RESUMEN
Thrombopoietin (THPO or TPO) is an essential cytokine for hematopoietic stem cell (HSC) maintenance and megakaryocyte differentiation. Here, we report the 3.4 Å resolution cryoelectron microscopy structure of the extracellular TPO-TPO receptor (TpoR or MPL) signaling complex, revealing the basis for homodimeric MPL activation and providing a structural rationalization for genetic loss-of-function thrombocytopenia mutations. The structure guided the engineering of TPO variants (TPOmod) with a spectrum of signaling activities, from neutral antagonists to partial- and super-agonists. Partial agonist TPOmod decoupled JAK/STAT from ERK/AKT/CREB activation, driving a bias for megakaryopoiesis and platelet production without causing significant HSC expansion in mice and showing superior maintenance of human HSCs in vitro. These data demonstrate the functional uncoupling of the two primary roles of TPO, highlighting the potential utility of TPOmod in hematology research and clinical HSC transplantation.
Asunto(s)
Receptores de Trombopoyetina , Trombopoyetina , Animales , Humanos , Ratones , Ciclo Celular , Microscopía por Crioelectrón , Receptores de Trombopoyetina/genética , Trombopoyesis , Metilación de ADNRESUMEN
The complement system is a critical part of our innate immune response, and the terminal products of this cascade, anaphylatoxins C3a and C5a, exert their physiological and pathophysiological responses primarily via two GPCRs, C3aR and C5aR1. However, the molecular mechanism of ligand recognition, activation, and signaling bias of these receptors remains mostly elusive. Here, we present nine cryo-EM structures of C3aR and C5aR1 activated by their natural and synthetic agonists, which reveal distinct binding pocket topologies of complement anaphylatoxins and provide key insights into receptor activation and transducer coupling. We also uncover the structural basis of a naturally occurring mechanism to dampen the inflammatory response of C5a via proteolytic cleavage of the terminal arginine and the G-protein signaling bias elicited by a peptide agonist of C3aR identified here. In summary, our study elucidates the innerworkings of the complement anaphylatoxin receptors and should facilitate structure-guided drug discovery to target these receptors in a spectrum of disorders.
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Anafilatoxinas , Receptores de Complemento , Transducción de Señal , Anafilatoxinas/metabolismo , Complemento C3a/metabolismo , Inmunidad Innata , Receptores de Complemento/metabolismo , Humanos , Animales , RatonesRESUMEN
Angiotensin II type 1 receptor (AT(1)R) is a G protein-coupled receptor that serves as a primary regulator for blood pressure maintenance. Although several anti-hypertensive drugs have been developed as AT(1)R blockers (ARBs), the structural basis for AT(1)R ligand-binding and regulation has remained elusive, mostly due to the difficulties of growing high-quality crystals for structure determination using synchrotron radiation. By applying the recently developed method of serial femtosecond crystallography at an X-ray free-electron laser, we successfully determined the room-temperature crystal structure of the human AT(1)R in complex with its selective antagonist ZD7155 at 2.9-Å resolution. The AT(1)R-ZD7155 complex structure revealed key structural features of AT(1)R and critical interactions for ZD7155 binding. Docking simulations of the clinically used ARBs into the AT(1)R structure further elucidated both the common and distinct binding modes for these anti-hypertensive drugs. Our results thereby provide fundamental insights into AT(1)R structure-function relationship and structure-based drug design.
