RESUMEN
Transglutaminase 2 (TG2) is a GTP-binding, protein-crosslinking enzyme that has been investigated as a therapeutic target for Celiac disease, neurological disorders, and aggressive cancers. TG2 has been suggested to adopt two conformational states that regulate its functions: a GTP-bound, closed conformation, and a calcium-bound, crosslinking-active open conformation. TG2 mutants that constitutively adopt an open conformation are cytotoxic to cancer cells. Thus, small molecules that bind and stabilize the open conformation of TG2 could offer a new therapeutic strategy. Here, we investigate TG2, using static and time-resolved small-angle X-ray scattering (SAXS) and single-particle cryoelectron microscopy (cryo-EM), to determine the conformational states responsible for conferring its biological effects. We also describe a newly developed TG2 inhibitor, LM11, that potently kills glioblastoma cells and use SAXS to investigate how LM11 affects the conformational states of TG2. Using SAXS and cryo-EM, we show that guanine nucleotides bind and stabilize a monomeric closed conformation while calcium binds to an open state that can form higher order oligomers. SAXS analysis suggests how a TG2 mutant that constitutively adopts the open state binds nucleotides through an alternative mechanism to wildtype TG2. Furthermore, we use time resolved SAXS to show that LM11 increases the ability of calcium to bind and stabilize an open conformation, which is not reversible by guanine nucleotides and is cytotoxic to cancer cells. Taken together, our findings demonstrate that the conformational dynamics of TG2 are more complex than previously suggested and highlight how conformational stabilization of TG2 by LM11 maintains TG2 in a cytotoxic conformational state.
Asunto(s)
Supervivencia Celular , Proteínas de Unión al GTP , Conformación Proteica , Proteína Glutamina Gamma Glutamiltransferasa 2 , Transglutaminasas , Proteína Glutamina Gamma Glutamiltransferasa 2/metabolismo , Humanos , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/genética , Transglutaminasas/metabolismo , Transglutaminasas/química , Transglutaminasas/genética , Supervivencia Celular/efectos de los fármacos , Microscopía por Crioelectrón , Línea Celular Tumoral , Muerte Celular/efectos de los fármacos , Dispersión del Ángulo Pequeño , Difracción de Rayos X , Calcio/metabolismoRESUMEN
Mammalian transglutaminases, a family of Ca2+-dependent proteins, are implicated in a variety of diseases. For example, celiac disease (CeD) is an autoimmune disorder whose pathogenesis requires transglutaminase 2 (TG2) to deamidate select glutamine residues in diet-derived gluten peptides. Deamidation involves the formation of transient γ-glutamyl thioester intermediates. Recent studies have revealed that in addition to the deamidated gluten peptides themselves, their corresponding thioester intermediates are also pathogenically relevant. A mechanistic understanding of this relevance is hindered by the absence of any structure of Ca2+-bound TG2. We report the X-ray crystallographic structure of human TG2 bound to an inhibitory gluten peptidomimetic and two Ca2+ ions in sites previously designated as S1 and S3. Together with additional structure-guided experiments, this structure provides a mechanistic explanation for how S1 regulates formation of an inhibitory disulfide bond in TG2, while also establishing that S3 is essential for γ-glutamyl thioester formation. Furthermore, our crystallographic findings and associated analyses have revealed that i) two interacting residues, H305 and E363, play a critical role in resolving the thioester intermediate into an isopeptide bond (transamidation) but not in thioester hydrolysis (deamidation); and ii) residues N333 and K176 stabilize preferred TG2 substrates and inhibitors via hydrogen bonding to nonreactive backbone atoms. Overall, the intermediate-state conformer of TG2 reported here represents a superior model to previously characterized conformers for both transition states of the TG2-catalyzed reaction.
