RESUMEN
Huntington's disease is caused by a polyglutamine (polyQ) expansion in the huntingtin protein. Huntingtin exon 1 (Httex1), as well as other naturally occurring N-terminal huntingtin fragments with expanded polyQ are prone to aggregation, forming potentially cytotoxic oligomers and fibrils. Antibodies and other N-terminal huntingtin binders are widely explored as biomarkers and possible aggregation-inhibiting therapeutics. A monoclonal antibody, MW1, is known to preferentially bind to huntingtin fragments with expanded polyQ lengths, but the molecular basis of the polyQ length specificity remains poorly understood. Using solution NMR, electron paramagnetic resonance, and other biophysical methods, we investigated the structural features of the Httex1-MW1 interaction. Rather than recognizing residual α-helical structure, which is promoted by expanded Q-lengths, MW1 caused the formation of a new, non-native, conformation in which the entire polyQ is largely extended. This non-native polyQ structure allowed the formation of large mixed Httex1-MW1 multimers (600-2900 kD), when Httex1 with pathogenic Q-length (Q46) was used. We propose that these multivalent, entropically favored interactions, are available only to proteins with longer Q-lengths and represent a major factor governing the Q-length preference of MW1. The present study reveals that it is possible to target proteins with longer Q-lengths without having to stabilize a natively favored conformation. Such mechanisms could be exploited in the design of other Q-length specific binders.
Asunto(s)
Anticuerpos Monoclonales , Proteína Huntingtina , Humanos , Anticuerpos Monoclonales/metabolismo , Exones/genética , Proteína Huntingtina/química , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Enfermedad de Huntington/genética , Conformación Proteica en Hélice alfa/genética , Unión Proteica , Espectroscopía de Resonancia Magnética , Multimerización de Proteína/genéticaRESUMEN
Candida auris is a multidrug-resistant yeast pathogen causing outbreaks in health care facilities worldwide, and the emergence of echinocandin-resistant C. auris is a concern. Currently used Clinical and Laboratory Standards Institute (CLSI) and commercial antifungal susceptibility tests (AFST) are phenotype-based, slow, and not scalable, limiting their effectiveness in the surveillance of echinocandin-resistant C. auris. The urgent need for accurate and rapid methods of assessment of echinocandin resistance cannot be overstated, as this class of antifungal drugs is preferred for patient management. We report the development and validation of a TaqMan chemistry probe-based fluorescence melt curve analysis (FMCA) following asymmetric polymerase chain reaction (PCR) to assess mutations within the hot spot one (HS1) region of FKS1, the gene responsible for encoding 1,3-ß-d-glucan synthase that is a target for echinocandins. The assay correctly identified F635C, F635Y, F635del, F635S, S639F or S639Y, S639P, and D642H/R645T mutations. Of these mutations, F635S and D642H/R645T were not involved in echinocandin resistance, while the rest were, as confirmed by AFST. Of 31 clinical cases, the predominant mutation conferring echinocandin resistance was S639F/Y (20 cases) followed by S639P (4 cases), F635del (4 cases), F635Y (2 cases), and F635C (1 case). The FMCA assay was highly specific and did not cross-react with closely and distantly related Candida and other yeast and mold species. Structural modeling of the Fks1 protein, its mutants, and docked conformations of three echinocandin drugs suggest a plausible Fks1 binding orientation for echinocandins. These findings lay the groundwork for future evaluations of additional FKS1 mutations and their impact on the development of drug resistance. The TaqMan chemistry probe-based FMCA would allow rapid, high throughput, and accurate detection of FKS1 mutations conferring echinocandin resistance in C. auris.