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Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Receptor de Angiotensina Tipo 1/química , Secuencia de Aminoácidos , Bloqueadores del Receptor Tipo 1 de Angiotensina II/química , Cristalografía por Rayos X , Humanos , Datos de Secuencia Molecular , Mutagénesis , Naftiridinas/química , Naftiridinas/farmacología , Receptor de Angiotensina Tipo 1/genética , Receptor de Angiotensina Tipo 1/metabolismo , Alineación de SecuenciaRESUMEN
γ-Aminobutyric acid (GABA) transporter 1 (GAT1)1 regulates neuronal excitation of the central nervous system by clearing the synaptic cleft of the inhibitory neurotransmitter GABA upon its release from synaptic vesicles. Elevating the levels of GABA in the synaptic cleft, by inhibiting GABA reuptake transporters, is an established strategy to treat neurological disorders, such as epilepsy2. Here we determined the cryo-electron microscopy structure of full-length, wild-type human GAT1 in complex with its clinically used inhibitor tiagabine3, with an ordered part of only 60 kDa. Our structure reveals that tiagabine locks GAT1 in the inward-open conformation, by blocking the intracellular gate of the GABA release pathway, and thus suppresses neurotransmitter uptake. Our results provide insights into the mixed-type inhibition of GAT1 by tiagabine, which is an important anticonvulsant medication. Its pharmacodynamic profile, confirmed by our experimental data, suggests initial binding of tiagabine to the substrate-binding site in the outward-open conformation, whereas our structure presents the drug stalling the transporter in the inward-open conformation, consistent with a two-step mechanism of inhibition4. The presented structure of GAT1 gives crucial insights into the biology and pharmacology of this important neurotransmitter transporter and provides blueprints for the rational design of neuromodulators, as well as moving the boundaries of what is considered possible in single-particle cryo-electron microscopy of challenging membrane proteins.
Asunto(s)
Proteínas Transportadoras de GABA en la Membrana Plasmática , Inhibidores de Recaptación de GABA , Ácido gamma-Aminobutírico , Anticonvulsivantes/química , Anticonvulsivantes/farmacología , Microscopía por Crioelectrón , Proteínas Transportadoras de GABA en la Membrana Plasmática/química , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Proteínas Transportadoras de GABA en la Membrana Plasmática/ultraestructura , Inhibidores de Recaptación de GABA/química , Inhibidores de Recaptación de GABA/farmacología , Humanos , Neurotransmisores/metabolismo , Conformación Proteica/efectos de los fármacos , Tiagabina/química , Tiagabina/metabolismo , Tiagabina/farmacología , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Metabotropic γ-aminobutyric acid receptors (GABAB) are involved in the modulation of synaptic responses in the central nervous system and have been implicated in neuropsychological conditions that range from addiction to psychosis1. GABAB belongs to class C of the G-protein-coupled receptors, and its functional entity comprises an obligate heterodimer that is composed of the GB1 and GB2 subunits2. Each subunit possesses an extracellular Venus flytrap domain, which is connected to a canonical seven-transmembrane domain. Here we present four cryo-electron microscopy structures of the human full-length GB1-GB2 heterodimer: one structure of its inactive apo state, two intermediate agonist-bound forms and an active form in which the heterodimer is bound to an agonist and a positive allosteric modulator. The structures reveal substantial differences, which shed light on the complex motions that underlie the unique activation mechanism of GABAB. Our results show that agonist binding leads to the closure of the Venus flytrap domain of GB1, triggering a series of transitions, first rearranging and bringing the two transmembrane domains into close contact along transmembrane helix 6 and ultimately inducing conformational rearrangements in the GB2 transmembrane domain via a lever-like mechanism to initiate downstream signalling. This active state is stabilized by a positive allosteric modulator binding at the transmembrane dimerization interface.
