RESUMO
We present a novel method to determine engagement and specificity of KRAS4B-targeting compounds in vitro. By employing top-down mass spectrometry (MS), which analyzes intact and modified protein molecules (proteoforms), we can directly visualize and confidently characterize each KRAS4B species within compound-treated samples. Moreover, by employing targeted MS2 fragmentation, we can precisely localize each compound molecule to a specific residue on a given KRAS4B proteoform. This method allows us to comprehensively evaluate compound specificity, clearly detect nonspecific binding events, and determine the order and frequency with which they occur. We provide two proof-of-concept examples of our method employing publicly available compounds, along with detailed protocols for sample preparation, top-down MS data acquisition, targeted proteoform MS2 fragmentation, and analysis of the resulting data. Our results demonstrate the concentration dependence of KRAS4B-compound engagement and highlight the ability of top-down MS to directly map compound binding location(s) without disrupting the KRAS4B primary structure. Our hope is that this novel method may help accelerate the identification of new successful targeted inhibitors for KRAS4B and other RAS isoforms.
Assuntos
Proteínas Proto-Oncogênicas p21(ras) , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Humanos , Espectrometria de Massas/métodos , Ligação Proteica , Espectrometria de Massas em Tandem/métodosRESUMO
The diversity of chemical and structural attributes of proteins makes it inherently difficult to produce a wide range of proteins in a single recombinant protein production system. The nature of the target proteins themselves, along with cost, ease of use, and speed, are typically cited as major factors to consider in production. Despite a wide variety of alternative expression systems, most recombinant proteins for research and therapeutics are produced in a limited number of systems: Escherichia coli, yeast, insect cells, and the mammalian cell lines HEK293 and CHO. Recent interest in Vibrio natriegens as a new bacterial recombinant protein expression host is due in part to its short doubling time of ≤ 10 min but also stems from the promise of compatibility with techniques and genetic systems developed for E. coli. We successfully incorporated V. natriegens as an additional bacterial expression system for recombinant protein production and report improvements to published protocols as well as new protocols that expand the versatility of the system. While not all proteins benefit from production in V. natriegens, we successfully produced several proteins that were difficult or impossible to produce in E. coli. We also show that in some cases, the increased yield is due to higher levels of properly folded protein. Additionally, we were able to adapt our enhanced isotope incorporation methods for use with V. natriegens. Taken together, these observations and improvements allowed production of proteins for structural biology, biochemistry, assay development, and structure-based drug design in V. natriegens that were impossible and/or unaffordable to produce in E. coli.
Assuntos
Proteínas Recombinantes , Vibrio , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Vibrio/genética , Vibrio/metabolismo , Escherichia coli/metabolismo , Escherichia coli/genética , HumanosRESUMO
The viral genome of SARS-CoV-2 is packaged by the nucleocapsid (N-)protein into ribonucleoprotein particles (RNPs), 38 ± 10 of which are contained in each virion. Their architecture has remained unclear due to the pleomorphism of RNPs, the high flexibility of N-protein intrinsically disordered regions, and highly multivalent interactions between viral RNA and N-protein binding sites in both N-terminal (NTD) and C-terminal domain (CTD). Here we explore critical interaction motifs of RNPs by applying a combination of biophysical techniques to ancestral and mutant proteins binding different nucleic acids in an in vitro assay for RNP formation, and by examining nucleocapsid protein variants in a viral assembly assay. We find that nucleic acid-bound N-protein dimers oligomerize via a recently described protein-protein interface presented by a transient helix in its long disordered linker region between NTD and CTD. The resulting hexameric complexes are stabilized by multivalent protein-nucleic acid interactions that establish crosslinks between dimeric subunits. Assemblies are stabilized by the dimeric CTD of N-protein offering more than one binding site for stem-loop RNA. Our study suggests a model for RNP assembly where N-protein scaffolding at high density on viral RNA is followed by cooperative multimerization through protein-protein interactions in the disordered linker.
