RESUMEN
A key capability of ion channels is the facilitation of selective permeation of certain ionic species across cellular membranes at high rates. Due to their physiological significance, ion channels are of great pharmaceutical interest as drug targets. The polymodal signal-detecting Transient Receptor Potential (TRP) superfamily of ion channels forms a particularly promising group of drug targets. While most members of this family permeate a broad range of cations including Ca2+, TRPM4 and TRPM5 are unique due to their strong monovalent-selectivity and impermeability for divalent cations. Here, we investigated the mechanistic basis for their unique monovalent-selectivity by in silico electrophysiology simulations of TRPM5. Our simulations reveal an unusual mechanism of cation selectivity, which is underpinned by the function of the central channel cavity alongside the selectivity filter. Our results suggest that a subtle hydrophobic barrier at the cavity entrance ("hydrophobic funnel") enables monovalent, but not divalent cations to pass and occupy the cavity at physiologically relevant membrane voltages. Monovalent cations then permeate efficiently by a co-operative, distant knock-on mechanism between two binding regions in the extracellular pore vestibule and the central cavity. By contrast, divalent cations do not enter or interact favorably with the channel cavity due to its raised hydrophobicity. Hydrophilic mutations in the transition zone between the selectivity filter and the central channel cavity abolish the barrier for divalent cations, enabling both monovalent and divalent cations to traverse TRPM5.
RESUMEN
Protein-ligand binding prediction typically relies on docking methodologies and associated scoring functions to propose the binding mode of a ligand in a biological target. Significant challenges are associated with this approach, including the flexibility of the protein-ligand system, solvent-mediated interactions, and associated entropy changes. In addition, scoring functions are only weakly accurate due to the short time required for calculating enthalpic and entropic binding interactions. The workflow described here attempts to address these limitations by combining supervised molecular dynamics with dynamical averaging quantum mechanics fragment molecular orbital. This combination significantly increased the ability to predict the experimental binding structure of protein-ligand complexes independent from the starting position of the ligands or the binding site conformation. We found that the predictive power could be enhanced by combining the residence time and interaction energies as descriptors in a novel scoring function named the P-score. This is illustrated using six different protein-ligand targets as case studies.
Asunto(s)
Simulación de Dinámica Molecular , Unión Proteica , Proteínas , Ligandos , Proteínas/química , Proteínas/metabolismo , Sitios de Unión , Teoría Cuántica , TermodinámicaRESUMEN
The selective exchange of ions across cellular membranes is a vital biological process. Ca2+-mediated signaling is implicated in a broad array of physiological processes in cells, while elevated intracellular concentrations of Ca2+ are cytotoxic. Due to the significance of this cation, strict Ca2+ concentration gradients are maintained across the plasma and organelle membranes. Therefore, Ca2+ signaling relies on permeation through selective ion channels that control the flux of Ca2+ ions. A key family of Ca2+-permeable membrane channels is the polymodal signal-detecting transient receptor potential (TRP) ion channels. TRP channels are activated by a wide variety of cues including temperature, small molecules, transmembrane voltage, and mechanical stimuli. While most members of this family permeate a broad range of cations non-selectively, TRPV5 and TRPV6 are unique due to their strong Ca2+ selectivity. Here, we address the question of how some members of the TRPV subfamily show a high degree of Ca2+ selectivity while others conduct a wider spectrum of cations. We present results from all-atom molecular dynamics simulations of ion permeation through two Ca2+-selective and two non-selective TRPV channels. Using a new method to quantify permeation cooperativity based on mutual information, we show that Ca2+-selective TRPV channel permeation occurs by a three-binding site knock-on mechanism, whereas a two-binding site knock-on mechanism is observed in non-selective TRPV channels. Each of the ion binding sites involved displayed greater affinity for Ca2+ over Na+. As such, our results suggest that coupling to an extra binding site in the Ca2+-selective TRPV channels underpins their increased selectivity for Ca2+ over Na+ ions. Furthermore, analysis of all available TRPV channel structures shows that the selectivity filter entrance region is wider for the non-selective TRPV channels, slightly destabilizing ion binding at this site, which is likely to underlie mechanistic decoupling.
Asunto(s)
Calcio , Canales de Potencial de Receptor Transitorio , Calcio/metabolismo , Canales Catiónicos TRPV/metabolismo , Cationes/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Simulación de Dinámica Molecular , Sodio/metabolismoRESUMEN
The efficient permeation across the Gram-negative bacterial membrane is an important step in the overall process of antibacterial action of a molecule and the one that has posed a significant hurdle on the way toward approved antibiotics. Predicting the permeability for a large library of molecules and assessing the effect of different molecular transformations on permeation rates of a given molecule is critical to the development of effective antibiotics. We present a computational approach for obtaining estimates of molecular permeability through a porin channel in a matter of hours using a Brownian dynamics approach. The fast sampling using a temperature acceleration scheme enables the approximate estimation of permeability using the inhomogeneous solubility diffusion model. Although the method is a significant approximation to similar all-atom approaches tested previously, we show that the present approach predicts permeabilities that correlate fairly well with the respective experimental permeation rates from liposome swelling experiments and accumulation rates from antibiotic accumulation assays, and is significantly, i.e., about 14 times, faster compared with a previously reported approach. The possible applications of the scheme in high-throughput screening for fast permeators are discussed.