Asunto(s)
Microscopía por Crioelectrón , Receptores de GABA-B/química , Receptores de GABA-B/ultraestructura , Regulación Alostérica/efectos de los fármacos , Apoproteínas/química , Apoproteínas/metabolismo , Apoproteínas/ultraestructura , Sitios de Unión/efectos de los fármacos , Agonistas de Receptores GABA-B/química , Agonistas de Receptores GABA-B/metabolismo , Agonistas de Receptores GABA-B/farmacología , Humanos , Modelos Moleculares , Dominios Proteicos/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Receptores de GABA-B/metabolismo , Transducción de Señal , Relación Estructura-ActividadRESUMEN
Lipopolysaccharide (LPS) resides in the outer membrane of Gram-negative bacteria where it is responsible for barrier function1,2. LPS can cause death as a result of septic shock, and its lipid A core is the target of polymyxin antibiotics3,4. Despite the clinical importance of polymyxins and the emergence of multidrug resistant strains5, our understanding of the bacterial factors that regulate LPS biogenesis is incomplete. Here we characterize the inner membrane protein PbgA and report that its depletion attenuates the virulence of Escherichia coli by reducing levels of LPS and outer membrane integrity. In contrast to previous claims that PbgA functions as a cardiolipin transporter6-9, our structural analyses and physiological studies identify a lipid A-binding motif along the periplasmic leaflet of the inner membrane. Synthetic PbgA-derived peptides selectively bind to LPS in vitro and inhibit the growth of diverse Gram-negative bacteria, including polymyxin-resistant strains. Proteomic, genetic and pharmacological experiments uncover a model in which direct periplasmic sensing of LPS by PbgA coordinates the biosynthesis of lipid A by regulating the stability of LpxC, a key cytoplasmic biosynthetic enzyme10-12. In summary, we find that PbgA has an unexpected but essential role in the regulation of LPS biogenesis, presents a new structural basis for the selective recognition of lipids, and provides opportunities for future antibiotic discovery.
Asunto(s)
Membrana Celular/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/química , Escherichia coli/patogenicidad , Lipopolisacáridos/química , Lipopolisacáridos/metabolismo , Amidohidrolasas/química , Amidohidrolasas/metabolismo , Secuencias de Aminoácidos , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/metabolismo , Sitios de Unión , Membrana Celular/metabolismo , Estabilidad de Enzimas , Escherichia coli/citología , Escherichia coli/efectos de los fármacos , Genes Esenciales , Hidrolasas/química , Hidrolasas/metabolismo , Lípido A/química , Lípido A/metabolismo , Lipopolisacáridos/biosíntesis , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Modelos Moleculares , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Periplasma/química , Periplasma/metabolismo , Unión Proteica , VirulenciaRESUMEN
The human MT1 and MT2 melatonin receptors1,2 are G-protein-coupled receptors (GPCRs) that help to regulate circadian rhythm and sleep patterns3. Drug development efforts have targeted both receptors for the treatment of insomnia, circadian rhythm and mood disorders, and cancer3, and MT2 has also been implicated in type 2 diabetes4,5. Here we report X-ray free electron laser (XFEL) structures of the human MT2 receptor in complex with the agonists 2-phenylmelatonin (2-PMT) and ramelteon6 at resolutions of 2.8 Å and 3.3 Å, respectively, along with two structures of function-related mutants: H2085.46A (superscripts represent the Ballesteros-Weinstein residue numbering nomenclature7) and N862.50D, obtained in complex with 2-PMT. Comparison of the structures of MT2 with a published structure8 of MT1 reveals that, despite conservation of the orthosteric ligand-binding site residues, there are notable conformational variations as well as differences in [3H]melatonin dissociation kinetics that provide insights into the selectivity between melatonin receptor subtypes. A membrane-buried lateral ligand entry channel is observed in both MT1 and MT2, but in addition the MT2 structures reveal a narrow opening towards the solvent in the extracellular part of the receptor. We provide functional and kinetic data that support a prominent role for intramembrane ligand entry in both receptors, and suggest that there might also be an extracellular entry path in MT2. Our findings contribute to a molecular understanding of melatonin receptor subtype selectivity and ligand access modes, which are essential for the design of highly selective melatonin tool compounds and therapeutic agents.