Asunto(s)
Calcio , Proteínas de Unión al GTP , Proteína Glutamina Gamma Glutamiltransferasa 2 , Transglutaminasas , Transglutaminasas/metabolismo , Transglutaminasas/química , Proteína Glutamina Gamma Glutamiltransferasa 2/metabolismo , Humanos , Calcio/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/química , Cristalografía por Rayos X , Glútenes/metabolismo , Glútenes/química , Modelos Moleculares , Conformación Proteica , Enfermedad Celíaca/metabolismo , Unión ProteicaRESUMEN
Human tissue transglutaminase (tTG) is an intriguing multifunctional enzyme involved in various diseases, including celiac disease and neurological disorders. Although a number of tTG inhibitors have been developed, the molecular determinants governing ligand binding remain incomplete due to the lack of high-resolution structural data in the vicinity of its active site. In this study, we obtained the complete high-resolution model of tTG by in silico methods based on available PDB structures. We discovered significant differences in the active site architecture between our and known tTG models, revealing an additional loop which affects the ligand binding affinity. We assembled a library of new potential tTG inhibitors based on the obtained complete model of the enzyme. Our library substantially expands the spectrum of possible drug candidates targeting tTG and encompasses twelve molecular scaffolds, eleven of which are novel and exhibit higher binding affinity then already known ones, according to our in silico studies. The results of this study open new directions for structure-based drug design of tTG inhibitors, offering the complete protein model and suggesting a wide range of new compounds for further experimental validation.
Asunto(s)
Dominio Catalítico , Proteínas de Unión al GTP , Proteína Glutamina Gamma Glutamiltransferasa 2 , Transglutaminasas , Transglutaminasas/metabolismo , Transglutaminasas/química , Transglutaminasas/antagonistas & inhibidores , Humanos , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/antagonistas & inhibidores , Simulación por Computador , Unión Proteica , Modelos Moleculares , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Simulación del Acoplamiento Molecular , Ligandos , Conformación ProteicaRESUMEN
G protein-coupled receptors (GPCRs) regulate cellular signaling processes by coupling to diverse combinations of heterotrimeric G proteins composed of Gα, Gß, and Gγ subunits. Biosensors based on bioluminescence resonance energy transfer (BRET) have advanced our understanding of GPCR functional selectivity. Some BRET biosensors monitor ligand-induced conformational changes in the receptor or G proteins, whereas others monitor the recruitment of downstream effectors to sites of G protein activation. Here, we compared the ability of conformation-and activation-based BRET biosensors to assess the coupling of various class A and B GPCRs to specific Gα proteins in cultured cells. These GPCRs included serotonin 5-HT2A and 5-HT7 receptors, the GLP-1 receptor (GLP-1R), and the M3 muscarinic receptor. We observed different signaling profiles between the two types of sensors, highlighting how data interpretation could be affected by the nature of the biosensor. We also found that the identity of the Gßγ subunits used in the assay could differentially influence the selectivity of a receptor toward Gα subtypes, emphasizing the importance of the receptor-Gßγ pairing in determining Gα coupling specificity. Last, the addition of epitope tags to the receptor could affect stoichiometry and coupling selectivity and yield artifactual findings. These results highlight the need for careful sensor selection and experimental design when probing GPCR-G protein coupling.
Asunto(s)
Transferencia de Energía por Resonancia de Bioluminiscencia , Técnicas Biosensibles , Receptores Acoplados a Proteínas G , Humanos , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/química , Transferencia de Energía por Resonancia de Bioluminiscencia/métodos , Células HEK293 , Técnicas Biosensibles/métodos , Conformación Proteica , Transducción de Señal , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/genéticaRESUMEN
TG2 is a unique member of the transglutaminase family as it undergoes a dramatic conformational change, allowing its mutually exclusive function as either a cross-linking enzyme or a G-protein. The enzyme's dysregulated activity has been implicated in a variety of pathologies (e.g., celiac disease, fibrosis, cancer), leading to the development of a wide range of inhibitors. Our group has primarily focused on the development of peptidomimetic targeted covalent inhibitors, the nature and size of which were thought to be important features to abolish TG2's conformational dynamism and ultimately inhibit both its activities. However, we recently demonstrated that the enzyme was unable to bind guanosine triphosphate (GTP) when catalytically inactivated by small molecule inhibitors. In this study, we designed a library of models targeting covalent inhibitors of progressively smaller sizes (15 to 4 atoms in length). We evaluated their ability to inactivate TG2 by measuring their respective kinetic parameters kinact and KI. Their impact on the enzyme's ability to bind GTP was then evaluated and subsequently correlated to the conformational state of the enzyme, as determined via native PAGE and capillary electrophoresis. All irreversible inhibitors evaluated herein locked TG2 in its open conformation and precluded GTP binding. Therefore, we conclude that steric bulk and structural complexity are not necessary factors to consider when designing TG2 inhibitors to abolish G-protein activity.