Assuntos
Proteínas do Nucleocapsídeo de Coronavírus , Multimerização Proteica , RNA Viral , SARS-CoV-2 , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , SARS-CoV-2/química , Proteínas do Nucleocapsídeo de Coronavírus/química , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Proteínas do Nucleocapsídeo de Coronavírus/genética , RNA Viral/metabolismo , RNA Viral/química , RNA Viral/genética , Ligação Proteica , Sítios de Ligação , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Montagem de Vírus/genética , Humanos , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/metabolismo , Proteínas do Nucleocapsídeo/genética , Modelos Moleculares , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , COVID-19/virologiaRESUMO
Prior analysis of intact and modified protein forms (proteoforms) of KRAS4B isolated from cell lines and tumor samples by top-down mass spectrometry revealed the presence of novel posttranslational modifications (PTMs) and potential evidence of context-specific KRAS4B modifications. However, low endogenous proteoform signal resulted in ineffective characterization, making it difficult to visualize less abundant PTMs or perform follow-up PTM validation using standard proteomic workflows. The NCI RAS Initiative has developed a model system, whereby KRAS4B bearing an N-terminal FLAG tag can be stably expressed within a panel of cancer cell lines. Herein, we present a method for combining immunoprecipitation with complementary proteomic methods to directly analyze N-terminally FLAG-tagged KRAS4B proteoforms and PTMs. We provide detailed protocols for FLAG-KRAS4B purification, proteoform analysis by targeted top-down LC-MS/MS, and validation of abundant PTMs by bottom-up LC-MS/MS with example results.
Assuntos
Proteômica , Espectrometria de Massas em Tandem , Cromatografia Líquida , Espectrometria de Massas em Tandem/métodos , Proteômica/métodos , Processamento de Proteína Pós-Traducional , Espectrometria de Massa com Cromatografia LíquidaRESUMO
The tobacco etch virus (TEV) protease is a commonly used reagent for removal of solubility and purification tags from recombinant proteins and is cited as being highly specific for its canonical cleavage site. Flexibility in some amino acids within this recognition sequence has been described in the literature but researchers generally assume few native human proteins will carry off-target sequences for TEV cleavage. We report here the aberrant cleavage of three human proteins with non-canonical TEV protease cleavage sites and identify broader sequence specificity rules that can be used to predict unwanted cleavage of recombinant proteins. Using these rules, 456 human proteins were identified that could be substrates for unwanted TEV protease cleavage.
Assuntos
Endopeptidases , Humanos , Endopeptidases/química , Endopeptidases/metabolismo , Endopeptidases/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteólise , Especificidade por SubstratoRESUMO
Development of new targeted inhibitors for oncogenic KRAS mutants may benefit from insight into how a given mutation influences the accessibility of protein residues and how compounds interact with mutant or wild-type KRAS proteins. Targeted proteomic analysis, a key validation step in the KRAS inhibitor development process, typically involves both intact mass- and peptide-based methods to confirm compound localization or quantify binding. However, these methods may not always provide a clear picture of the compound binding affinity for KRAS, how specific the compound is to the target KRAS residue, and how experimental conditions may impact these factors. To address this, we have developed a novel top-down proteomic assay to evaluate in vitro KRAS4B-compound engagement while assessing relative quantitation in parallel. We present two applications to demonstrate the capabilities of our assay: maleimide-biotin labeling of a KRAS4BG12D cysteine mutant panel and treatment of three KRAS4B proteins (WT, G12C, and G13C) with small molecule compounds. Our results show the time- or concentration-dependence of KRAS4B-compound engagement in context of the intact protein molecule while directly mapping the compound binding site.
Assuntos
Proteômica , Proteínas Proto-Oncogênicas p21(ras) , Proteínas Proto-Oncogênicas p21(ras)/genética , Mutação , Sítios de LigaçãoRESUMO
RAF kinases are integral to the RAS-MAPK signaling pathway, and proper RAF1 folding relies on its interaction with the chaperone HSP90 and the cochaperone CDC37. Understanding the intricate molecular interactions governing RAF1 folding is crucial for comprehending this process. Here, we present a cryo-EM structure of the closed-state RAF1-HSP90-CDC37 complex, where the C-lobe of the RAF1 kinase domain binds to one side of the HSP90 dimer, and an unfolded N-lobe segment of the RAF1 kinase domain threads through the center of the HSP90 dimer. CDC37 binds to the kinase C-lobe, mimicking the N-lobe with its HxNI motif. We also describe structures of HSP90 dimers without RAF1 and CDC37, displaying only N-terminal and middle domains, which we term the semi-open state. Employing 1 µs atomistic simulations, energetic decomposition, and comparative structural analysis, we elucidate the dynamics and interactions within these complexes. Our quantitative analysis reveals that CDC37 bridges the HSP90-RAF1 interaction, RAF1 binds HSP90 asymmetrically, and that HSP90 structural elements engage RAF1's unfolded region. Additionally, N- and C-terminal interactions stabilize HSP90 dimers, and molecular interactions in HSP90 dimers rearrange between the closed and semi-open states. Our findings provide valuable insight into the contributions of HSP90 and CDC37 in mediating client folding.