Asunto(s)
Electrones , Rayos Láser , Modelos Moleculares , Receptor de Melatonina MT2/química , Receptor de Melatonina MT2/metabolismo , Cristalización , Diabetes Mellitus Tipo 2/genética , Humanos , Indenos/química , Indenos/metabolismo , Ligandos , Melatonina/análogos & derivados , Melatonina/química , Melatonina/metabolismo , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Mutación , Receptor de Melatonina MT1/química , Receptor de Melatonina MT1/metabolismo , Receptor de Melatonina MT2/genética , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone that maintains circadian rhythms1 by synchronization to environmental cues and is involved in diverse physiological processes2 such as the regulation of blood pressure and core body temperature, oncogenesis, and immune function3. Melatonin is formed in the pineal gland in a light-regulated manner4 by enzymatic conversion from 5-hydroxytryptamine (5-HT or serotonin), and modulates sleep and wakefulness5 by activating two high-affinity G-protein-coupled receptors, type 1A (MT1) and type 1B (MT2)3,6. Shift work, travel, and ubiquitous artificial lighting can disrupt natural circadian rhythms; as a result, sleep disorders affect a substantial population in modern society and pose a considerable economic burden7. Over-the-counter melatonin is widely used to alleviate jet lag and as a safer alternative to benzodiazepines and other sleeping aids8,9, and is one of the most popular supplements in the United States10. Here, we present high-resolution room-temperature X-ray free electron laser (XFEL) structures of MT1 in complex with four agonists: the insomnia drug ramelteon11, two melatonin analogues, and the mixed melatonin-serotonin antidepressant agomelatine12,13. The structure of MT2 is described in an accompanying paper14. Although the MT1 and 5-HT receptors have similar endogenous ligands, and agomelatine acts on both receptors, the receptors differ markedly in the structure and composition of their ligand pockets; in MT1, access to the ligand pocket is tightly sealed from solvent by extracellular loop 2, leaving only a narrow channel between transmembrane helices IV and V that connects it to the lipid bilayer. The binding site is extremely compact, and ligands interact with MT1 mainly by strong aromatic stacking with Phe179 and auxiliary hydrogen bonds with Asn162 and Gln181. Our structures provide an unexpected example of atypical ligand entry for a non-lipid receptor, lay the molecular foundation of ligand recognition by melatonin receptors, and will facilitate the design of future tool compounds and therapeutic agents, while their comparison to 5-HT receptors yields insights into the evolution and polypharmacology of G-protein-coupled receptors.
Asunto(s)
Electrones , Rayos Láser , Modelos Moleculares , Receptor de Melatonina MT1/química , Receptor de Melatonina MT1/metabolismo , Acetamidas/química , Acetamidas/metabolismo , Secuencia de Aminoácidos , Antidepresivos/química , Antidepresivos/metabolismo , Cristalización , Humanos , Indenos/química , Indenos/metabolismo , Ligandos , Melatonina/análogos & derivados , Melatonina/química , Simulación del Acoplamiento Molecular , Mutación , Receptor de Melatonina MT1/agonistas , Receptor de Melatonina MT1/genética , Receptor de Serotonina 5-HT2C/química , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Change history: In this Letter, the rotation signs around 90°, 135° and 15° were missing and in the HTML, Extended Data Tables 2 and 3 were the wrong tables; these errors have been corrected online.
RESUMEN
Genetic variants in the SLC6A1 gene can cause a broad phenotypic disease spectrum by altering the protein function. Thus, systematically curated clinically relevant genotype-phenotype associations are needed to understand the disease mechanism and improve therapeutic decision-making. We aggregated genetic and clinical data from 172 individuals with likely pathogenic/pathogenic (lp/p) SLC6A1 variants and functional data for 184 variants (14.1% lp/p). Clinical and functional data were available for a subset of 126 individuals. We explored the potential associations of variant positions on the GAT1 3D structure with variant pathogenicity, altered molecular function and phenotype severity using bioinformatic approaches. The GAT1 transmembrane domains 1, 6 and extracellular loop 4 (EL4) were enriched for patient over population variants. Across functionally tested missense variants (n = 156), the spatial proximity from the ligand was associated with loss-of-function in the GAT1 transporter activity. For variants with complete loss of in vitro GABA uptake, we found a 4.6-fold enrichment in patients having severe disease versus non-severe disease (P = 2.9 × 10-3, 95% confidence interval: 1.5-15.3). In summary, we delineated associations between the 3D structure and variant pathogenicity, variant function and phenotype in SLC6A1-related disorders. This knowledge supports biology-informed variant interpretation and research on GAT1 function. All our data can be interactively explored in the SLC6A1 portal (https://slc6a1-portal.broadinstitute.org/).
