RESUMEN
Balanced pan-class I phosphoinositide 3-kinase inhibition as an approach to cancer treatment offers the prospect of treating a broad range of tumor types and/or a way to achieve greater efficacy with a single inhibitor. Taking buparlisib as the starting point, the balanced pan-class I PI3K inhibitor 40 (NVP-CLR457) was identified with what was considered to be a best-in-class profile. Key to the optimization to achieve this profile was eliminating a microtubule stabilizing off-target activity, balancing the pan-class I PI3K inhibition profile, minimizing CNS penetration, and developing an amorphous solid dispersion formulation. A rationale for the poor tolerability profile of 40 in a clinical study is discussed.
Asunto(s)
Antineoplásicos , Fosfatidilinositol 3-Quinasas , Aminopiridinas/farmacología , Antineoplásicos/farmacología , Línea Celular Tumoral , Compuestos Orgánicos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
The transcription factor PAX8 is critical for the development of the thyroid and urogenital system. Comprehensive genomic screens furthermore indicate an additional oncogenic role for PAX8 in renal and ovarian cancers. While a plethora of PAX8-regulated genes in different contexts have been proposed, we still lack a mechanistic understanding of how PAX8 engages molecular complexes to drive disease-relevant oncogenic transcriptional programs. Here we show that protein isoforms originating from the MECOM locus form a complex with PAX8. These include MDS1-EVI1 (also called PRDM3) for which we map its interaction with PAX8 in vitro and in vivo. We show that PAX8 binds a large number of genomic sites and forms transcriptional hubs. At a subset of these, PAX8 together with PRDM3 regulates a specific gene expression module involved in adhesion and extracellular matrix. This gene module correlates with PAX8 and MECOM expression in large scale profiling of cell lines, patient-derived xenografts (PDXs) and clinical cases and stratifies gynecological cancer cases with worse prognosis. PRDM3 is amplified in ovarian cancers and we show that the MECOM locus and PAX8 sustain in vivo tumor growth, further supporting that the identified function of the MECOM locus underlies PAX8-driven oncogenic functions in ovarian cancer.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Proteína del Locus del Complejo MDS1 y EV11/genética , Neoplasias Ováricas/genética , Factor de Transcripción PAX8/genética , Animales , Línea Celular Tumoral , Femenino , Células HEK293 , Humanos , Proteína del Locus del Complejo MDS1 y EV11/metabolismo , Ratones Desnudos , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Factor de Transcripción PAX8/metabolismo , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Carga Tumoral/genética , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Bruton's tyrosine kinase (BTK), a cytoplasmic tyrosine kinase, plays a central role in immunity and is considered an attractive target for treating autoimmune diseases. The use of currently marketed covalent BTK inhibitors is limited to oncology indications based on their suboptimal kinase selectivity. We describe the discovery and preclinical profile of LOU064 (remibrutinib, 25), a potent, highly selective covalent BTK inhibitor. LOU064 exhibits an exquisite kinase selectivity due to binding to an inactive conformation of BTK and has the potential for a best-in-class covalent BTK inhibitor for the treatment of autoimmune diseases. It demonstrates potent in vivo target occupancy with an EC90 of 1.6 mg/kg and dose-dependent efficacy in rat collagen-induced arthritis. LOU064 is currently being tested in phase 2 clinical studies for chronic spontaneous urticaria and Sjoegren's syndrome.