Assuntos
Proteínas de Ciclo Celular , Chaperoninas , Humanos , Proteínas de Ciclo Celular/metabolismo , Ligação Proteica , Chaperoninas/química , Chaperonas Moleculares/metabolismo , Proteínas de Choque Térmico HSP90RESUMO
Fusion-positive rhabdomyosarcoma (FP-RMS) is an aggressive pediatric sarcoma driven primarily by the PAX3-FOXO1 fusion oncogene, for which therapies targeting PAX3-FOXO1 are lacking. Here, we screen 62,643 compounds using an engineered cell line that monitors PAX3-FOXO1 transcriptional activity identifying a hitherto uncharacterized compound, P3FI-63. RNA-seq, ATAC-seq, and docking analyses implicate histone lysine demethylases (KDMs) as its targets. Enzymatic assays confirm the inhibition of multiple KDMs with the highest selectivity for KDM3B. Structural similarity search of P3FI-63 identifies P3FI-90 with improved solubility and potency. Biophysical binding of P3FI-90 to KDM3B is demonstrated using NMR and SPR. P3FI-90 suppresses the growth of FP-RMS in vitro and in vivo through downregulating PAX3-FOXO1 activity, and combined knockdown of KDM3B and KDM1A phenocopies P3FI-90 effects. Thus, we report KDM inhibitors P3FI-63 and P3FI-90 with the highest specificity for KDM3B. Their potent suppression of PAX3-FOXO1 activity indicates a possible therapeutic approach for FP-RMS and other transcriptionally addicted cancers.
Assuntos
Rabdomiossarcoma Alveolar , Rabdomiossarcoma , Criança , Humanos , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Rabdomiossarcoma Alveolar/genética , Linhagem Celular Tumoral , Rabdomiossarcoma/tratamento farmacológico , Rabdomiossarcoma/genética , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Regulação Neoplásica da Expressão Gênica , Fator de Transcrição PAX3/genética , Fator de Transcrição PAX3/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Histona Desmetilases/metabolismoRESUMO
The small GTPase Rat sarcoma virus proteins (RAS) are key regulators of cell growth and involved in 20-30% of cancers. RAS switches between its active state and inactive state via exchange of GTP (active) and GDP (inactive). Therefore, to study active protein, it needs to undergo nucleotide exchange to a non-hydrolysable GTP analog. Calf intestine alkaline phosphatase bound to agarose beads (CIP-agarose) is regularly used in a nucleotide exchange protocol to replace GDP with a non-hydrolysable analog. Due to pandemic supply problems and product shortages, we found the need for an alternative to this commercially available product. Here we describe how we generated a bacterial alkaline phosphatase (BAP) with an affinity tag bound to an agarose bead. This BAP completely exchanges the nucleotide in our samples, thereby demonstrating an alternative to the commercially available product using generally available laboratory equipment.
Assuntos
Proteínas Monoméricas de Ligação ao GTP , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Nucleotídeos , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Sefarose , Guanosina Trifosfato/metabolismo , Guanosina Difosfato/metabolismoRESUMO
KRAS, the most frequently mutated oncogene in human cancer, produces two isoforms, KRAS4a and KRAS4b, through alternative splicing. These isoforms differ in exon 4, which encodes the final 15 residues of the G-domain and hypervariable regions (HVRs), vital for trafficking and membrane localization. While KRAS4b has been extensively studied, KRAS4a has been largely overlooked. Our multidisciplinary study compared the structural and functional characteristics of KRAS4a and KRAS4b, revealing distinct structural properties and thermal stability. Position 151 influences KRAS4a's thermal stability, while position 153 affects binding to RAF1 CRD protein. Nuclear magnetic resonance analysis identified localized structural differences near sequence variations and provided a solution-state conformational ensemble. Notably, KRAS4a exhibits substantial transcript abundance in bile ducts, liver, and stomach, with transcript levels approaching KRAS4b in the colon and rectum. Functional disparities were observed in full-length KRAS variants, highlighting the impact of HVR variations on interaction with trafficking proteins and downstream effectors like RAF and PI3K within cells.
