ABSTRACT
Many viruses utilize ringed packaging ATPases to translocate double-stranded DNA into procapsids during replication. A critical step in the mechanochemical cycle of such ATPases is ATP binding, which causes a subunit within the motor to grip DNA tightly. Here, we probe the underlying molecular mechanism by which ATP binding is coupled to DNA gripping and show that a glutamate-switch residue found in AAA+ enzymes is central to this coupling in viral packaging ATPases. Using free-energy landscapes computed through molecular dynamics simulations, we determined the stable conformational state of the ATPase active site in ATP- and ADP-bound states. Our results show that the catalytic glutamate residue transitions from an active to an inactive pose upon ATP hydrolysis and that a residue assigned as the glutamate switch is necessary for regulating this transition. Furthermore, we identified via mutual information analyses the intramolecular signaling pathway mediated by the glutamate switch that is responsible for coupling ATP binding to conformational transitions of DNA-gripping motifs. We corroborated these predictions with both structural and functional experimental measurements. Specifically, we showed that the crystal structure of the ADP-bound P74-26 packaging ATPase is consistent with the structural coupling predicted from simulations, and we further showed that disrupting the predicted signaling pathway indeed decouples ATPase activity from DNA translocation activity in the φ29 DNA packaging motor. Our work thus establishes a signaling pathway that couples chemical and mechanical events in viral DNA packaging motors.
Subject(s)
Adenosine Triphosphatases/metabolism , Glutamic Acid/metabolism , Molecular Dynamics Simulation , Viral Genome Packaging , Signal TransductionABSTRACT
Tailed bacteriophages use a DNA-packaging motor to encapsulate their genome during viral particle assembly. The small terminase (TerS) component of this DNA-packaging machinery acts as a molecular matchmaker that recognizes both the viral genome and the main motor component, the large terminase (TerL). However, how TerS binds DNA and the TerL protein remains unclear. Here we identified gp83 of the thermophilic bacteriophage P74-26 as the TerS protein. We found that TerSP76-26 oligomerizes into a nonamer that binds DNA, stimulates TerL ATPase activity, and inhibits TerL nuclease activity. A cryo-EM structure of TerSP76-26 revealed that it forms a ring with a wide central pore and radially arrayed helix-turn-helix domains. The structure further showed that these helix-turn-helix domains, which are thought to bind DNA by wrapping the double helix around the ring, are rigidly held in an orientation distinct from that seen in other TerS proteins. This rigid arrangement of the putative DNA-binding domain imposed strong constraints on how TerSP76-26 can bind DNA. Finally, the TerSP76-26 structure lacked the conserved C-terminal ß-barrel domain used by other TerS proteins for binding TerL. This suggests that a well-ordered C-terminal ß-barrel domain is not required for TerSP76-26 to carry out its matchmaking function. Our work highlights a thermophilic system for studying the role of small terminase proteins in viral maturation and presents the structure of TerSP76-26, revealing key differences between this thermophilic phage and its mesophilic counterparts.
Subject(s)
Adenosine Triphosphatases/metabolism , Bacteriophages/metabolism , Endodeoxyribonucleases/metabolism , Virus Assembly/physiology , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/genetics , Cryoelectron Microscopy , DNA, Viral/chemistry , DNA, Viral/metabolism , Endodeoxyribonucleases/chemistry , Endodeoxyribonucleases/genetics , Molecular Dynamics Simulation , Mutagenesis , Protein Binding , Protein Conformation, alpha-Helical , Protein Structure, Quaternary , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Static ElectricityABSTRACT
Many viruses use a powerful terminase motor to pump their genome inside an empty procapsid shell during virus maturation. The large terminase (TerL) protein contains both enzymatic activities necessary for packaging in such viruses: the adenosine triphosphatase (ATPase) that powers DNA translocation and an endonuclease that cleaves the concatemeric genome at both initiation and completion of genome packaging. However, how TerL binds DNA during translocation and cleavage remains mysterious. Here we investigate DNA binding and cleavage using TerL from the thermophilic phage P74-26. We report the structure of the P74-26 TerL nuclease domain, which allows us to model DNA binding in the nuclease active site. We screened a large panel of TerL variants for defects in binding and DNA cleavage, revealing that the ATPase domain is the primary site for DNA binding, and is required for nuclease activity. The nuclease domain is dispensable for DNA binding but residues lining the active site guide DNA for cleavage. Kinetic analysis of DNA cleavage suggests flexible tethering of the nuclease domains during DNA cleavage. We propose that interactions with the procapsid during DNA translocation conformationally restrict the nuclease domain, inhibiting cleavage; TerL release from the capsid upon completion of packaging unlocks the nuclease domains to cleave DNA.