Asunto(s)
Antifúngicos , Candida auris , Farmacorresistencia Fúngica Múltiple , Equinocandinas , Proteínas Fúngicas , Glucosiltransferasas , Reacción en Cadena en Tiempo Real de la Polimerasa , Candida auris/efectos de los fármacos , Candida auris/genética , Candida auris/aislamiento & purificación , Equinocandinas/farmacología , Antifúngicos/farmacología , Sondas Moleculares/química , Farmacorresistencia Fúngica Múltiple/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Desnaturalización de Ácido Nucleico , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Glucosiltransferasas/química , Glucosiltransferasas/genética , Conformación Proteica en Hélice alfa/genética , Mutación , Candidiasis Invasiva/diagnóstico , Candidiasis Invasiva/microbiología , Fluorescencia , Análisis Mutacional de ADN/métodosRESUMEN
BACKGROUND AND AIMS: Hepatitis E virus (HEV) is one of the most common causes of acute hepatitis worldwide. Its positive-strand RNA genome encodes three open reading frames (ORF). ORF1 is translated into a large protein composed of multiple domains and is known as the viral replicase. The RNA-dependent RNA polymerase (RDRP) domain is responsible for the synthesis of viral RNA. APPROACH AND RESULTS: Here, we identified a highly conserved α-helix located in the RDRP thumb subdomain. Nuclear magnetic resonance demonstrated an amphipathic α-helix extending from amino acids 1628 to 1644 of the ORF1 protein. Functional analyses revealed a dual role of this helix in HEV RNA replication and virus production, including assembly and release. Mutations on the hydrophobic side of the amphipathic α-helix impaired RNA replication and resulted in the selection of a second-site compensatory change in the RDRP palm subdomain. Other mutations enhanced RNA replication but impaired virus assembly and/or release. CONCLUSIONS: Structure-function analyses identified a conserved amphipathic α-helix in the thumb subdomain of the HEV RDRP with a dual role in viral RNA replication and infectious particle production. This study provides structural insights into a key segment of the ORF1 protein and describes the successful use of reverse genetics in HEV, revealing functional interactions between the RDRP thumb and palm subdomains. On a broader scale, it demonstrates that the HEV replicase, similar to those of other positive-strand RNA viruses, is also involved in virus production.
Asunto(s)
Virus de la Hepatitis E/patogenicidad , Hepatitis E/virología , ARN Polimerasa Dependiente del ARN/metabolismo , Replicación Viral/genética , Células Hep G2 , Virus de la Hepatitis E/genética , Humanos , Mutación , Conformación Proteica en Hélice alfa/genética , ARN Viral/metabolismo , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/ultraestructura , Relación Estructura-ActividadRESUMEN
TMEM16A, the calcium-activated chloride channel, is broadly expressed and plays pivotal roles in diverse physiological processes. To understand the structural and functional relationships of TMEM16A, it is necessary to fully clarify the structural basis of the gating of the TMEM16A channel. Herein, we performed the protein electrostatic analysis and molecular dynamics simulation on the TMEM16A in the presence and absence of Ca2+. Data showed that the separation of TM4 and TM6 causes pore expansion, and Q646 may be a key residue for the formation of π-helix in the middle segment of TM6. Moreover, E705 was found to form a group of H-bond interactions with D554/K588/K645 below the hydrophobic gate to stabilize the closed conformation of the pore in the Ca2+-free state. Interestingly, in the Ca2+ bound state, the E705 side chain swings 100o to serve as Ca2+-binding coordination and released K645. K645 is closer to the hydrophobic gate in the calcium-bound state, which facilitates the provision of electrostatic forces for chloride ions as the ions pass through the hydrophobic gate. Our findings provide the structural-based insights to understanding the mechanisms of gating of TMEM16A.
Asunto(s)
Anoctamina-1/ultraestructura , Comunicación Celular/genética , Conformación Proteica en Hélice alfa/genética , Conformación Proteica , Anoctamina-1/química , Anoctamina-1/genética , Calcio/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Simulación de Dinámica Molecular , Electricidad Estática , Relación Estructura-ActividadRESUMEN
Rapid and sensitive detection of Salmonella is a critical step in routine food quality control, outbreak investigation, and food recalls. Although various genes have been the targets in the design of rapid molecular detection methods for Salmonella, there is limited information on the diversity of these target genes at the level of DNA sequence and the encoded protein structures. In this study, we investigated the diversity of ten target genes (invA, fimA, phoP, spvC, and agfA; ttrRSBCA operon including 5 genes) commonly used in the detection and identification of Salmonella. To this end, we performed whole genome sequencing of 143 isolates of Salmonella serotypes (Enteritidis, Typhimurium, and Heidelberg) obtained from poultry (eggs and chicken). Phylogenetic analysis showed that Salmonella ser. Typhimurium was more diverse than either Enteritidis or Heidelberg. Forty-five non-synonymous mutations were identified in the target genes from the 143 isolates, with the two most common mutations as T â C (15 times) and A â G (13 times). The gene spvC was primarily present in Salmonella ser. Enteritidis isolates and absent from Heidelberg isolates, whereas ttrR was more conserved (0 non-synonymous mutations) than ttrS, ttrB, ttrC, and ttrA (7, 2, 2, and 7 non-synonymous mutations, respectively). Notably, we found one non-synonymous mutation (fimA-Mut.6) across all Salmonella ser. Enteritidis and Salmonella ser. Heidelberg, C â T (496 nt postion), resulting in the change at AA 166 position, Glutamine (Q) â Stop condon (TAG), suggesting that the fimA gene has questionable sites as a target for detection. Using Phyre2 and SWISS-MODEL software, we predicted the structures of the proteins encoded by some of the target genes, illustrating the positions of these non-synonymous mutations that mainly located on the α-helix and ß-sheet which are key elements for maintaining the conformation of proteins. These results will facilitate the development of sensitive molecular detection methods for Salmonella.