Assuntos
Neoplasias , Proteínas Proto-Oncogênicas p21(ras) , Humanos , Conformação Molecular , Isoformas de Proteínas/genética , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
Individual oncogenic KRAS mutants confer distinct differences in biochemical properties and signaling for reasons that are not well understood. KRAS activity is closely coupled to protein dynamics and is regulated through two interconverting conformations: state 1 (inactive, effector binding deficient) and state 2 (active, effector binding enabled). Here, we use 31P NMR to delineate the differences in state 1 and state 2 populations present in WT and common KRAS oncogenic mutants (G12C, G12D, G12V, G13D, and Q61L) bound to its natural substrate GTP or a commonly used nonhydrolyzable analog GppNHp (guanosine-5'-[(ß,γ)-imido] triphosphate). Our results show that GppNHp-bound proteins exhibit significant state 1 population, whereas GTP-bound KRAS is primarily (90% or more) in state 2 conformation. This observation suggests that the predominance of state 1 shown here and in other studies is related to GppNHp and is most likely nonexistent in cells. We characterize the impact of this differential conformational equilibrium of oncogenic KRAS on RAF1 kinase effector RAS-binding domain and intrinsic hydrolysis. Through a KRAS G12C drug discovery, we have identified a novel small-molecule inhibitor, BBO-8956, which is effective against both GDP- and GTP-bound KRAS G12C. We show that binding of this inhibitor significantly perturbs state 1-state 2 equilibrium and induces an inactive state 1 conformation in GTP-bound KRAS G12C. In the presence of BBO-8956, RAF1-RAS-binding domain is unable to induce a signaling competent state 2 conformation within the ternary complex, demonstrating the mechanism of action for this novel and active-conformation inhibitor.
Assuntos
Proteínas Proto-Oncogênicas p21(ras) , Proteínas ras , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas ras/metabolismo , Guanosina Trifosfato/metabolismo , Espectroscopia de Ressonância Magnética , Transdução de Sinais , MutaçãoRESUMO
The viral genome of SARS-CoV-2 is packaged by the nucleocapsid (N-) protein into ribonucleoprotein particles (RNPs), 38±10 of which are contained in each virion. Their architecture has remained unclear due to the pleomorphism of RNPs, the high flexibility of N-protein intrinsically disordered regions, and highly multivalent interactions between viral RNA and N-protein binding sites in both N-terminal (NTD) and C-terminal domain (CTD). Here we explore critical interaction motifs of RNPs by applying a combination of biophysical techniques to mutant proteins binding different nucleic acids in an in vitro assay for RNP formation, and by examining mutant proteins in a viral assembly assay. We find that nucleic acid-bound N-protein dimers oligomerize via a recently described protein-protein interface presented by a transient helix in its long disordered linker region between NTD and CTD. The resulting hexameric complexes are stabilized by multi-valent protein-nucleic acid interactions that establish crosslinks between dimeric subunits. Assemblies are stabilized by the dimeric CTD of N-protein offering more than one binding site for stem-loop RNA. Our study suggests a model for RNP assembly where N-protein scaffolding at high density on viral RNA is followed by cooperative multimerization through protein-protein interactions in the disordered linker.
RESUMO
Aim: Ferroptosis is a non-apoptotic form of cell death caused by lethal lipid peroxidation. Several small molecule ferroptosis inducers (FINs) have been reported, yet little information is available regarding their interaction with the ATP-binding cassette (ABC) transporters P-glycoprotein (P-gp, ABCB1) and ABCG2. We thus sought to characterize the interactions of FINs with P-gp and ABCG2, which may provide information regarding oral bioavailability and brain penetration and predict drug-drug interactions. Methods: Cytotoxicity assays with ferroptosis-sensitive A673 cells transfected to express P-gp or ABCG2 were used to determine the ability of the transporters to confer resistance to FINs; confirmatory studies were performed in OVCAR8 and NCI/ADR-RES cells. The ability of FINs to inhibit P-gp or ABCG2 was determined using the fluorescent substrates rhodamine 123 or purpuin-18, respectively. Results: P-gp overexpression conferred resistance to FIN56 and the erastin derivatives imidazole ketone erastin and piperazine erastin. P-gp-mediated resistance to imidazole ketone erastin and piperazine erastin was also reversed in UO-31 renal cancer cells by CRISPR-mediated knockout of ABCB1. The FINs ML-162, GPX inhibitor 26a, and PACMA31 at 10 µM were able to increase intracellular rhodamine 123 fluorescence over 10-fold in P-gp-expressing MDR-19 cells. GPX inhibitor 26a was able to increase intracellular purpurin-18 fluorescence over 4-fold in ABCG2-expressing R-5 cells. Conclusion: Expression of P-gp may reduce the efficacy of these FINs in cancers that express the transporter and may prevent access to sanctuary sites such as the brain. The ability of some FINs to inhibit P-gp and ABCG2 suggests potential drug-drug interactions.