Subject(s)
Adenosine Triphosphatases/chemistry , DNA, Viral/metabolism , Endodeoxyribonucleases/chemistry , Viral Proteins/chemistry , Adenosine Triphosphatases/metabolism , Bacteriophages/enzymology , Bacteriophages/genetics , Binding Sites , DNA Cleavage , Endodeoxyribonucleases/metabolism , Models, Molecular , Protein Domains , Viral Proteins/metabolism , Virus AssemblyABSTRACT
Many viruses package their genomes into procapsids using an ATPase machine that is among the most powerful known biological motors. However, how this motor couples ATP hydrolysis to DNA translocation is still unknown. Here, we introduce a model system with unique properties for studying motor structure and mechanism. We describe crystal structures of the packaging motor ATPase domain that exhibit nucleotide-dependent conformational changes involving a large rotation of an entire subdomain. We also identify the arginine finger residue that catalyzes ATP hydrolysis in a neighboring motor subunit, illustrating that previous models for motor structure need revision. Our findings allow us to derive a structural model for the motor ring, which we validate using small-angle X-ray scattering and comparisons with previously published data. We illustrate the model's predictive power by identifying the motor's DNA-binding and assembly motifs. Finally, we integrate our results to propose a mechanistic model for DNA translocation by this molecular machine.
Subject(s)
Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/metabolism , Bacteriophages/enzymology , Bacteriophages/genetics , DNA Packaging , Genome, Viral , Virus Assembly , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Arginine/metabolism , Biophysical Phenomena , Electrophoresis, Polyacrylamide Gel , Models, Biological , Models, Molecular , Molecular Sequence Data , Protein Structure, Tertiary , Reproducibility of Results , Thermus thermophilus/virology , Viral Proteins/chemistry , Viral Proteins/metabolismABSTRACT
C-terminal Binding Protein (CtBP) is a transcriptional co-regulator that downregulates the expression of many tumor-suppressor genes. Utilizing a crystal structure of CtBP with its substrate 4-methylthio-2-oxobutyric acid (MTOB) and NAD(+) as a guide, we have designed, synthesized, and tested a series of small molecule inhibitors of CtBP. From our first round of compounds, we identified 2-(hydroxyimino)-3-phenylpropanoic acid as a potent CtBP inhibitor (IC50=0.24µM). A structure-activity relationship study of this compound further identified the 4-chloro- (IC50=0.18µM) and 3-chloro- (IC50=0.17µM) analogues as additional potent CtBP inhibitors. Evaluation of the hydroxyimine analogues in a short-term cell growth/viability assay showed that the 4-chloro- and 3-chloro-analogues are 2-fold and 4-fold more potent, respectively, than the MTOB control. A functional cellular assay using a CtBP-specific transcriptional readout revealed that the 4-chloro- and 3-chloro-hydroxyimine analogues were able to block CtBP transcriptional repression activity. This data suggests that substrate-competitive inhibition of CtBP dehydrogenase activity is a potential mechanism to reactivate tumor-suppressor gene expression as a therapeutic strategy for cancer.
Subject(s)
Alcohol Oxidoreductases/antagonists & inhibitors , DNA-Binding Proteins/antagonists & inhibitors , Oximes/chemistry , Oximes/pharmacology , Phenylpropionates/chemistry , Phenylpropionates/pharmacology , Alcohol Oxidoreductases/metabolism , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , DNA-Binding Proteins/metabolism , Drug Design , Halogenation , Humans , Methionine/analogs & derivatives , Methionine/metabolism , Models, Molecular , Neoplasms/drug therapy , Neoplasms/metabolism , Oximes/chemical synthesis , Phenylpropionates/chemical synthesis , Structure-Activity RelationshipABSTRACT
Virus capsids are protein shells that protect the viral genome from environmental assaults, while maintaining the high internal pressure of the tightly packaged genome. To elucidate how capsids maintain stability under harsh conditions, we investigated the capsid components of the hyperthermophilic phage P74-26. We determined the structure of capsid protein gp87 and show that it has the same fold as decoration proteins in many other phages, despite lacking significant sequence homology. We also find that gp87 is significantly more stable than mesophilic homologs. Our analysis of the gp87 structure reveals that the core "ß tulip" domain is conserved in trimeric capsid components across numerous double-stranded DNA viruses, including Herpesviruses. Moreover, this ß barrel domain is found in anti-CRISPR protein AcrIIC1, suggesting a mechanism for the evolution of this Cas9 inhibitor. Our work illustrates the principles for increased stability of gp87, and extends the evolutionary reach of the ß tulip domain.
