RESUMEN
Cysteine-focused chemical proteomic platforms have accelerated the clinical development of covalent inhibitors for a wide range of targets in cancer. However, how different oncogenic contexts influence cysteine targeting remains unknown. To address this question, we have developed "DrugMap," an atlas of cysteine ligandability compiled across 416 cancer cell lines. We unexpectedly find that cysteine ligandability varies across cancer cell lines, and we attribute this to differences in cellular redox states, protein conformational changes, and genetic mutations. Leveraging these findings, we identify actionable cysteines in NF-κB1 and SOX10 and develop corresponding covalent ligands that block the activity of these transcription factors. We demonstrate that the NF-κB1 probe blocks DNA binding, whereas the SOX10 ligand increases SOX10-SOX10 interactions and disrupts melanoma transcriptional signaling. Our findings reveal heterogeneity in cysteine ligandability across cancers, pinpoint cell-intrinsic features driving cysteine targeting, and illustrate the use of covalent probes to disrupt oncogenic transcription-factor activity.
Asunto(s)
Cisteína , Neoplasias , Animales , Humanos , Ratones , Línea Celular Tumoral , Cisteína/metabolismo , Cisteína/química , Ligandos , Melanoma/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , FN-kappa B/química , FN-kappa B/metabolismo , Oxidación-Reducción , Transducción de Señal , Factores de Transcripción SOXE/química , Factores de Transcripción SOXE/metabolismoRESUMEN
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.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Ácido Glutámico/metabolismo , Simulación de Dinámica Molecular , Empaquetamiento del Genoma Viral , Transducción de SeñalRESUMEN
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.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Bacteriófagos/metabolismo , Endodesoxirribonucleasas/metabolismo , Ensamble de Virus/fisiología , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Microscopía por Crioelectrón , ADN Viral/química , ADN Viral/metabolismo , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/genética , Simulación de Dinámica Molecular , Mutagénesis , Unión Proteica , Conformación Proteica en Hélice alfa , Estructura Cuaternaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Electricidad EstáticaRESUMEN
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.
Asunto(s)
Adenosina Trifosfatasas/química , ADN Viral/metabolismo , Endodesoxirribonucleasas/química , Proteínas Virales/química , Adenosina Trifosfatasas/metabolismo , Bacteriófagos/enzimología , Bacteriófagos/genética , Sitios de Unión , División del ADN , Endodesoxirribonucleasas/metabolismo , Modelos Moleculares , Dominios Proteicos , Proteínas Virales/metabolismo , Ensamble de VirusRESUMEN
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.
Asunto(s)
Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Bacteriófagos/enzimología , Bacteriófagos/genética , Empaquetamiento del ADN , Genoma Viral , Ensamble de Virus , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Arginina/metabolismo , Fenómenos Biofísicos , Electroforesis en Gel de Poliacrilamida , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Reproducibilidad de los Resultados , Thermus thermophilus/virología , Proteínas Virales/química , Proteínas Virales/metabolismoRESUMEN
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.
Asunto(s)
Oxidorreductasas de Alcohol/antagonistas & inhibidores , Proteínas de Unión al ADN/antagonistas & inhibidores , Oximas/química , Oximas/farmacología , Fenilpropionatos/química , Fenilpropionatos/farmacología , Oxidorreductasas de Alcohol/metabolismo , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Diseño de Fármacos , Halogenación , Humanos , Metionina/análogos & derivados , Metionina/metabolismo , Modelos Moleculares , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Oximas/síntesis química , Fenilpropionatos/síntesis química , Relación Estructura-ActividadRESUMEN
Phosphoinositide 3-kinase α (PIK3CA) is one of the most mutated genes across cancers, especially breast, gynecologic, and head and neck squamous cell carcinoma tumors. Mutations occur throughout the gene, but hotspot mutations in the helical and kinase domains predominate. The therapeutic benefit of isoform-selective PI3Kα inhibition was established with alpelisib, which displays equipotent activity against the wild-type and mutant enzyme. Inhibition of wild-type PI3Kα is associated with severe hyperglycemia and rash, which limits alpelisib use and suggests that selectively targeting mutant PI3Kα could reduce toxicity and improve efficacy. Here we describe STX-478, an allosteric PI3Kα inhibitor that selectively targets prevalent PI3Kα helical- and kinase-domain mutant tumors. STX-478 demonstrated robust efficacy in human tumor xenografts without causing the metabolic dysfunction observed with alpelisib. Combining STX-478 with fulvestrant and/or cyclin-dependent kinase 4/6 inhibitors was well tolerated and provided robust and durable tumor regression in ER+HER2- xenograft tumor models. SIGNIFICANCE: These preclinical data demonstrate that the mutant-selective, allosteric PI3Kα inhibitor STX-478 provides robust efficacy while avoiding the metabolic dysfunction associated with the nonselective inhibitor alpelisib. Our results support the ongoing clinical evaluation of STX-478 in PI3Kα-mutated cancers, which is expected to expand the therapeutic window and mitigate counterregulatory insulin release. See related commentary by Kearney and Vasan, p. 2313. This article is featured in Selected Articles from This Issue, p. 2293.