RESUMO
Due to a combination of asymptomatic or undiagnosed infections, the proportion of the United States population infected with SARS-CoV-2 was unclear from the beginning of the pandemic. We previously established a platform to screen for SARS-CoV-2 positivity across a representative proportion of the US population, from which we reported that almost 17 million Americans were estimated to have had undocumented infections in the Spring of 2020. Since then, vaccine rollout and prevalence of different SARS-CoV-2 variants have further altered seropositivity trends within the United States population. To explore the longitudinal impacts of the pandemic and vaccine responses on seropositivity, we re-enrolled participants from our baseline study in a 6- and 12- month follow-up study to develop a longitudinal antibody profile capable of representing seropositivity within the United States during a critical period just prior to and during the initiation of vaccine rollout. Initial measurements showed that, since July 2020, seropositivity elevated within this population from 4.8% at baseline to 36.2% and 89.3% at 6 and 12 months, respectively. We also evaluated nucleocapsid seropositivity and compared to spike seropositivity to identify trends in infection versus vaccination relative to baseline. These data serve as a window into a critical timeframe within the COVID-19 pandemic response and serve as a resource that could be used in subsequent respiratory illness outbreaks.
RESUMO
Localized dynamics of RAS, including regions distal to the nucleotide-binding site, is of high interest for elucidating the mechanisms by which RAS proteins interact with effectors and regulators and for designing inhibitors. Among several oncogenic mutants, methyl relaxation dispersion experiments reveal highly synchronized conformational dynamics in the active (GMPPNP-bound) KRASG13D, which suggests an exchange between two conformational states in solution. Methyl and 31P NMR spectra of active KRASG13D in solution confirm a two-state ensemble interconverting on the millisecond timescale, with a major Pγ atom peak corresponding to the dominant State 1 conformation and a secondary peak indicating an intermediate state different from the known State 2 conformation recognized by RAS effectors. High-resolution crystal structures of active KRASG13D and KRASG13D-RAF1 RBD complex provide snapshots of the State 1 and 2 conformations, respectively. We use residual dipolar couplings to solve and cross-validate the structure of the intermediate state of active KRASG13D, showing a conformation distinct from those of States 1 and 2 outside the known flexible switch regions. The dynamic coupling between the conformational exchange in the effector lobe and the breathing motion in the allosteric lobe is further validated by a secondary mutation in the allosteric lobe, which affects the conformational population equilibrium.
Assuntos
Proteínas Proto-Oncogênicas p21(ras) , Proteínas ras , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Sítios de Ligação , Proteínas ras/metabolismo , Conformação Proteica , Espectroscopia de Ressonância MagnéticaRESUMO
Ferroptosis is a form of cell death caused by direct or indirect inhibition of glutathione peroxidase 4 that leads to lethal lipid peroxidation. Several small molecule ferroptosis inducers (FINs) have been reported, yet little information is available regarding resistance mechanisms, particularly their interaction with the ATP-binding cassette (ABC) transporters P-glycoprotein (P-gp, ABCB1) and ABCG2. Given the role that ABC transporters play in absorption, distribution, and excretion of many drugs, characterizing these interactions could provide information regarding oral bioavailability and brain penetration and may predict drug-drug interactions. Using ferroptosis-sensitive A673 cells transfected to express P-gp or ABCG2, we found that P-gp overexpression was able to confer resistance to FIN56 and the erastin derivatives imidazole ketone erastin and piperazine erastin. Results were confirmed with OVCAR8-derived NCI/ADR-RES cells that overexpress P-gp, where the P-gp inhibitor valspodar completely inhibited resistance to the FINs. P-gp-mediated resistance to imidazole ketone erastin and piperazine erastin was also reversed in UO-31 renal cancer cells by CRISPR-mediated knockout of ABCB1. At a concentration of 10 µM, the FINs ML-162, GPX inhibitor 26a, and PACMA31 were able to increase intracellular rhodamine 123 fluorescence over 10-fold in P-gp-expressing MDR-19 cells and GPX inhibitor 26a was able to increase intracellular purpurin-18 fluorescence over 4-fold in ABCG2-expressing R-5 cells. Expression of P-gp may reduce the efficacy of these FINs in cancers that express the transporter and may prevent access to sanctuary sites such as the brain. The ability of some FINs to inhibit P-gp and ABCG2 suggests potential drug-drug interactions.