Subject(s)
Bacteriophages/metabolism , Capsid Proteins/chemistry , Herpesviridae/metabolism , Bacteriophages/chemistry , CRISPR-Associated Protein 9/antagonists & inhibitors , Evolution, Molecular , Herpesviridae/chemistry , Models, Molecular , Protein Domains , Protein Folding , Protein Stability , Protein Structure, SecondaryABSTRACT
Nucleic acid editing enzymes are essential components of the immune system that lethally mutate viral pathogens and somatically mutate immunoglobulins, and contribute to the diversification and lethality of cancers. Among these enzymes are the seven human APOBEC3 deoxycytidine deaminases, each with unique target sequence specificity and subcellular localization. While the enzymology and biological consequences have been extensively studied, the mechanism by which APOBEC3s recognize and edit DNA remains elusive. Here we present the crystal structure of a complex of a cytidine deaminase with ssDNA bound in the active site at 2.2 Å. This structure not only visualizes the active site poised for catalysis of APOBEC3A, but pinpoints the residues that confer specificity towards CC/TC motifs. The APOBEC3A-ssDNA complex defines the 5'-3' directionality and subtle conformational changes that clench the ssDNA within the binding groove, revealing the architecture and mechanism of ssDNA recognition that is likely conserved among all polynucleotide deaminases, thereby opening the door for the design of mechanistic-based therapeutics.
Subject(s)
Catalytic Domain , Cytidine Deaminase/chemistry , Cytidine/chemistry , DNA, Single-Stranded/chemistry , Proteins/chemistry , Amino Acid Sequence , Crystallography, X-Ray , Cytidine/metabolism , Cytidine Deaminase/genetics , Cytidine Deaminase/metabolism , DNA, Single-Stranded/genetics , DNA, Single-Stranded/metabolism , Deamination , Humans , Models, Molecular , Protein Binding , Protein Domains , Protein Structure, Secondary , Proteins/genetics , Proteins/metabolism , Sequence Homology, Amino Acid , Substrate SpecificityABSTRACT
The heavy chain IGHV1-69 germline gene exhibits a high level of polymorphism and shows biased use in protective antibody (Ab) responses to infections and vaccines. It is also highly expressed in several B cell malignancies and autoimmune diseases. G6 is an anti-idiotypic monoclonal Ab that selectively binds to IGHV1-69 heavy chain germline gene 51p1 alleles that have been implicated in these Ab responses and disease processes. Here, we determine the co-crystal structure of humanized G6 (hG6.3) in complex with anti-influenza hemagglutinin stem-directed broadly neutralizing Ab D80. The core of the hG6.3 idiotope is a continuous string of CDR-H2 residues starting with M53 and ending with N58. G6 binding studies demonstrate the remarkable breadth of binding to 51p1 IGHV1-69 Abs with diverse CDR-H3, light chain, and antigen binding specificities. These studies detail the broad expression of the G6 cross-reactive idiotype (CRI) that further define its potential role in precision medicine.
Subject(s)
Antibodies, Anti-Idiotypic/chemistry , Antibodies, Monoclonal, Humanized/chemistry , Antibodies, Neutralizing/chemistry , Antibodies, Viral/chemistry , Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Receptors, Antigen, B-Cell/chemistry , Amino Acid Sequence , Antibodies, Anti-Idiotypic/genetics , Antibodies, Anti-Idiotypic/immunology , Antibodies, Monoclonal, Humanized/genetics , Antibodies, Monoclonal, Humanized/immunology , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/immunology , Antibodies, Viral/genetics , Antibodies, Viral/immunology , Antibody Specificity , Binding Sites , Cloning, Molecular , Crystallography, X-Ray , Gene Expression , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Humans , Models, Molecular , Orthomyxoviridae/chemistry , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Receptors, Antigen, B-Cell/genetics , Receptors, Antigen, B-Cell/immunology , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
The eukaryotic DNA polymerase sliding clamp, proliferating cell nuclear antigen or PCNA, is a ring-shaped protein complex that surrounds DNA to act as a sliding platform for increasing processivity of cellular replicases and for coordinating various cellular pathways with DNA replication. A single point mutation, Ser228Ile, in the human PCNA gene was recently identified to cause a disease whose symptoms resemble those of DNA damage and repair disorders. The mutation lies near the binding site for most PCNA-interacting proteins. However, the structural consequences of the S228I mutation are unknown. Here, we describe the structure of the disease-causing variant, which reveals a large conformational change that dramatically transforms the binding pocket for PCNA client proteins. We show that the mutation markedly alters the binding energetics for some client proteins, while another, p21(CIP1), is only mildly affected. Structures of the disease variant bound to peptides derived from two PCNA partner proteins reveal that the binding pocket can adjust conformation to accommodate some ligands, indicating that the binding site is dynamic and pliable. Our work has implications for the plasticity of the binding site in PCNA and reveals how a disease mutation selectively alters interactions to a promiscuous binding site that is critical for DNA metabolism.