Asunto(s)
Alquilantes , Dominio Catalítico , Proteínas de Unión al GTP , Proteína Glutamina Gamma Glutamiltransferasa 2 , Transglutaminasas , Transglutaminasas/química , Transglutaminasas/metabolismo , Transglutaminasas/antagonistas & inhibidores , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/metabolismo , Humanos , Alquilantes/química , Alquilantes/farmacología , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/farmacología , Conformación Proteica , Cinética , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacologíaRESUMEN
G-protein-coupled receptors (GPCRs) transmit downstream signals predominantly via G-protein pathways. However, the conformational basis of selective coupling of primary G-protein remains elusive. Histamine receptors H2R and H3R couple with Gs- or Gi-proteins respectively. Here, three cryo-EM structures of H2R-Gs and H3R-Gi complexes are presented at a global resolution of 2.6-2.7 Å. These structures reveal the unique binding pose for endogenous histamine in H3R, wherein the amino group interacts with E2065.46 of H3R instead of the conserved D1143.32 of other aminergic receptors. Furthermore, comparative analysis of the H2R-Gs and H3R-Gi complexes reveals that the structural geometry of TM5/TM6 determines the primary G-protein selectivity in histamine receptors. Machine learning (ML)-based structuromic profiling and functional analysis of class A GPCR-G-protein complexes illustrate that TM5 length, TM5 tilt, and TM6 outward movement are key determinants of the Gs and Gi/o selectivity among the whole Class A family. Collectively, the findings uncover the common structural geometry within class A GPCRs that determines the primary Gs- and Gi/o-coupling selectivity.
Asunto(s)
Microscopía por Crioelectrón , Receptores Acoplados a Proteínas G , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Humanos , Microscopía por Crioelectrón/métodos , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/genética , Histamina/metabolismo , Histamina/química , Receptores Histamínicos H2/metabolismo , Receptores Histamínicos H2/genética , Receptores Histamínicos H2/química , Receptores Histamínicos H3/metabolismo , Receptores Histamínicos H3/química , Receptores Histamínicos H3/genética , Transducción de SeñalRESUMEN
All living organisms deploy cell-autonomous defenses to combat infection. In plants and animals, large supramolecular complexes often activate immune proteins for protection. In this work, we resolved the native structure of a massive host-defense complex that polymerizes 30,000 guanylate-binding proteins (GBPs) over the surface of gram-negative bacteria inside human cells. Construction of this giant nanomachine took several minutes and remained stable for hours, required guanosine triphosphate hydrolysis, and recruited four GBPs plus caspase-4 and Gasdermin D as a cytokine and cell death immune signaling platform. Cryo-electron tomography suggests that GBP1 can adopt an extended conformation for bacterial membrane insertion to establish this platform, triggering lipopolysaccharide release that activated coassembled caspase-4. Our "open conformer" model provides a dynamic view into how the human GBP1 defense complex mobilizes innate immunity to infection.