RESUMO
The majority of pathogenic mutations in the neurofibromatosis type I (NF1) gene reduce total neurofibromin protein expression through premature truncation or microdeletion, but it is less well understood how loss-of-function missense variants drive NF1 disease. We have found that patient variants in codons 844 to 848, which correlate with a severe phenotype, cause protein instability and exert an additional dominant-negative action whereby wild-type neurofibromin also becomes destabilized through protein dimerization. We have used our neurofibromin cryogenic electron microscopy structure to predict and validate other patient variants that act through a similar mechanism. This provides a foundation for understanding genotype-phenotype correlations and has important implications for patient counseling, disease management, and therapeutics.
Assuntos
Neurofibromatose 1 , Neurofibromina 1 , Humanos , Neurofibromina 1/metabolismo , Neurofibromatose 1/genética , Dimerização , Mutação , Mutação de Sentido IncorretoRESUMO
SHOC2 acts as a strong synthetic lethal interactor with MEK inhibitors in multiple KRAS cancer cell lines. SHOC2 forms a heterotrimeric complex with MRAS and PP1C that is essential for regulating RAF and MAPK-pathway activation by dephosphorylating a specific phosphoserine on RAF kinases. Here we present the high-resolution crystal structure of the SHOC2-MRAS-PP1C (SMP) complex and apo-SHOC2. Our structures reveal that SHOC2, MRAS, and PP1C form a stable ternary complex in which all three proteins synergistically interact with each other. Our results show that dephosphorylation of RAF substrates by PP1C is enhanced upon interacting with SHOC2 and MRAS. The SMP complex forms only when MRAS is in an active state and is dependent on SHOC2 functioning as a scaffolding protein in the complex by bringing PP1C and MRAS together. Our results provide structural insights into the role of the SMP complex in RAF activation and how mutations found in Noonan syndrome enhance complex formation, and reveal new avenues for therapeutic interventions.
Assuntos
Síndrome de Noonan , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Síndrome de Noonan/genética , Síndrome de Noonan/metabolismo , Fosfosserina/metabolismo , Proteína Fosfatase 1 , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Quinases raf/genética , Quinases raf/metabolismo , Proteínas ras/metabolismoRESUMO
Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants.
Assuntos
Bacteriófagos , COVID-19 , Anticorpos de Domínio Único , Anticorpos Neutralizantes , Anticorpos Antivirais , Bacteriófagos/metabolismo , Humanos , Ligação Proteica , SARS-CoV-2 , Glicoproteína da Espícula de CoronavírusRESUMO
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is a trimer of S1/S2 heterodimers with three receptor-binding domains (RBDs) at the S1 subunit for human angiotensin-converting enzyme 2 (hACE2). Due to their small size, nanobodies can recognize protein cavities that are not accessible to conventional antibodies. To isolate high-affinity nanobodies, large libraries with great diversity are highly desirable. Dromedary camels (Camelus dromedarius) are natural reservoirs of coronaviruses like Middle East respiratory syndrome CoV (MERS-CoV) that are transmitted to humans. Here, we built large dromedary camel VHH phage libraries to isolate nanobodies that broadly neutralize SARS-CoV-2 variants. We isolated two VHH nanobodies, NCI-CoV-7A3 (7A3) and NCI-CoV-8A2 (8A2), which have a high affinity for the RBD via targeting nonoverlapping epitopes and show broad neutralization activity against SARS-CoV-2 and its emerging variants of concern. Cryoelectron microscopy (cryo-EM) complex structures revealed that 8A2 binds the RBD in its up mode with a long CDR3 loop directly involved in the ACE2 binding residues and that 7A3 targets a deeply buried region that uniquely extends from the S1 subunit to the apex of the S2 subunit regardless of the conformational state of the RBD. At a dose of ≥5 mg/kg, 7A3 efficiently protected transgenic mice expressing hACE2 from the lethal challenge of variants B.1.351 or B.1.617.2, suggesting its therapeutic use against COVID-19 variants. The dromedary camel VHH phage libraries could be helpful as a unique platform ready for quickly isolating potent nanobodies against future emerging viruses.