Asunto(s)
Proteínas/genética , Salmonella enteritidis/genética , Animales , Codón de Terminación/genética , Mutación/genética , Operón/genética , Filogenia , Aves de Corral/microbiología , Conformación Proteica en Hélice alfa/genética , Conformación Proteica en Lámina beta/genética , Serogrupo , Secuenciación Completa del Genoma/métodosRESUMEN
The elongasome, or Rod system, is a protein complex that controls cell wall formation in rod-shaped bacteria. MreC is a membrane-associated elongasome component that co-localizes with the cytoskeletal element MreB and regulates the activity of cell wall biosynthesis enzymes, in a process that may be dependent on MreC self-association. Here, we use electron cryo-microscopy and X-ray crystallography to determine the structure of a self-associated form of MreC from Pseudomonas aeruginosa in atomic detail. MreC monomers interact in head-to-tail fashion. Longitudinal and lateral interfaces are essential for oligomerization in vitro, and a phylogenetic analysis of proteobacterial MreC sequences indicates the prevalence of the identified interfaces. Our results are consistent with a model where MreC's ability to alternate between self-association and interaction with the cell wall biosynthesis machinery plays a key role in the regulation of elongasome activity.
Asunto(s)
Proteínas Bacterianas/metabolismo , Pared Celular/metabolismo , Pseudomonas aeruginosa/metabolismo , Secuencia de Aminoácidos/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/ultraestructura , Pared Celular/ultraestructura , Secuencia Conservada/genética , Microscopía por Crioelectrón , Cristalografía por Rayos X , Mutagénesis , Filogenia , Conformación Proteica en Hélice alfa/genética , Conformación Proteica en Lámina beta/genética , Dominios Proteicos/genética , Multimerización de Proteína , Pseudomonas aeruginosa/citología , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/ultraestructura , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructuraRESUMEN
Coiled-coil (CC) dimers are widely used in protein design because of their modularity and well-understood sequence-structure relationship. In CC protein origami design, a polypeptide chain is assembled from a defined sequence of CC building segments that determine the self-assembly of protein cages into polyhedral shapes, such as the tetrahedron, triangular prism, or four-sided pyramid. However, a targeted functionalization of the CC modules could significantly expand the versatility of protein origami scaffolds. Here, we describe a panel of single-chain camelid antibodies (nanobodies) directed against different CC modules of a de novo designed protein origami tetrahedron. We show that these nanobodies are able to recognize the same CC modules in different polyhedral contexts, such as isolated CC dimers, tetrahedra, triangular prisms, or trigonal bipyramids, thereby extending the ability to functionalize polyhedra with nanobodies in a desired stoichiometry. Crystal structures of five nanobody-CC complexes in combination with small-angle X-ray scattering show binding interactions between nanobodies and CC dimers forming the edges of a tetrahedron with the nanobody entering the tetrahedral cavity. Furthermore, we identified a pair of allosteric nanobodies in which the binding to the distant epitopes on the antiparallel homodimeric APH CC is coupled via a strong positive cooperativity. A toolbox of well-characterized nanobodies specific for CC modules provides a unique tool to target defined sites in the designed protein structures, thus opening numerous opportunities for the functionalization of CC protein origami polyhedra or CC-based bionanomaterials.