Asunto(s)
Bacterias , Infecciones Bacterianas , Membrana Celular , Proteínas de Unión al GTP , Reconocimiento de Inmunidad Innata , Humanos , Citocinas/química , Tomografía con Microscopio Electrónico , Proteínas de Unión al GTP/química , Guanosina Trifosfato/química , Hidrólisis , Inmunidad Celular , Microscopía por Crioelectrón , Gasderminas/química , Proteínas de Unión a Fosfato/química , Conformación Proteica , Membrana Celular/química , Membrana Celular/inmunología , Caspasas Iniciadoras/química , Infecciones Bacterianas/inmunología , Bacterias/inmunologíaRESUMEN
The molecular basis of receptor bias in G protein-coupled receptors (GPCRs) caused by mutations that preferentially activate specific intracellular transducers over others remains poorly understood. Two experimentally identified biased variants of ß2-adrenergic receptors (ß2AR), a prototypical GPCR, are a triple mutant (T68F, Y132A, and Y219A) and a single mutant (Y219A); the former bias the receptor toward the ß-arrestin pathway by disfavoring G protein engagement, while the latter induces G protein signaling explicitly due to selection against GPCR kinases (GRKs) that phosphorylate the receptor as a prerequisite of ß-arrestin binding. Though rigorous characterizations have revealed functional implications of these mutations, the atomistic origin of the observed transducer selectivity is not clear. In this study, we investigated the allosteric mechanism of receptor bias in ß2AR using microseconds of all-atom Gaussian accelerated molecular dynamics (GaMD) simulations. Our observations reveal distinct rearrangements in transmembrane helices, intracellular loop 3, and critical residues R1313.50 and Y3267.53 in the conserved motifs D(E)RY and NPxxY for the mutant receptors, leading to their specific transducer interactions. Moreover, partial dissociation of G protein from the receptor core is observed in the simulations of the triple mutant in contrast to the single mutant and wild-type receptor. The reorganization of allosteric communications from the extracellular agonist BI-167107 to the intracellular receptor-transducer interfaces drives the conformational rearrangements responsible for receptor bias in the single and triple mutants. The molecular insights into receptor bias of ß2AR presented here could improve the understanding of biased signaling in GPCRs, potentially opening new avenues for designing novel therapeutics with fewer side-effects and superior efficacy.
Asunto(s)
Simulación de Dinámica Molecular , Transducción de Señal , beta-Arrestinas/metabolismo , Proteínas de Unión al GTP/química , Receptores Adrenérgicos/metabolismo , Receptores Adrenérgicos beta 2/química , Receptores Acoplados a Proteínas G/químicaRESUMEN
G proteins are the major signal proteins of â¼800 receptors for medicines, hormones, neurotransmitters, tastants and odorants. GproteinDb offers integrated genomic, structural, and pharmacological data and tools for analysis, visualization and experiment design. Here, we present the first major update of GproteinDb greatly expanding its coupling data and structural templates, adding AlphaFold2 structure models of GPCR-G protein complexes and advancing the interactive analysis tools for their interfaces underlying coupling selectivity. We present insights on coupling agreement across datasets and parameters, including constitutive activity, agonist-induced activity and kinetics. GproteinDb is accessible at https://gproteindb.org.
Asunto(s)
Bases de Datos de Proteínas , Proteínas de Unión al GTP , Receptores Acoplados a Proteínas G , Biología Computacional , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/genética , Internet , Modelos Moleculares , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , HumanosRESUMEN
Transglutaminase 2 (TG2) plays a vital role in stabilizing extracellular matrix (ECM) proteins through enzymatic crosslinking during tissue growth, repair, and inflammation. TG2 also binds non-covalently to fibronectin (FN), an essential component of the ECM, facilitating cell adhesion, migration, proliferation, and survival. However, the interaction between TG2 and fibrillar FN remains poorly understood, as most studies have focused on soluble or surface-adsorbed FN or FN fragments, which differ in their conformations from insoluble FN fibers. Using a well-established in vitro FN fiber stretch assay, we discovered that the binding of a crosslinking enzyme to ECM fibers is mechano-regulated. TG2 binding to FN is tuned by the mechanical tension of FN fibers, whereby TG2 predominantly co-localizes to low-tension FN fibers, while fiber stretching reduces their affinity for TG2. This mechano-regulated binding relies on the proximity between the N-terminal ß-sandwich and C-terminal ß-barrels of TG2. Crosslinking mass spectrometry (XL-MS) revealed a novel TG2-FN synergy site within TG2's C-terminal ß-barrels that interacts with FN regions located outside of the canonical gelatin binding domain, specifically FNI2 and FNIII14-15. Combining XL-MS distance restraints with molecular docking revealed the mechano-regulated binding mechanism between TG2 and modules FNI7-9 by which mechanical forces regulate TG2-FN interactions. This highlights a previously unrecognized role of TG2 as a tension sensor for FN fibers. This novel interaction mechanism has significant implications in physiology and mechanobiology, including how forces regulate cell adhesion, spreading, migration, phenotype modulation, depending on the tensional state of ECM fibers. Data are available via ProteomeXchange with identifier PXD043976.