Asunto(s)
Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Agammaglobulinemia Tirosina Quinasa/metabolismo , Descubrimiento de Drogas/métodos , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Agammaglobulinemia Tirosina Quinasa/química , Animales , Benzamidas/química , Benzamidas/metabolismo , Benzamidas/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Compuestos Bicíclicos Heterocíclicos con Puentes/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Cristalografía por Rayos X/métodos , Perros , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Ratones , Unión Proteica/fisiología , Inhibidores de Proteínas Quinasas/química , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ratas , Ratas Endogámicas Lew , OvinosRESUMEN
Bruton's tyrosine kinase (BTK) is a member of the TEC kinase family and is selectively expressed in a subset of immune cells. It is a key regulator of antigen receptor signaling in B cells and of Fc receptor signaling in mast cells and macrophages. A BTK inhibitor will likely have a positive impact on autoimmune diseases which are caused by autoreactive B cells and immune-complex driven inflammation. We report the design, optimization, and characterization of potent and selective covalent BTK inhibitors. Starting from the selective reversible inhibitor 3 binding to an inactive conformation of BTK, we designed covalent irreversible compounds by attaching an electrophilic warhead to reach Cys481. The first prototype 4 covalently modified BTK and showed an excellent kinase selectivity including several Cys-containing kinases, validating the design concept. In addition, this compound blocked FcγR-mediated hypersensitivity in vivo. Optimization of whole blood potency and metabolic stability resulted in compounds such as 8, which maintained the excellent kinase selectivity and showed improved BTK occupancy in vivo.
RESUMEN
The predominant expression of phosphoinositide 3-kinase δ (PI3Kδ) in leukocytes and its critical role in B and T cell functions led to the hypothesis that selective inhibitors of this isoform would have potential as therapeutics for the treatment of allergic and inflammatory disease. Targeting specifically PI3Kδ should avoid potential side effects associated with the ubiquitously expressed PI3Kα and ß isoforms. We disclose how morphing the heterocyclic core of previously discovered 4,6-diaryl quinazolines to a significantly less lipophilic 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine, followed by replacement of one of the phenyl groups with a pyrrolidine-3-amine, led to a compound series with an optimal on-target profile and good ADME properties. A final lipophilicity adjustment led to the discovery of CDZ173 (leniolisib), a potent PI3Kδ selective inhibitor with suitable properties and efficacy for clinical development as an anti-inflammatory therapeutic. In vitro, CDZ173 inhibits a large spectrum of immune cell functions, as demonstrated in B and T cells, neutrophils, monocytes, basophils, plasmocytoid dendritic cells, and mast cells. In vivo, CDZ173 inhibits B cell activation in rats and monkeys in a concentration- and time-dependent manner. After prophylactic or therapeutic dosing, CDZ173 potently inhibited antigen-specific antibody production and reduced disease symptoms in a rat collagen-induced arthritis model. Structurally, CDZ173 differs significantly from the first generation of PI3Kδ and PI3Kγδ-selective clinical compounds. Therefore, CDZ173 could differentiate by a more favorable safety profile. CDZ173 is currently in clinical studies in patients suffering from primary Sjögren's syndrome and in APDS/PASLI, a disease caused by gain-of-function mutations of PI3Kδ.
RESUMEN
Inhibition of the lipid kinase PI3Kδ is a promising principle to treat B and T cell driven inflammatory diseases. Using a scaffold deconstruction-reconstruction strategy, we identified 4-aryl quinazolines that were optimized into potent PI3Kδ isoform selective analogues with good pharmacokinetic properties. With compound 11, we illustrate that biochemical PI3Kδ inhibition translates into modulation of isoform-dependent immune cell function (human, rat, and mouse). After oral administration of compound 11 to rats, proximal PD markers are inhibited, and dose-dependent efficacy in a mechanistic plaque forming cell assay could be demonstrated.
RESUMEN
Cancer Osaka thyroid (COT) kinase is an important regulator of pro-inflammatory cytokines in macrophages. Thus, pharmacologic inhibition of COT should be a valid approach to therapeutically intervene in the pathogenesis of macrophage-driven inflammatory diseases such as rheumatoid arthritis. We report the discovery and chemical optimization of a novel series of COT kinase inhibitors, with unprecedented nanomolar potency for the inhibition of TNFα. Pharmacological profiling in vivo revealed a high metabolism of these compounds in rats which was demonstrated to be predominantly attributed to aldehyde oxidase. Due to the very low activity of hepatic AO in the dog, the selected candidate 32 displayed significant blood exposure in dogs which resulted in a clear prevention of inflammation-driven lameness. Taken together, the described compounds both potently and selectively inhibit COT kinase in primary human cells and ameliorate inflammatory pathologies in vivo, supporting the notion that COT is an appropriate therapeutic target for inflammatory diseases.
