suggesting adaptability of ZF arrays to sequence variations. Despite conservation in mammals, mutations at Igf2 and ZFP568 reduce their binding affinity in chimpanzee and humans. Our studies provide important insights into the evolutionary and structural dynamics of ZF-DNA interactions that play a key role in mammalian development and evolution.