Asunto(s)
Conformación Proteica en Hélice alfa/fisiología , Ingeniería de Proteínas/métodos , Anticuerpos de Dominio Único/química , Dimerización , Modelos Moleculares , Péptidos/química , Polímeros/metabolismo , Conformación Proteica en Hélice alfa/genética , Dominios Proteicos/genética , Dominios Proteicos/fisiología , Pliegue de Proteína , Multimerización de Proteína , Proteínas/química , Anticuerpos de Dominio Único/metabolismoRESUMEN
The stability of proteins is an important factor for industrial and medical applications. Improving protein stability is one of the main subjects in protein engineering. In a previous study, we improved the stability of a four-helix bundle dimeric de novo protein (WA20) by five mutations. The stabilised mutant (H26L/G28S/N34L/V71L/E78L, SUWA) showed an extremely high denaturation midpoint temperature (Tm). Although SUWA is a remarkably hyperstable protein, in protein design and engineering, it is an attractive challenge to rationally explore more stable mutants. In this study, we predicted stabilising mutations of WA20 by in silico saturation mutagenesis and molecular dynamics simulation, and experimentally confirmed three stabilising mutations of WA20 (N22A, N22E, and H86K). The stability of a double mutant (N22A/H86K, rationally optimised WA20, ROWA) was greatly improved compared with WA20 (ΔTm = 10.6 °C). The model structures suggested that N22A enhances the stability of the α-helices and N22E and H86K contribute to salt-bridge formation for protein stabilisation. These mutations were also added to SUWA and improved its Tm. Remarkably, the most stable mutant of SUWA (N22E/H86K, rationally optimised SUWA, ROSA) showed the highest Tm (129.0 °C). These new thermostable mutants will be useful as a component of protein nanobuilding blocks to construct supramolecular protein complexes.
Asunto(s)
Conformación Proteica en Hélice alfa/genética , Ingeniería de Proteínas/métodos , Estructura Secundaria de Proteína/genética , Secuencia de Aminoácidos/genética , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida/métodos , Desnaturalización Proteica , Estabilidad Proteica , Estructura Secundaria de Proteína/fisiología , Proteínas/metabolismoRESUMEN
The human zinc transporter ZnT8 provides the granules of pancreatic ß-cells with zinc (II) ions for assembly of insulin hexamers for storage. Until recently, the structure and function of human ZnTs have been modelled on the basis of the 3D structures of bacterial zinc exporters, which form homodimers with each monomer having six transmembrane α-helices harbouring the zinc transport site and a cytosolic domain with an α,ß structure and additional zinc-binding sites. However, there are important differences in function as the bacterial proteins export an excess of zinc ions from the bacterial cytoplasm, whereas ZnT8 exports zinc ions into subcellular vesicles when there is no apparent excess of cytosolic zinc ions. Indeed, recent structural investigations of human ZnT8 show differences in metal binding in the cytosolic domain when compared to the bacterial proteins. Two common variants, one with tryptophan (W) and the other with arginine (R) at position 325, have generated considerable interest as the R-variant is associated with a higher risk of developing type 2 diabetes. Since the mutation is at the apex of the cytosolic domain facing towards the cytosol, it is not clear how it can affect zinc transport through the transmembrane domain. We expressed the cytosolic domain of both variants of human ZnT8 and have begun structural and functional studies. We found that (i) the metal binding of the human protein is different from that of the bacterial proteins, (ii) the human protein has a C-terminal extension with three cysteine residues that bind a zinc(II) ion, and (iii) there are small differences in stability between the two variants. In this investigation, we employed nickel(II) ions as a probe for the spectroscopically silent Zn(II) ions and utilised colorimetric and fluorimetric indicators for Ni(II) ions to investigate metal binding. We established Ni(II) coordination to the C-terminal cysteines and found differences in metal affinity and coordination in the two ZnT8 variants. These structural differences are thought to be critical for the functional differences regarding the diabetes risk. Further insight into the assembly of the metal centres in the cytosolic domain was gained from potentiometric investigations of zinc binding to synthetic peptides corresponding to N-terminal and C-terminal sequences of ZnT8 bearing the metal-coordinating ligands. Our work suggests the involvement of the C-terminal cysteines, which are part of the cytosolic domain, in a metal chelation and/or acquisition mechanism and, as now supported by the high-resolution structural work, provides the first example of metal-thiolate coordination chemistry in zinc transporters.
Asunto(s)
Proteínas Portadoras/ultraestructura , Insulina/genética , Relación Estructura-Actividad , Transportador 8 de Zinc/ultraestructura , Proteínas Portadoras/química , Proteínas Portadoras/genética , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patología , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Conformación Molecular , Níquel/química , Conformación Proteica en Hélice alfa/genética , Dominios Proteicos/genética , Zinc/química , Transportador 8 de Zinc/química , Transportador 8 de Zinc/genéticaRESUMEN
POU3F3 proteins are eukaryotic transcription factors and contribute to the processes in the development of brain and kidney. Pathogenic POU3F3 variants cause a neurodevelopmental disorder called Snijders Blok-Fisher syndrome (SNIBFIS). This article reports a new SNIBFIS case harboring a novel heterozygous c.1018_1019delCAinsTT (p.Gln340Leu) variant in the POU3F3 gene. This variant affects the α2 helix of POU-S domain and is predicted to be "pathogenic" by multiple in-silico tools. The proband had severe intellectual disability, hypotonia, autistic features, sleep disturbances, and dysmorphic features. The association with epilepsy and hemangioma like two of the three previously reported patients with mutations in the POU-S domain was also a remarkable finding to understand the importance of POU-S domain. This clinical report also highlights the interest of reinterpretation of molecular data and brings a new perspective to the genotype-phenotype relationship in "Snijders Blok-Fisher syndrome".