Asunto(s)
Descubrimiento de Drogas , Imidazoles/farmacología , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Quinolinas/farmacología , Animales , Cristalografía por Rayos X , Perros , Relación Dosis-Respuesta a Droga , Humanos , Imidazoles/síntesis química , Imidazoles/química , Quinasas Quinasa Quinasa PAM/metabolismo , Masculino , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Proteínas Proto-Oncogénicas/metabolismo , Quinolinas/síntesis química , Quinolinas/química , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
The discovery of inhibitors targeting novel allosteric kinase sites is very challenging. Such compounds, however, once identified could offer exquisite levels of selectivity across the kinome. Herein we report our structure-based optimization strategy of a dibenzodiazepine hit 1, discovered in a fragment-based screen, yielding highly potent and selective inhibitors of PAK1 such as 2 and 3. Compound 2 was cocrystallized with PAK1 to confirm binding to an allosteric site and to reveal novel key interactions. Compound 3 modulated PAK1 at the cellular level and due to its selectivity enabled valuable research to interrogate biological functions of the PAK1 kinase.
RESUMEN
Macrophages are important cellular effectors in innate immune responses and play a major role in autoimmune diseases such as rheumatoid arthritis. Cancer Osaka thyroid (COT) kinase, also known as mitogen-activated protein kinase kinase kinase 8 (MAP3K8) and tumor progression locus 2 (Tpl-2), is a serine-threonine (ST) kinase and is a key regulator in the production of pro-inflammatory cytokines in macrophages. Due to its pivotal role in immune biology, COT kinase has been identified as an attractive target for pharmaceutical research that is directed at the discovery of orally available, selective, and potent inhibitors for the treatment of autoimmune disorders and cancer. The production of monomeric, recombinant COT kinase has proven to be very difficult, and issues with solubility and stability of the enzyme have hampered the discovery and optimization of potent and selective inhibitors. We developed a protocol for the production of recombinant human COT kinase that yields pure and highly active enzyme in sufficient yields for biochemical and structural studies. The quality of the enzyme allowed us to establish a robust in vitro phosphorylation assay for the efficient biochemical characterization of COT kinase inhibitors and to determine the x-ray co-crystal structures of the COT kinase domain in complex with two ATP-binding site inhibitors. The structures presented in this study reveal two distinct ligand binding modes and a unique kinase domain architecture that has not been observed previously. The structurally versatile active site significantly impacts the design of potent, low molecular weight COT kinase inhibitors.
Asunto(s)
Quinasas Quinasa Quinasa PAM/química , Pliegue de Proteína , Proteínas Proto-Oncogénicas/química , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Conformación Proteica , Proteínas Recombinantes/químicaRESUMEN
Bone marrow kinase on chromosome X (BMX) is a cytosolic tyrosine kinase and a member of the TEC kinase family. BMX is expressed in hematopoietic cells of the myeloid lineage where it participates in the immune response. It is also involved in the response to ischemia and pressure overload in the endocardium and the cardiac endothelium. Moreover, BMX is expressed in several types of cancers and very recently has been shown to mediate the survival and tumorigenicity of glioblastoma cancer stem cells. In the inflammatory response BMX regulates the secretion of proinflammatory cytokines induced by TNFα, IL-1ß, and TLR agonists. It is required for the activation of the MAP kinase and NFκB pathways and acts at the level of the essential TAK1/TAB complex. Cellular regulation of the IL-8 promoter by BMX is dependent on membrane localization mediated by its pleckstrin homology domain, as well as on BMX kinase activity. BMX deficiency confers protection from arthritis in a mouse model known to be dependent on macrophages and IL-1ß. Genetic replacement of BMX with a kinase-inactive allele surprisingly restored susceptibility to arthritis, suggesting that in vivo BMX kinase activity can be dispensable. This review summarizes recent advances in the knowledge of BMX biology and their relevance for translational medicine.