Asunto(s)
Proteínas Transportadoras de GABA en la Membrana Plasmática , Estudios de Asociación Genética , Mutación Missense , Humanos , Proteínas Transportadoras de GABA en la Membrana Plasmática/genética , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , FenotipoRESUMEN
Prostaglandin D2 (PGD2) signals through the G protein-coupled receptor (GPCR) CRTH2 to mediate various inflammatory responses. CRTH2 is the only member of the prostanoid receptor family that is phylogenetically distant from others, implying a nonconserved mechanism of lipid action on CRTH2. Here, we report a crystal structure of human CRTH2 bound to a PGD2 derivative, 15R-methyl-PGD2 (15mPGD2), by serial femtosecond crystallography. The structure revealed a "polar group in"-binding mode of 15mPGD2 contrasting the "polar group out"-binding mode of PGE2 in its receptor EP3. Structural comparison analysis suggested that these two lipid-binding modes, associated with distinct charge distributions of ligand-binding pockets, may apply to other lipid GPCRs. Molecular dynamics simulations together with mutagenesis studies also identified charged residues at the ligand entry port that function to capture lipid ligands of CRTH2 from the lipid bilayer. Together, our studies suggest critical roles of charge environment in lipid recognition by GPCRs.
Asunto(s)
Receptores Inmunológicos/química , Receptores Inmunológicos/metabolismo , Receptores de Prostaglandina/química , Receptores de Prostaglandina/metabolismo , Cristalografía por Rayos X/métodos , Humanos , Metabolismo de los Lípidos , Simulación de Dinámica Molecular , Mutación , Prostaglandina D2/química , Prostaglandina D2/metabolismo , Conformación Proteica , Receptores Inmunológicos/genética , Receptores de Prostaglandina/genéticaRESUMEN
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common blood disorder, presenting multiple symptoms, including hemolytic anemia. It affects 400 million people worldwide, with more than 160 single mutations reported in G6PD. The most severe mutations (about 70) are classified as class I, leading to more than 90% loss of activity of the wild-type G6PD. The crystal structure of G6PD reveals these mutations are located away from the active site, concentrating around the noncatalytic NADP+-binding site and the dimer interface. However, the molecular mechanisms of class I mutant dysfunction have remained elusive, hindering the development of efficient therapies. To resolve this, we performed integral structural characterization of five G6PD mutants, including four class I mutants, associated with the noncatalytic NADP+ and dimerization, using crystallography, small-angle X-ray scattering (SAXS), cryogenic electron microscopy (cryo-EM), and biophysical analyses. Comparisons with the structure and properties of the wild-type enzyme, together with molecular dynamics simulations, bring forward a universal mechanism for this severe G6PD deficiency due to the class I mutations. We highlight the role of the noncatalytic NADP+-binding site that is crucial for stabilization and ordering two ß-strands in the dimer interface, which together communicate these distant structural aberrations to the active site through a network of additional interactions. This understanding elucidates potential paths for drug development targeting G6PD deficiency.
Asunto(s)
Coenzimas/química , Glucosafosfato Deshidrogenasa/química , Leucina/química , Mutación , NADP/química , Prolina/química , Sitios de Unión , Clonación Molecular , Coenzimas/metabolismo , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Glucosafosfato Deshidrogenasa/genética , Glucosafosfato Deshidrogenasa/metabolismo , Deficiencia de Glucosafosfato Deshidrogenasa/enzimología , Deficiencia de Glucosafosfato Deshidrogenasa/genética , Deficiencia de Glucosafosfato Deshidrogenasa/patología , Humanos , Cinética , Leucina/metabolismo , Modelos Moleculares , Simulación de Dinámica Molecular , NADP/metabolismo , Prolina/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Misoprostol is a life-saving drug in many developing countries for women at risk of post-partum hemorrhaging owing to its affordability, stability, ease of administration and clinical efficacy. However, misoprostol lacks receptor and tissue selectivities, and thus its use is accompanied by a number of serious side effects. The development of pharmacological agents combining the advantages of misoprostol with improved selectivity is hindered by the absence of atomic details of misoprostol action in labor induction. Here, we present the 2.5 Å resolution crystal structure of misoprostol free-acid form bound to the myometrium labor-inducing prostaglandin E2 receptor 3 (EP3). The active state structure reveals a completely enclosed binding pocket containing a structured water molecule that coordinates misoprostol's ring structure. Modeling of selective agonists in the EP3 structure reveals rationales for selectivity. These findings will provide the basis for the next generation of uterotonic drugs that will be suitable for administration in low resource settings.