Asunto(s)
Fibronectinas , Proteína Glutamina Gamma Glutamiltransferasa 2 , Fibronectinas/metabolismo , Transglutaminasas/genética , Transglutaminasas/química , Transglutaminasas/metabolismo , Simulación del Acoplamiento Molecular , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/metabolismo , Proteínas de la Matriz Extracelular/metabolismoRESUMEN
Receptor-G protein promiscuity is frequently observed in class A G protein-coupled receptors (GPCRs). In particular, GPCRs can couple with G proteins from different families (Gαs, Gαq/11, Gαi/o, and Gα12/13) or the same family subtypes. The molecular basis underlying the selectivity/promiscuity is not fully revealed. We recently reported the structures of kappa opioid receptor (KOR) in complex with the Gi/o family subtypes [Gαi1, GαoA, Gαz, and Gustducin (Gαg)] determined by cryo-electron microscopy (cryo-EM). The structural analysis, in combination with pharmacological studies, provides insights into Gi/o subtype selectivity. Given the conserved sequence identity and activation mechanism between different G protein families, the findings within Gi/o subtypes could be likely extended to other families. Understanding the KOR-Gi/o or GPCR-G protein selectivity will facilitate the development of more precise therapeutics targeting a specific G protein subtype.
Asunto(s)
Microscopía por Crioelectrón , Receptores Acoplados a Proteínas G , Receptores Opioides kappa , Humanos , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Opioides kappa/metabolismo , Receptores Opioides kappa/química , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/genética , Unión Proteica , Animales , Conformación Proteica , Modelos MolecularesRESUMEN
Guanylate-binding proteins (GBPs) are essential interferon-γ-activated large GTPases that play a crucial role in host defense against intracellular bacteria and parasites. While their protective functions rely on protein polymerization, our understanding of the structural intricacies of these multimerized states remains limited. To bridge this knowledge gap, we present dimer models for human GBP1 (hGBP1) and murine GBP2 and 7 (mGBP2 and mGBP7) using an integrative approach, incorporating the crystal structure of hGBP1's GTPase domain dimer, crosslinking mass spectrometry, small-angle X-ray scattering, protein-protein docking, and molecular dynamics simulations. Our investigation begins by comparing the protein dynamics of hGBP1, mGBP2, and mGBP7. We observe that the M/E domain in all three proteins exhibits significant mobility and hinge motion, with mGBP7 displaying a slightly less pronounced motion but greater flexibility in its GTPase domain. These dynamic distinctions can be attributed to variations in the sequences of mGBP7 and hGBP1/mGBP2, resulting in different dimerization modes. Unlike hGBP1 and its close ortholog mGBP2, which exclusively dimerize through their GTPase domains, we find that mGBP7 exhibits three equally probable alternative dimer structures. The GTPase domain of mGBP7 is only partially involved in its dimerization, primarily due to an accumulation of negative charge, allowing mGBP7 to dimerize independently of GTP. Instead, mGBP7 exhibits a strong tendency to dimerize in an antiparallel arrangement across its stalks. The results of this work go beyond the sequence-structure-function relationship, as the sequence differences in mGBP7 and mGBP2/hGBP1 do not lead to different structures, but to different protein dynamics and dimerization. The distinct GBP dimer structures are expected to encode specific functions crucial for disrupting pathogen membranes.
Asunto(s)
Proteínas Portadoras , Proteínas de Unión al GTP , Animales , Ratones , Humanos , Proteínas Portadoras/metabolismo , Proteínas de Unión al GTP/química , GTP Fosfohidrolasas/metabolismo , Unión Proteica , DimerizaciónRESUMEN
G protein-coupled receptors (GPCRs) play a key role in regulating the homeostasis of the internal environment and are closely associated with tumour progression as major mediators of cellular signalling. As a diverse and multifunctional group of proteins, the G protein signalling regulator (RGS) family was proven to be involved in the cellular transduction of GPCRs. Growing evidence has revealed dysregulation of RGS proteins as a common phenomenon and highlighted the key roles of these proteins in human cancers. Furthermore, their differential expression may be a potential biomarker for tumour diagnosis, treatment and prognosis. Most importantly, there are few systematic reviews on the functional/mechanistic characteristics and clinical application of RGS family members at present. In this review, we focus on the G-protein signalling regulator (RGS) family, which includes more than 20 family members. We analysed the classification, basic structure, and major functions of the RGS family members. Moreover, we summarize the expression changes of each RGS family member in various human cancers and their important roles in regulating cancer cell proliferation, stem cell maintenance, tumorigenesis and cancer metastasis. On this basis, we outline the molecular signalling pathways in which some RGS family members are involved in tumour progression. Finally, their potential application in the precise diagnosis, prognosis and treatment of different types of cancers and the main possible problems for clinical application at present are discussed. Our review provides a comprehensive understanding of the role and potential mechanisms of RGS in regulating tumour progression. Video Abstract.