Asunto(s)
Enfermedades Cardiovasculares/metabolismo , Inflamación/metabolismo , Neoplasias/metabolismo , Proteínas Tirosina Quinasas/fisiología , Animales , Artritis/metabolismo , Línea Celular , Humanos , Ratones , Modelos Moleculares , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal/fisiologíaRESUMEN
An easy to use and robust approach for amino acid type selective isotope labeling in insect cells is presented. It relies on inexpensive commercial media and can be implemented in laboratories without sophisticated infrastructure. In contrast to previous protocols, where either high protein amounts or high incorporation ratios were obtained, here we achieve both at the same time. By supplementing media with a well considered amount of yeast extract, similar protein amounts as with full media are obtained, without compromising on isotope incorporation. In single and dual amino acid labeling experiments incorporation ratios are consistently ≥90% for all amino acids tested. This enables NMR studies of eukaryotic proteins and their interactions even for proteins with low expression levels. We show applications with human kinases, where protein-ligand interactions are characterized by 2D [(15)N, (1)H]- and [(13)C, (1)H]-HSQC spectra.
Asunto(s)
Marcaje Isotópico/métodos , Proteínas Proto-Oncogénicas c-akt/análisis , Proteínas Proto-Oncogénicas c-akt/química , Aminoácidos/análisis , Aminoácidos/química , Aminoácidos/metabolismo , Animales , Baculoviridae/química , Células Cultivadas , Humanos , Insectos , Resonancia Magnética Nuclear Biomolecular/métodosRESUMEN
Radiation damage is the major impediment for obtaining structural information from biological samples by using ionizing radiation such as x-rays or electrons. The knowledge of underlying processes especially at cryogenic temperatures is still fragmentary, and a consistent mechanism has not been found yet. By using a combination of single-crystal x-ray diffraction, small-angle scattering, and qualitative and quantitative radiolysis experiments, we show that hydrogen gas, formed inside the sample during irradiation, rather than intramolecular bond cleavage between non-hydrogen atoms, is mainly responsible for the loss of high-resolution information and contrast in diffraction experiments and microscopy. The experiments that are presented in this paper cover a temperature range between 5 and 160 K and reveal that the commonly used temperature in x-ray crystallography of 100 K is not optimal in terms of minimizing radiation damage and thereby increasing the structural information obtainable in a single experiment. At 50 K, specific radiation damage to disulfide bridges is reduced by a factor of 4 compared to 100 K, and samples can tolerate a factor of 2.6 and 3.9 higher dose, as judged by the increase of R(free) values of elastase and cubic insulin crystals, respectively.
Asunto(s)
Frío , Rayos X , Cristalografía por Rayos XRESUMEN
Baculoviruses are ubiquitous insect viruses well known for their use as bioinsecticides, gene therapy vectors, and protein expression systems. Overexpression of recombinant proteins in insect cell culture utilizes the strong promoter of the polyhedrin gene. In infected larvae, the polyhedrin protein forms robust intracellular crystals called polyhedra, which protect encased virions for prolonged periods in the environment. Polyhedra are produced by two unrelated families of insect viruses, baculoviruses and cypoviruses. The atomic structure of cypovirus polyhedra revealed an intricate packing of trimers, which are interconnected by a projecting N-terminal helical arm of the polyhedrin molecule. Baculovirus and cypovirus polyhedra share nearly identical lattices, and the N-terminal region of the otherwise unrelated baculovirus polyhedrin protein sequence is also predicted to be alpha-helical. These results suggest homology between the proteins and a common structural basis for viral polyhedra. Here, we present the 2.2-A structure of baculovirus polyhedra determined by x-ray crystallography from microcrystals produced in vivo. We show that the underlying molecular organization is, in fact, very different. Although both polyhedra have nearly identical unit cell dimensions and share I23 symmetry, the polyhedrin molecules are structurally unrelated and pack differently in the crystals. In particular, disulfide bonds and domain-swapped N-terminal domains stabilize the building blocks of baculovirus polyhedra and interlocking C-terminal arms join unit cells together. We show that the N-terminal projecting helical arms have different structural roles in baculovirus and cypovirus polyhedra and conclude that there is no structural evidence for a common evolutionary origin for both classes of polyhedra.