Asunto(s)
Neoplasias de la Mama , Neoplasias , Humanos , Femenino , Xenoinjertos , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Fosfatidilinositol 3-Quinasa Clase I/genéticaRESUMEN
Cysteine-focused chemical proteomic platforms have accelerated the clinical development of covalent inhibitors of a wide-range of targets in cancer. However, how different oncogenic contexts influence cysteine targeting remains unknown. To address this question, we have developed DrugMap , an atlas of cysteine ligandability compiled across 416 cancer cell lines. We unexpectedly find that cysteine ligandability varies across cancer cell lines, and we attribute this to differences in cellular redox states, protein conformational changes, and genetic mutations. Leveraging these findings, we identify actionable cysteines in NFκB1 and SOX10 and develop corresponding covalent ligands that block the activity of these transcription factors. We demonstrate that the NFκB1 probe blocks DNA binding, whereas the SOX10 ligand increases SOX10-SOX10 interactions and disrupts melanoma transcriptional signaling. Our findings reveal heterogeneity in cysteine ligandability across cancers, pinpoint cell-intrinsic features driving cysteine targeting, and illustrate the use of covalent probes to disrupt oncogenic transcription factor activity.
RESUMEN
The CRISPR-Cas type V-I is a family of Cas12i-containing programmable nuclease systems guided by a short crRNA without requirement for a tracrRNA. Here we present an engineered Type V-I CRISPR system (Cas12i), ABR-001, which utilizes a tracr-less guide RNA. The compact Cas12i effector is capable of self-processing pre-crRNA and cleaving dsDNA targets, which facilitates versatile delivery options and multiplexing, respectively. We apply an unbiased mutational scanning approach to enhance initially low editing activity of Cas12i2. The engineered variant, ABR-001, exhibits broad genome editing capability in human cell lines, primary T cells, and CD34+ hematopoietic stem and progenitor cells, with both robust efficiency and high specificity. In addition, ABR-001 achieves a high level of genome editing when delivered via AAV vector to HEK293T cells. This work establishes ABR-001 as a versatile, specific, and high-performance platform for ex vivo and in vivo gene therapy.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Sistemas CRISPR-Cas/genética , Endonucleasas/genética , Endonucleasas/metabolismo , Edición Génica/métodos , Células HEK293 , Humanos , ARN/metabolismo , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismoRESUMEN
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.
Asunto(s)
Bacteriófagos/metabolismo , Proteínas de la Cápside/química , Herpesviridae/metabolismo , Bacteriófagos/química , Proteína 9 Asociada a CRISPR/antagonistas & inhibidores , Evolución Molecular , Herpesviridae/química , Modelos Moleculares , Dominios Proteicos , Pliegue de Proteína , Estabilidad Proteica , Estructura Secundaria de ProteínaRESUMEN
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.
Asunto(s)
Dominio Catalítico , Citidina Desaminasa/química , Citidina/química , ADN de Cadena Simple/química , Proteínas/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Citidina/metabolismo , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Desaminación , Humanos , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Estructura Secundaria de Proteína , Proteínas/genética , Proteínas/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Anticuerpos Antiidiotipos/química , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Neutralizantes/química , Anticuerpos Antivirales/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Receptores de Antígenos de Linfocitos B/química , Secuencia de Aminoácidos , Anticuerpos Antiidiotipos/genética , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Monoclonales Humanizados/genética , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Expresión Génica , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Humanos , Modelos Moleculares , Orthomyxoviridae/química , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
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.
Asunto(s)
Enfermedades Genéticas Congénitas/patología , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación Puntual , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Mutación Missense , Antígeno Nuclear de Célula en Proliferación/química , Unión Proteica , Conformación Proteica , Mapeo de Interacción de ProteínasRESUMEN
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.
Asunto(s)
Oxidorreductasas de Alcohol/química , Proteínas de Unión al ADN/química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Proteínas del Tejido Nervioso/química , Oxidorreductasas de Alcohol/antagonistas & inhibidores , Oxidorreductasas de Alcohol/metabolismo , Sitios de Unión , Proteínas Co-Represoras , Cristalografía por Rayos X , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Inhibidores Enzimáticos/metabolismo , Humanos , Hidroxilaminas/química , Hidroxilaminas/metabolismo , Hidroxilaminas/farmacología , Ligandos , Modelos Moleculares , NAD/química , NAD/metabolismo , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/metabolismo , Fenilpropionatos/química , Fenilpropionatos/metabolismo , Fenilpropionatos/farmacología , Ácidos Fenilpirúvicos/química , Ácidos Fenilpirúvicos/metabolismo , Ácidos Fenilpirúvicos/farmacología , Conformación Proteica , Relación Estructura-Actividad , TermodinámicaRESUMEN
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.