Subject(s)
Genetic Diseases, Inborn/pathology , Mutant Proteins/genetics , Mutant Proteins/metabolism , Point Mutation , Proliferating Cell Nuclear Antigen/genetics , Proliferating Cell Nuclear Antigen/metabolism , Binding Sites , Crystallography, X-Ray , Humans , Models, Molecular , Mutation, Missense , Proliferating Cell Nuclear Antigen/chemistry , Protein Binding , Protein Conformation , Protein Interaction MappingABSTRACT
Oncogenic transcriptional coregulators C-terminal Binding Protein (CtBP) 1 and 2 possess regulatory d-isomer specific 2-hydroxyacid dehydrogenase (D2-HDH) domains that provide an attractive target for small molecule intervention. Findings that the CtBP substrate 4-methylthio 2-oxobutyric acid (MTOB) can interfere with CtBP oncogenic activity in cell culture and in mice confirm that such inhibitors could have therapeutic benefit. Recent crystal structures of CtBP 1 and 2 revealed that MTOB binds in an active site containing a dominant tryptophan and a hydrophilic cavity, neither of which are present in other D2-HDH family members. Here, we demonstrate the effectiveness of exploiting these active site features for the design of high affinity inhibitors. Crystal structures of two such compounds, phenylpyruvate (PPy) and 2-hydroxyimino-3-phenylpropanoic acid (HIPP), show binding with favorable ring stacking against the CtBP active site tryptophan and alternate modes of stabilizing the carboxylic acid moiety. Moreover, ITC experiments show that HIPP binds to CtBP with an affinity greater than 1000-fold over that of MTOB, and enzymatic assays confirm that HIPP substantially inhibits CtBP catalysis. These results, thus, provide an important step, and additional insights, for the development of highly selective antineoplastic CtBP inhibitors.
Subject(s)
Alcohol Oxidoreductases/chemistry , DNA-Binding Proteins/chemistry , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Nerve Tissue Proteins/chemistry , Alcohol Oxidoreductases/antagonists & inhibitors , Alcohol Oxidoreductases/metabolism , Binding Sites , Co-Repressor Proteins , Crystallography, X-Ray , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/metabolism , Enzyme Inhibitors/metabolism , Humans , Hydroxylamines/chemistry , Hydroxylamines/metabolism , Hydroxylamines/pharmacology , Ligands , Models, Molecular , NAD/chemistry , NAD/metabolism , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/metabolism , Phenylpropionates/chemistry , Phenylpropionates/metabolism , Phenylpropionates/pharmacology , Phenylpyruvic Acids/chemistry , Phenylpyruvic Acids/metabolism , Phenylpyruvic Acids/pharmacology , Protein Conformation , Structure-Activity Relationship , ThermodynamicsABSTRACT
The oncogenic corepressors C-terminal Binding Protein (CtBP) 1 and 2 harbor regulatory d-isomer specific 2-hydroxyacid dehydrogenase (d2-HDH) domains. 4-Methylthio 2-oxobutyric acid (MTOB) exhibits substrate inhibition and can interfere with CtBP oncogenic activity in cell culture and mice. Crystal structures of human CtBP1 and CtBP2 in complex with MTOB and NAD(+) revealed two key features: a conserved tryptophan that likely contributes to substrate specificity and a hydrophilic cavity that links MTOB with an NAD(+) phosphate. Neither feature is present in other d2-HDH enzymes. These structures thus offer key opportunities for the development of highly selective anti-neoplastic CtBP inhibitors.