Asunto(s)
Discapacidades del Desarrollo/genética , Epilepsia/genética , Hemangioma/genética , Factores del Dominio POU/genética , Encéfalo/crecimiento & desarrollo , Encéfalo/patología , Discapacidades del Desarrollo/complicaciones , Discapacidades del Desarrollo/diagnóstico , Discapacidades del Desarrollo/patología , Epilepsia/complicaciones , Epilepsia/diagnóstico , Epilepsia/patología , Estudios de Asociación Genética , Hemangioma/complicaciones , Hemangioma/diagnóstico , Hemangioma/patología , Humanos , Riñón/crecimiento & desarrollo , Riñón/patología , Factores del Dominio POU/ultraestructura , Conformación Proteica en Hélice alfa/genéticaRESUMEN
Low complexity regions (LCRs) are very frequent in protein sequences, generally having a lower propensity to form structured domains and tending to be much less evolutionarily conserved than globular domains. Their higher abundance in eukaryotes and in species with more cellular types agrees with a growing number of reports on their function in protein interactions regulated by post-translational modifications. LCRs facilitate the increase of regulatory and network complexity required with the emergence of organisms with more complex tissue distribution and development. Although the low conservation and structural flexibility of LCRs complicate their study, evolutionary studies of proteins across species have been used to evaluate their significance and function. To investigate how to apply this evolutionary approach to the study of LCR function in protein-protein interactions, we performed a detailed analysis for Huntingtin (HTT), a large protein that is a hub for interaction with hundreds of proteins, has a variety of LCRs, and for which partial structural information (in complex with HAP40) is available. We hypothesize that proteins RASA1, SYN2, and KAT2B may compete with HAP40 for their attachment to the core of HTT using similar LCRs. Our results illustrate how evolution might favor the interplay of LCRs with domains, and the possibility of detecting multiple modes of LCR-mediated protein-protein interactions with a large hub such as HTT when enough protein interaction data is available.
Asunto(s)
Evolución Molecular , Proteína Huntingtina/metabolismo , Proteínas Nucleares/metabolismo , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos/genética , Animales , Humanos , Proteína Huntingtina/química , Proteína Huntingtina/genética , Proteína Huntingtina/ultraestructura , Microscopía Electrónica , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/ultraestructura , Unión Proteica/genética , Conformación Proteica en Hélice alfa/genética , Dominios Proteicos/genética , Mapeo de Interacción de Proteínas , Mapas de Interacción de Proteínas , Alineación de Secuencia , Sinapsinas/química , Sinapsinas/metabolismo , Proteína Activadora de GTPasa p120/química , Proteína Activadora de GTPasa p120/metabolismo , Factores de Transcripción p300-CBP/química , Factores de Transcripción p300-CBP/metabolismoRESUMEN
Herpesviruses infect a majority of the human population, establishing lifelong latent infections for which there is no cure. Periodic viral reactivation spreads infection to new hosts while causing various disease states particularly detrimental in the immunocompromised. Efficient viral replication, and ultimately the spread of infection, is dependent on the nuclear egress complex (NEC), a conserved viral heterodimer that helps translocate viral capsids from the nucleus to the cytoplasm where they mature into infectious virions. Here, we have identified peptides, derived from the capsid protein UL25, that are capable of inhibiting the membrane-budding activity of the NEC from herpes simplex virus type 1 in vitro. We show that the inhibitory ability of the peptides depends on their length and the propensity to form an α-helix but not on the exact amino acid sequence. Current therapeutics that target viral DNA replication machinery are rendered ineffective by drug resistance due to viral mutations. Our results establish a basis for the development of an alternative class of inhibitors against nuclear egress, an essential step in herpesvirus replication, potentially expanding the current repertoire of available therapeutics.