Asunto(s)
Nucleopoliedrovirus/química , Nucleopoliedrovirus/ultraestructura , Reoviridae/química , Reoviridae/ultraestructura , Proteínas Estructurales Virales/química , Proteínas Estructurales Virales/ultraestructura , Animales , Línea Celular , Cristalización , Microscopía Electrónica de Rastreo , Modelos Moleculares , Mariposas Nocturnas , Complejos Multiproteicos/química , Complejos Multiproteicos/ultraestructura , Nucleopoliedrovirus/genética , Proteínas de la Matriz de Cuerpos de Oclusión , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/ultraestructura , Especificidad de la Especie , Spodoptera , Proteínas Estructurales Virales/genéticaRESUMEN
Radiation damage is one of the major impediments in obtaining high-resolution structural information utilizing ionizing radiation. From electron microscopy it is known that electron irradiation of biological samples results in the formation of molecular hydrogen. In the present work radiation-induced structural changes of the polypeptide cyclosporine A were observed at a temperature of 100 K. Bond length changes are thought to be due to radiation-induced hydrogen abstraction which chemically modifies the molecules in an irreversible way. The resulting formation of molecular hydrogen might explain the observed increase of the crystal mosaicity, which has also been reported in many previous radiation damage studies.
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Ciclosporina/química , Ciclosporina/efectos de la radiación , Hidrógeno/química , Hidrógeno/efectos de la radiación , Modelos Químicos , Modelos Moleculares , Simulación por Computador , Cristalización/métodos , Conformación Molecular/efectos de la radiación , Dosis de RadiaciónRESUMEN
Compartmentalization is an important organizational feature of life. It occurs at varying levels of complexity ranging from eukaryotic organelles and the bacterial microcompartments, to the molecular reaction chambers formed by enzyme assemblies. The structural basis of enzyme encapsulation in molecular compartments is poorly understood. Here we show, using X-ray crystallographic, biochemical and EM experiments, that a widespread family of conserved bacterial proteins, the linocin-like proteins, form large assemblies that function as a minimal compartment to package enzymes. We refer to this shell-forming protein as 'encapsulin'. The crystal structure of such a particle from Thermotoga maritima determined at 3.1-angstroms resolution reveals that 60 copies of the monomer assemble into a thin, icosahedral shell with a diameter of 240 angstroms. The interior of this nanocompartment is lined with conserved binding sites for short polypeptide tags present as C-terminal extensions of enzymes involved in oxidative-stress response.