Asunto(s)
Neoplasias , Proteínas RGS , Humanos , Transducción de Señal , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/metabolismo , Proteínas RGS/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Trace-amine-associated receptors (TAARs), a group of biogenic amine receptors, have essential roles in neurological and metabolic homeostasis1. They recognize diverse endogenous trace amines and subsequently activate a range of G-protein-subtype signalling pathways2,3. Notably, TAAR1 has emerged as a promising therapeutic target for treating psychiatric disorders4,5. However, the molecular mechanisms underlying its ability to recognize different ligands remain largely unclear. Here we present nine cryo-electron microscopy structures, with eight showing human and mouse TAAR1 in a complex with an array of ligands, including the endogenous 3-iodothyronamine, two antipsychotic agents, the psychoactive drug amphetamine and two identified catecholamine agonists, and one showing 5-HT1AR in a complex with an antipsychotic agent. These structures reveal a rigid consensus binding motif in TAAR1 that binds to endogenous trace amine stimuli and two extended binding pockets that accommodate diverse chemotypes. Combined with mutational analysis, functional assays and molecular dynamic simulations, we elucidate the structural basis of drug polypharmacology and identify the species-specific differences between human and mouse TAAR1. Our study provides insights into the mechanism of ligand recognition and G-protein selectivity by TAAR1, which may help in the discovery of ligands or therapeutic strategies for neurological and metabolic disorders.
Asunto(s)
Proteínas de Unión al GTP , Receptores Acoplados a Proteínas G , Animales , Humanos , Ratones , Aminas/metabolismo , Anfetamina/metabolismo , Antipsicóticos/química , Antipsicóticos/metabolismo , Sitios de Unión , Catecolaminas/agonistas , Catecolaminas/química , Catecolaminas/metabolismo , Microscopía por Crioelectrón , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/ultraestructura , Ligandos , Simulación de Dinámica Molecular , Mutación , Polifarmacología , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/ultraestructura , Especificidad de la Especie , Especificidad por SustratoRESUMEN
Guanylate-binding proteins (GBPs) are a family of intracellular proteins which have diverse biological functions, including pathogen sensing and host defense against infectious disease. These proteins are expressed in response to interferon (IFN) stimulation and can localize and target intracellular microbes (e.g., bacteria and viruses) by protein trafficking and membrane binding. These properties contribute to the ability of GBPs to induce inflammasome activation, inflammation, and cell death, and to directly disrupt pathogen membranes. Recent biochemical studies have revealed that human GBP1, GBP2, and GBP3 can directly bind to the lipopolysaccharide (LPS) of Gram-negative bacteria. In this review we discuss emerging data highlighting the functional versatility of GBPs, with a focus on their molecular mechanisms of pattern recognition and antimicrobial activity.
Asunto(s)
Antiinfecciosos , Proteínas Portadoras , Humanos , Proteínas de Unión al GTP/química , Inflamasomas/metabolismo , Bacterias/metabolismo , Antiinfecciosos/farmacologíaRESUMEN
G-protein metallochaperones are essential for the proper maturation of numerous metalloenzymes. The G-protein chaperone MMAA in humans (MeaB in bacteria) uses GTP hydrolysis to facilitate the delivery of adenosylcobalamin (AdoCbl) to AdoCbl-dependent methylmalonyl-CoA mutase, an essential metabolic enzyme. This G-protein chaperone also facilitates the removal of damaged cobalamin (Cbl) for repair. Although most chaperones are standalone proteins, isobutyryl-CoA mutase fused (IcmF) has a G-protein domain covalently attached to its target mutase. We previously showed that dimeric MeaB undergoes a 180° rotation to reach a state capable of GTP hydrolysis (an active G-protein state), in which so-called switch III residues of one protomer contact the G-nucleotide of the other protomer. However, it was unclear whether other G-protein chaperones also adopted this conformation. Here, we show that the G-protein domain in a fused system forms a similar active conformation, requiring IcmF oligomerization. IcmF oligomerizes both upon Cbl damage and in the presence of the nonhydrolyzable GTP analog, guanosine-5'-[(ß,γ)-methyleno]triphosphate, forming supramolecular complexes observable by mass photometry and EM. Cryo-EM structural analysis reveals that the second protomer of the G-protein intermolecular dimer props open the mutase active site using residues of switch III as a wedge, allowing for AdoCbl insertion or damaged Cbl removal. With the series of structural snapshots now available, we now describe here the molecular basis of G-protein-assisted AdoCbl-dependent mutase maturation, explaining how GTP binding prepares a mutase for cofactor delivery and how GTP hydrolysis allows the mutase to capture the cofactor.