Asunto(s)
Núcleo Celular/genética , Herpesvirus Humano 1/genética , Proteínas Nucleares/genética , Péptidos/genética , Proteínas Virales/genética , Secuencia de Aminoácidos , Proteínas de la Cápside/genética , Citoplasma/genética , Replicación del ADN/genética , ADN Viral/genética , Mutación/genética , Membrana Nuclear/genética , Conformación Proteica en Hélice alfa/genética , Replicación Viral/genéticaRESUMEN
Nanophthalmos-4 is a rare autosomal dominant disorder caused by two known variations in TMEM98. An Austrian Caucasian pedigree was identified suffering from nanophthalmos and late onset angle-closure glaucoma and premature loss of visual acuity. Whole exome sequencing identified segregation of a c.602G > C transversion in TMEM98 (p.Arg201Pro) as potentially causative. A protein homology model generated showed a TMEM98 structure comprising α4, α5/6, α7 and α8 antiparallel helix bundles and two predicted transmembrane domains in α1 and α7 that have been confirmed in vitro. Both p.Arg201Pro and the two missense variations representing proline insertions identified previously to cause nanophthalmos-4 (p.Ala193Pro and p.His196Pro) are located in the charge polarized helix α8 (p.183-p210). Stability of the C-terminal alpha helical structure of TMEM98 is therefore essential to prevent the development of human nanophthalmos-4. Precise molecular diagnosis could lead to the development of tailored therapies for patients with orphan ocular disease.
Asunto(s)
Glaucoma de Ángulo Cerrado/genética , Hiperopía/genética , Proteínas de la Membrana/genética , Microftalmía/genética , Mutación Missense , Trastornos de la Visión/genética , Agudeza Visual/fisiología , Adulto , Anciano de 80 o más Años , Sustitución de Aminoácidos , Arginina , Femenino , Cirugía Filtrante , Glaucoma de Ángulo Cerrado/fisiopatología , Glaucoma de Ángulo Cerrado/cirugía , Humanos , Hiperopía/fisiopatología , Hiperopía/cirugía , Implantación de Lentes Intraoculares , Masculino , Microftalmía/fisiopatología , Microftalmía/cirugía , Microscopía Acústica , Persona de Mediana Edad , Linaje , Facoemulsificación , Prolina , Conformación Proteica en Hélice alfa/genética , Microscopía con Lámpara de Hendidura , Trastornos de la Visión/fisiopatología , Secuenciación del ExomaRESUMEN
The ASCC3 subunit of the activating signal co-integrator complex is a dual-cassette Ski2-like nucleic acid helicase that provides single-stranded DNA for alkylation damage repair by the α-ketoglutarate-dependent dioxygenase AlkBH3. Other ASCC components integrate ASCC3/AlkBH3 into a complex DNA repair pathway. We mapped and structurally analyzed interacting ASCC2 and ASCC3 regions. The ASCC3 fragment comprises a central helical domain and terminal, extended arms that clasp the compact ASCC2 unit. ASCC2-ASCC3 interfaces are evolutionarily highly conserved and comprise a large number of residues affected by somatic cancer mutations. We quantified contributions of protein regions to the ASCC2-ASCC3 interaction, observing that changes found in cancers lead to reduced ASCC2-ASCC3 affinity. Functional dissection of ASCC3 revealed similar organization and regulation as in the spliceosomal RNA helicase Brr2. Our results delineate functional regions in an important DNA repair complex and suggest possible molecular disease principles.
Asunto(s)
ADN Helicasas/genética , Reparación del ADN , Neoplasias/genética , Proteínas Nucleares/genética , Secuencia de Aminoácidos , Secuencia Conservada/genética , ADN Helicasas/aislamiento & purificación , ADN Helicasas/metabolismo , Células HEK293 , Humanos , Mutación , Proteínas Nucleares/aislamiento & purificación , Proteínas Nucleares/metabolismo , Unión Proteica/genética , Conformación Proteica en Hélice alfa/genética , Dominios Proteicos/genética , ARN Helicasas/genética , ARN Helicasas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/genética , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Empalmosomas/metabolismoRESUMEN
High order oligomers are crucial for normal cell physiology, and protein function perturbed by missense mutations underlies several autosomal dominant diseases. Dynamin-2 is one of such protein forming helical oligomers that catalyze membrane fission. Mutations in this protein, where R465W is the most frequent, cause dominant centronuclear myopathy, but the molecular mechanisms underpinning the functional modifications remain to be investigated. To unveil the structural impact of this mutation in dynamin-2, we used full-atom molecular dynamics simulations and coarse-grained models and built dimers and helices of wild-type (WT) monomers, mutant monomers, or both WT and mutant monomers combined. Our results show that the mutation R465W causes changes in the interactions with neighbor amino acids that propagate through the oligomer. These new interactions perturb the contact between monomers and favor an extended conformation of the bundle signaling element (BSE), a dynamin region that transmits the conformational changes from the GTPase domain to the rest of the protein. This extended configuration of the BSE that is only relevant in the helices illustrates how a small change in the microenvironment surrounding a single residue can propagate through the oligomer structures of dynamin explaining how dominance emerges in large protein complexes.