Asunto(s)
Thermotoga maritima/enzimología , Thermotoga maritima/ultraestructura , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Brevibacterium/enzimología , Brevibacterium/genética , Brevibacterium/ultraestructura , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multiproteicos/química , Orgánulos/enzimología , Orgánulos/ultraestructura , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homología de Secuencia de Aminoácido , Thermotoga maritima/genéticaRESUMEN
Cypoviruses and baculoviruses are notoriously difficult to eradicate because the virus particles are embedded in micrometre-sized protein crystals called polyhedra. The remarkable stability of polyhedra means that, like bacterial spores, these insect viruses remain infectious for years in soil. The environmental persistence of polyhedra is the cause of significant losses in silkworm cocoon harvests but has also been exploited against pests in biological alternatives to chemical insecticides. Although polyhedra have been extensively characterized since the early 1900s, their atomic organization remains elusive. Here we describe the 2 A crystal structure of both recombinant and infectious silkworm cypovirus polyhedra determined using crystals 5-12 micrometres in diameter purified from insect cells. These are the smallest crystals yet used for de novo X-ray protein structure determination. We found that polyhedra are made of trimers of the viral polyhedrin protein and contain nucleotides. Although the shape of these building blocks is reminiscent of some capsid trimers, polyhedrin has a new fold and has evolved to assemble in vivo into three-dimensional cubic crystals rather than icosahedral shells. The polyhedrin trimers are extensively cross-linked in polyhedra by non-covalent interactions and pack with an exquisite molecular complementarity similar to that of antigen-antibody complexes. The resulting ultrastable and sealed crystals shield the virus particles from environmental damage. The structure suggests that polyhedra can serve as the basis for the development of robust and versatile nanoparticles for biotechnological applications such as microarrays and biopesticides.
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Cuerpos de Inclusión Viral/química , Reoviridae/química , Proteínas Virales/química , Animales , Bombyx/virología , Cristalización , Cristalografía por Rayos X , Cuerpos de Inclusión Viral/ultraestructura , Modelos Moleculares , Estructura Cuaternaria de Proteína , Reoviridae/genética , Reoviridae/fisiología , Reoviridae/ultraestructura , Proteínas Virales/metabolismo , Esparcimiento de Virus/fisiologíaRESUMEN
Ribosomes translate genetic information encoded by mRNAs into protein chains with high fidelity. Truncated mRNAs lacking a stop codon will cause the synthesis of incomplete peptide chains and stall translating ribosomes. In bacteria, a ribonucleoprotein complex composed of tmRNA, a molecule that combines the functions of tRNAs and mRNAs, and small protein B (SmpB) rescues stalled ribosomes. The SmpB-tmRNA complex binds to the stalled ribosome, allowing translation to resume at a short internal tmRNA open reading frame that encodes a protein degradation tag. The aberrant protein is released when the ribosome reaches the stop codon at the end of the tmRNA open reading frame and the fused peptide tag targets it for degradation by cellular proteases. The recently determined NMR structures of SmpB, the crystal structure of the SmpB-tmRNA complex and the cryo-EM structure of the SmpB-tmRNA-EF-Tu-ribosome complex have provided first detailed insights into the intricate mechanisms involved in ribosome rescue.
Asunto(s)
Biosíntesis de Proteínas/genética , ARN Bacteriano/genética , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Alanina/metabolismo , Unión Proteica , Conformación Proteica , ARN Bacteriano/química , ARN Mensajero/química , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Ribosomas/química , Relación Estructura-ActividadRESUMEN
Accurate translation of genetic information into protein sequence depends on complete messenger RNA molecules. Truncated mRNAs cause synthesis of defective proteins, and arrest ribosomes at the end of their incomplete message. In bacteria, a hybrid RNA molecule that combines the functions of both transfer and messenger RNAs (called tmRNA) rescues stalled ribosomes, and targets aberrant, partially synthesized, proteins for proteolytic degradation. Here we report the 3.2-A-resolution structure of the tRNA-like domain of tmRNA (tmRNA(Delta)) in complex with small protein B (SmpB), a protein essential for biological functions of tmRNA. We find that the flexible RNA molecule adopts an open L-shaped conformation and SmpB binds to its elbow region, stabilizing the single-stranded D-loop in an extended conformation. The most striking feature of the structure of tmRNA(Delta) is a 90 degrees rotation of the TPsiC-arm around the helical axis. Owing to this unusual conformation, the SmpB-tmRNA(Delta) complex positioned into the A-site of the ribosome orients SmpB towards the small ribosomal subunit, and directs tmRNA towards the elongation-factor binding region of the ribosome. On the basis of this structure, we propose a model for the binding of tmRNA on the ribosome.