Asunto(s)
Misoprostol/química , Subtipo EP3 de Receptores de Prostaglandina E/química , Subtipo EP3 de Receptores de Prostaglandina E/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Dinoprostona/análogos & derivados , Dinoprostona/química , Dinoprostona/metabolismo , Humanos , Misoprostol/metabolismo , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Subtipo EP3 de Receptores de Prostaglandina E/agonistas , Subtipo EP3 de Receptores de Prostaglandina E/genética , Transducción de Señal , Agua/químicaRESUMEN
In the version of this article originally published, the present address for Petr Popov was incorrectly listed as 'Koltech Institute of Science & Technology, Moscow, Russia'. The correct present address is 'Skolkovo Institute of Science and Technology, Moscow, Russia'. The error has been corrected in the HTML and PDF versions of the paper.
RESUMEN
G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a â¼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology.
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Arrestina/química , Arrestina/metabolismo , Rodopsina/química , Rodopsina/metabolismo , Animales , Sitios de Unión , Cristalografía por Rayos X , Disulfuros/química , Disulfuros/metabolismo , Humanos , Rayos Láser , Ratones , Modelos Moleculares , Complejos Multiproteicos/biosíntesis , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Unión Proteica , Reproducibilidad de los Resultados , Transducción de Señal , Rayos XRESUMEN
The ribosome translates nucleotide sequences of messenger RNA to proteins through selection of cognate transfer RNA according to the genetic code. To date, structural studies of ribosomal decoding complexes yielding high-resolution data have predominantly relied on experiments performed at cryogenic temperatures. New light sources like the X-ray free electron laser (XFEL) have enabled data collection from macromolecular crystals at ambient temperature. Here, we report an X-ray crystal structure of the Thermus thermophilus 30S ribosomal subunit decoding complex to 3.45 Å resolution using data obtained at ambient temperature at the Linac Coherent Light Source (LCLS). We find that this ambient-temperature structure is largely consistent with existing cryogenic-temperature crystal structures, with key residues of the decoding complex exhibiting similar conformations, including adenosine residues 1492 and 1493. Minor variations were observed, namely an alternate conformation of cytosine 1397 near the mRNA channel and the A-site. Our serial crystallography experiment illustrates the amenability of ribosomal microcrystals to routine structural studies at ambient temperature, thus overcoming a long-standing experimental limitation to structural studies of RNA and RNA-protein complexes at near-physiological temperatures.