Asunto(s)
Dinamina II/genética , Miopatías Estructurales Congénitas/patología , Dominios Proteicos/genética , Multimerización de Proteína/genética , Arginina/genética , Cristalografía por Rayos X , Dinamina II/metabolismo , Dinamina II/ultraestructura , Humanos , Simulación de Dinámica Molecular , Mutación Missense , Miopatías Estructurales Congénitas/genética , Conformación Proteica en Hélice alfa/genética , Triptófano/genéticaRESUMEN
DNA replication in eukaryotic cells initiates from replication origins that bind the Origin Recognition Complex (ORC). Origin establishment requires well-defined DNA sequence motifs in Saccharomyces cerevisiae and some other budding yeasts, but most eukaryotes lack sequence-specific origins. A 3.9 Å structure of S. cerevisiae ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) bound to origin DNA revealed that a loop within Orc2 inserts into a DNA minor groove and an α-helix within Orc4 inserts into a DNA major groove. Using a massively parallel origin selection assay coupled with a custom mutual-information-based modeling approach, and a separate analysis of whole-genome replication profiling, here we show that the Orc4 α-helix contributes to the DNA sequence-specificity of origins in S. cerevisiae and Orc4 α-helix mutations change genome-wide origin firing patterns. The DNA sequence specificity of replication origins, mediated by the Orc4 α-helix, has co-evolved with the gain of ORC-Sir4-mediated gene silencing and the loss of RNA interference.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Silenciador del Gen , Complejo de Reconocimiento del Origen/metabolismo , Origen de Réplica/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Replicación del ADN , ADN de Hongos/genética , Evolución Molecular , Mutación , Complejo de Reconocimiento del Origen/ultraestructura , Conformación Proteica en Hélice alfa/genética , Proteínas de Saccharomyces cerevisiae/ultraestructura , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Especificidad por Sustrato/genéticaRESUMEN
The potassium ion (K+) channel plays a fundamental role in controlling K+ permeation across the cell membrane and regulating cellular excitabilities. Mutations in the transmembrane pore reportedly affect the gating transitions of K+ channels, and are associated with the onset of neural disorders. However, due to the lack of structural and dynamic insights into the functions of K+ channels, the structural mechanism by which these mutations cause K+ channel dysfunctions remains elusive. Here, we used nuclear magnetic resonance spectroscopy to investigate the structural mechanism underlying the decreased K+-permeation caused by disease-related mutations, using the prokaryotic K+ channel KcsA. We demonstrated that the conformational equilibrium in the transmembrane region is shifted toward the non-conductive state with the closed intracellular K+-gate in the disease-related mutant. We also demonstrated that this equilibrium shift is attributable to the additional steric contacts in the open-conductive structure, which are evoked by the increased side-chain bulkiness of the residues lining the transmembrane helix. Our results suggest that the alteration in the conformational equilibrium of the intracellular K+-gate is one of the fundamental mechanisms underlying the dysfunctions of K+ channels caused by disease-related mutations.