Asunto(s)
Cobamidas , Metilmalonil-CoA Mutasa , Modelos Moleculares , Chaperonas Moleculares , Cobamidas/metabolismo , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/metabolismo , Guanosina Trifosfato/metabolismo , Isomerasas/química , Isomerasas/metabolismo , Metilmalonil-CoA Mutasa/química , Metilmalonil-CoA Mutasa/metabolismo , Chaperonas Moleculares/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Cupriavidus/química , Cupriavidus/enzimología , Estructura Cuaternaria de Proteína , Dominio Catalítico , Coenzimas/metabolismoRESUMEN
Robust and precise tools are needed to enhance the functionality and resilience of synthetic nanoarchitectures. Here, we have employed directed evolution and rational design to build a fast-acting molecular superglue from a bacterial adhesion protein. We have generated the SnoopLigase2 coupling system, a genetically encoded route for efficient transamidation between SnoopTag2 and DogTag2 peptides. Each peptide was selected for rapid reaction by phage display screening. The optimized set allows more than 99% completion and is compatible with diverse buffers, pH values, and temperatures, accelerating the reaction over 1000-fold. SnoopLigase2 directs a specific reaction in the mammalian secretory pathway, allowing covalent display on the plasma membrane. Transglutaminase 2 (TG2) has a network of interactions and substrates amidst the mammalian cell surface and extracellular matrix. We expressed a modified TG2 with resistance to oxidative inactivation and minimal self-reactivity. SnoopLigase2 enables TG2 functionalization with transforming growth factor alpha (TGFα) in routes that would be impossible through genetic fusion. The TG2:TGFα conjugate retained transamidase activity, stably anchored TGFα for signal activation in the extracellular environment, and reprogrammed cell behavior. This modular toolbox should create new opportunities for molecular assembly, both for novel biomaterials and complex cellular environments.
Asunto(s)
Factor de Crecimiento Transformador alfa , Transglutaminasas , Animales , Transglutaminasas/metabolismo , Factor de Crecimiento Transformador alfa/metabolismo , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Péptidos/química , Membrana Celular/metabolismo , Mamíferos/metabolismoRESUMEN
The septins are a conserved family of filament-forming guanine nucleotide binding proteins, often named the fourth component of the cytoskeleton. Correctly assembled septin structures are required for essential intracellular processes such as cytokinesis, vesicular transport, polarity establishment, and cellular adhesion. Structurally, septins belong to the P-Loop NTPases but they do not mediate signals to effectors through GTP binding and hydrolysis. GTP binding and hydrolysis are believed to contribute to septin complex integrity, but biochemical approaches addressing this topic are hampered by the stability of septin complexes after recombinant expression and the lack of nucleotide-depleted complexes. To overcome this limitation, we used a molecular dynamics-based approach to determine inter-subunit binding free energies in available human septin dimer structures and in their apo forms, which we generated in silico. The nucleotide in the GTPase active subunits SEPT2 and SEPT7, but not in SEPT6, was identified as a stabilizing element in the G interface. Removal of GDP from SEPT2 and SEPT7 results in flipping of a conserved Arg residue and disruption of an extensive hydrogen bond network in the septin unique element, concomitant with a decreased inter-subunit affinity. Based on these findings we propose a singular "lock-hydrolysis" mechanism stabilizing human septin filaments.