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Sustancias Macromoleculares/química , Conformación de Ácido Nucleico , Subunidades Ribosómicas Pequeñas Bacterianas/química , Ribosomas/química , Adenosina/química , Cristalografía por Rayos X , Código Genético , Rayos Láser , ARN Mensajero/química , ARN Mensajero/genética , Subunidades Ribosómicas Pequeñas Bacterianas/genética , Ribosomas/genética , Temperatura , Thermus thermophilus/química , Rayos XRESUMEN
The bacterial 30S ribosomal subunit is a primary antibiotic target. Despite decades of discovery, the mechanisms by which antibiotic binding induces ribosomal dysfunction are not fully understood. Ambient temperature crystallographic techniques allow more biologically relevant investigation of how local antibiotic binding site interactions trigger global subunit rearrangements that perturb protein synthesis. Here, the structural effects of 2-deoxystreptamine (paromomycin and sisomicin), a novel sisomicin derivative, N1-methyl sulfonyl sisomicin (N1MS) and the non-deoxystreptamine (streptomycin) aminoglycosides on the ribosome at ambient and cryogenic temperatures were examined. Comparative studies led to three main observations. First, individual aminoglycoside-ribosome interactions in the decoding center were similar for cryogenic versus ambient temperature structures. Second, analysis of a highly conserved GGAA tetraloop of h45 revealed aminoglycoside-specific conformational changes, which are affected by temperature only for N1MS. We report the h44-h45 interface in varying states, i.e. engaged, disengaged and in equilibrium. Third, we observe aminoglycoside-induced effects on 30S domain closure, including a novel intermediary closure state, which is also sensitive to temperature. Analysis of three ambient and five cryogenic crystallography datasets reveal a correlation between h44-h45 engagement and domain closure. These observations illustrate the role of ambient temperature crystallography in identifying dynamic mechanisms of ribosomal dysfunction induced by local drug-binding site interactions. Together, these data identify tertiary ribosomal structural changes induced by aminoglycoside binding that provides functional insight and targets for drug design.
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Aminoglicósidos/química , Conformación de Ácido Nucleico/efectos de los fármacos , ARN Ribosómico/química , Ribosomas/química , Aminoglicósidos/antagonistas & inhibidores , Antibacterianos/química , Antibacterianos/farmacología , Sitios de Unión , Escherichia coli/genética , Hexosaminas/química , Hexosaminas/farmacología , Humanos , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/química , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Ribosómico/efectos de los fármacos , Ribosomas/efectos de los fármacos , Estreptomicina/química , Estreptomicina/farmacologíaRESUMEN
To understand how molecules function in biological systems, new methods are required to obtain atomic resolution structures from biological material under physiological conditions. Intense femtosecond-duration pulses from X-ray free-electron lasers (XFELs) can outrun most damage processes, vastly increasing the tolerable dose before the specimen is destroyed. This in turn allows structure determination from crystals much smaller and more radiation sensitive than previously considered possible, allowing data collection from room temperature structures and avoiding structural changes due to cooling. Regardless, high-resolution structures obtained from XFEL data mostly use crystals far larger than 1 µm3 in volume, whereas the X-ray beam is often attenuated to protect the detector from damage caused by intense Bragg spots. Here, we describe the 2 Å resolution structure of native nanocrystalline granulovirus occlusion bodies (OBs) that are less than 0.016 µm3 in volume using the full power of the Linac Coherent Light Source (LCLS) and a dose up to 1.3 GGy per crystal. The crystalline shell of granulovirus OBs consists, on average, of about 9,000 unit cells, representing the smallest protein crystals to yield a high-resolution structure by X-ray crystallography to date. The XFEL structure shows little to no evidence of radiation damage and is more complete than a model determined using synchrotron data from recombinantly produced, much larger, cryocooled granulovirus granulin microcrystals. Our measurements suggest that it should be possible, under ideal experimental conditions, to obtain data from protein crystals with only 100 unit cells in volume using currently available XFELs and suggest that single-molecule imaging of individual biomolecules could almost be within reach.
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Cristalografía/métodos , Electrones , Granulovirus/ultraestructura , Péptidos y Proteínas de Señalización Intercelular/química , Rayos Láser , Cristalografía/instrumentación , Granulovirus/química , Modelos Moleculares , Progranulinas , Estructura Secundaria de Proteína , SincrotronesRESUMEN
We describe a concentric-flow electrokinetic injector for efficiently delivering microcrystals for serial femtosecond X-ray crystallography analysis that enables studies of challenging biological systems in their unadulterated mother liquor. We used the injector to analyze microcrystals of Geobacillus stearothermophilus thermolysin (2.2-Å structure), Thermosynechococcus elongatus photosystem II (<3-Å diffraction) and Thermus thermophilus small ribosomal subunit bound to the antibiotic paromomycin at ambient temperature (3.4-Å structure).