Asunto(s)
Proteínas Bacterianas/metabolismo , Activación del Canal Iónico/genética , Canales de Potasio/metabolismo , Potasio/metabolismo , Alanina/genética , Ataxia/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Epilepsia/genética , Humanos , Síndrome de QT Prolongado/genética , Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular , Mutación Missense , Canales de Potasio/genética , Canales de Potasio/aislamiento & purificación , Conformación Proteica en Hélice alfa/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Streptomyces lividans , Valina/genéticaRESUMEN
The most commonly used oral antidiabetic drug, metformin, is a substrate of the hepatic uptake transporter OCT1 (gene name SLC22A1). However, OCT1 deficiency leads to more pronounced reductions of metformin concentrations in mouse than in human liver. Similarly, the effects of OCT1 deficiency on the pharmacokinetics of thiamine were reported to differ between human and mouse. Here, we compared the uptake characteristics of metformin and thiamine between human and mouse OCT1 using stably transfected human embryonic kidney 293 cells. The affinity for metformin was 4.9-fold lower in human than in mouse OCT1, resulting in a 6.5-fold lower intrinsic clearance. Therefore, the estimated liver-to-blood partition coefficient is only 3.34 in human compared with 14.4 in mouse and may contribute to higher intrahepatic concentrations in mice. Similarly, the affinity for thiamine was 9.5-fold lower in human than in mouse OCT1. Using human-mouse chimeric OCT1, we showed that simultaneous substitution of transmembrane helices TMH2 and TMH3 resulted in the reversal of affinity for metformin. Using homology modeling, we suggest several explanations, of which a different interaction of Leu155 (human TMH2) compared with Val156 (mouse TMH2) with residues in TMH3 had the strongest experimental support. In conclusion, the contribution of human OCT1 to the cellular uptake of thiamine and especially of metformin may be much lower than that of mouse OCT1. This may lead to an overestimation of the effects of OCT1 on hepatic concentrations in humans when using mouse as a model. In addition, comparative analyses of human and mouse orthologs may help reveal mechanisms of OCT1 transport. SIGNIFICANCE STATEMENT: OCT1 is a major hepatic uptake transporter of metformin and thiamine, but this study reports strong differences in the affinity for both compounds between human and mouse OCT1. Consequently, intrahepatic metformin concentrations could be much higher in mice than in humans, impacting metformin actions and representing a strong limitation of using rodent animal models for predictions of OCT1-related pharmacokinetics and efficacy in humans. Furthermore, OCT1 transmembrane helices TMH2 and TMH3 were identified to confer the observed species-specific differences in metformin affinity.
Asunto(s)
Metformina/farmacocinética , Transportador 1 de Catión Orgánico/metabolismo , Tiamina/farmacocinética , Animales , Evaluación Preclínica de Medicamentos/métodos , Células HEK293 , Hepatocitos , Humanos , Hígado/enzimología , Masculino , Ratones , Transportador 1 de Catión Orgánico/genética , Transportador 1 de Catión Orgánico/ultraestructura , Conformación Proteica en Hélice alfa/genética , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/ultraestructura , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Relación Estructura-ActividadRESUMEN
The precise kinetic pathways of peptide clustering and fibril formation are not fully understood. Here we study the initial clustering kinetics and transient cluster morphologies during aggregation of the heptapeptide fragment GNNQQNY from the yeast prion protein Sup35. We use a mid-resolution coarse-grained molecular dynamics model of Bereau and Deserno to explore the aggregation pathways from the initial random distribution of free monomers to the formation of large clusters. By increasing the system size to 72 peptides we could follow directly the molecular events leading to the formation of stable fibril-like structures. To quantify those structures we developed a new cluster helicity parameter. We found that the formation of fibril-like structures is a cooperative processes that requires a critical number of monomers, Mâ≈25, in a cluster. The terminal tyrosine residue is the structural determinant in the formation of helical fibril-like structures. This work supports and quantifies the two-step aggregation model where the initially formed amorphous clusters grow and, when they are large enough, rearrange into mature twisted structures. However, in addition to the nucleated fibrillation, growing aggregates undergo further internal reorganization, which leads to more compact structures of large aggregates.
Asunto(s)
Amiloide/ultraestructura , Factores de Terminación de Péptidos/ultraestructura , Péptidos/química , Proteínas Priónicas/ultraestructura , Proteínas de Saccharomyces cerevisiae/ultraestructura , Amiloide/genética , Humanos , Cinética , Simulación de Dinámica Molecular , Factores de Terminación de Péptidos/genética , Péptidos/genética , Proteínas Priónicas/genética , Agregado de Proteínas/genética , Agregación Patológica de Proteínas/genética , Conformación Proteica , Conformación Proteica en Hélice alfa/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Identifying stabilising variants of membrane protein targets is often required for structure determination. Our new computational pipeline, the Integral Membrane Protein Stability Selector (IMPROvER) provides a rational approach to variant selection by employing three independent approaches: deep-sequence, model-based and data-driven. In silico tests using known stability data, and in vitro tests using three membrane protein targets with 7, 11 and 16 transmembrane helices provided measures of success. In vitro, individual approaches alone all identified stabilising variants at a rate better than expected by random selection. Low numbers of overlapping predictions between approaches meant a greater success rate was achieved (fourfold better than random) when approaches were combined and selections restricted to the highest ranked sites. The mix of information IMPROvER uses can be extracted for any helical membrane protein. We have developed the first general-purpose tool for selecting stabilising variants of [Formula: see text]-helical membrane proteins, increasing efficiency and reducing workload. IMPROvER can be accessed at http://improver.ddns.net/IMPROvER/ .