Asunto(s)
Antibacterianos , Simulación de Dinámica Molecular , Antibacterianos/farmacología , Permeabilidad de la Membrana Celular , Porinas/metabolismo , PermeabilidadRESUMEN
The complex cell envelope of Gram-negative bacteria creates a formidable barrier to antibiotic influx. Reduced drug uptake impedes drug development and contributes to a wide range of drug-resistant bacterial infections, including those caused by extremely resistant species prioritized by the World Health Organization. To develop new and efficient treatments, a better understanding of the molecular features governing Gram-negative permeability is essential. Here, we present a data-driven approach, using matched molecular pair analysis and machine learning on minimal inhibitory concentration data from Gram-positive and Gram-negative bacteria to uncover chemical features that influence Gram-negative bioactivity. We find recurring chemical moieties, of a wider range than previously known, that consistently improve activity and suggest that this insight can be used to optimize compounds for increased Gram-negative uptake. Our findings may help to expand the chemical space of broad-spectrum antibiotics and aid the search for new antibiotic compound classes.
Asunto(s)
Bacterias Gramnegativas , Infecciones por Bacterias Gramnegativas , Antibacterianos/química , Desarrollo de Medicamentos , Farmacorresistencia Bacteriana Múltiple , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , Bacterias Grampositivas , Humanos , Pruebas de Sensibilidad MicrobianaRESUMEN
Ammonium translocation through biological membranes, by the ubiquitous Amt-Mep-Rh family of transporters, plays a key role in all domains of life. Two highly conserved histidine residues protrude into the lumen of the pore of these transporters, forming the family's characteristic Twin-His motif. It has been hypothesized that the motif is essential to confer the selectivity of the transport mechanism. Here, using a combination of in vitro electrophysiology on Escherichia coli AmtB, in silico molecular dynamics simulations, and in vivo yeast functional complementation assays, we demonstrate that variations in the Twin-His motif trigger a mechanistic switch between a specific transporter, depending on ammonium deprotonation, to an unspecific ion channel activity. We therefore propose that there is no selective filter that governs specificity in Amt-Mep-Rh transporters, but the inherent mechanism of translocation, dependent on the fragmentation of the substrate, ensures the high specificity of the translocation. We show that coexistence of both mechanisms in single Twin-His variants of yeast Mep2 transceptors disrupts the signaling function and so impairs fungal filamentation. These data support a signaling process driven by the transport mechanism of the fungal Mep2 transceptors. IMPORTANCE Fungal infections represent a significant threat to human health and cause huge damage to crop yields worldwide. The dimorphic switch between yeast and filamentous growth is associated with the virulence of pathogenic fungi. Of note, fungal Mep2 proteins of the conserved Amt-Mep-Rh family play a transceptor role in the induction of filamentation; however, the signaling mechanism remains largely unknown. Amt-Mep-Rh proteins ensure the specific scavenging of NH4+ through a mechanism relying on substrate deprotonation, thereby preventing competition and translocation of similar-sized K+. Our multidisciplinary approaches using E. coli AmtB, Saccharomyces cerevisiae, and Candida albicans Mep2 show that double variation of the family-defining Twin-His motif triggers a mechanistic switch from a specific transporter to an unspecific ion channel with both mechanisms still coexisting in single variants. Moreover, we show that this mechanistic alteration is associated with loss of signaling ability of Mep2, supporting a transport mechanism-driven process in filamentation induction.
Asunto(s)
Compuestos de Amonio , Proteínas de Transporte de Catión , Proteínas de Escherichia coli , Proteínas de Saccharomyces cerevisiae , Candida albicans/genética , Proteínas de Transporte de Catión/genética , Escherichia coli , Proteínas de Escherichia coli/genética , Proteínas Fúngicas/genética , Humanos , Proteínas de Transporte de Membrana/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal , Translocación GenéticaRESUMEN
Potassium channels play critical roles in many physiological processes, providing a selective permeation route for K+ ions in and out of a cell, by employing a carefully designed selectivity filter, evolutionarily conserved from viruses to mammals. The structure of the selectivity filter was determined at atomic resolution by x-ray crystallography, showing a tight coordination of desolvated K+ ions by the channel. However, the molecular mechanism of K+ ions permeation through potassium channels remains unclear, with structural, functional and computational studies often providing conflicting data and interpretations. In this review, we will present the proposed mechanisms, discuss their origins, and will critically assess them against all available data. General properties shared by all potassium channels are introduced first, followed by the introduction of two main mechanisms of ion permeation: soft and direct knock-on. Then, we will discuss critical computational and experimental studies that shaped the field. We will especially focus on molecular dynamics (MD) simulations, that provided mechanistic and energetic aspects of K+ permeation, but at the same time created long-standing controversies. Further challenges and possible solutions are presented as well.
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Canales de Potasio/metabolismo , Potasio/metabolismo , Humanos , Activación del Canal Iónico/fisiología , Simulación de Dinámica Molecular , PermeabilidadRESUMEN
Gram-negative bacteria cause the majority of highly drug-resistant bacterial infections. To cross the outer membrane of the complex Gram-negative cell envelope, antibiotics permeate through porins, trimeric channel proteins that enable the exchange of small polar molecules. Mutations in porins contribute to the development of drug-resistant phenotypes. In this work, we show that a single point mutation in the porin PorB from Neisseria meningitidis, the causative agent of bacterial meningitis, can strongly affect the binding and permeation of beta-lactam antibiotics. Using X-ray crystallography, high-resolution electrophysiology, atomistic biomolecular simulation, and liposome swelling experiments, we demonstrate differences in drug binding affinity, ion selectivity and drug permeability of PorB. Our work further reveals distinct interactions between the transversal electric field in the porin eyelet and the zwitterionic drugs, which manifest themselves under applied electric fields in electrophysiology and are altered by the mutation. These observations may apply more broadly to drug-porin interactions in other channels. Our results improve the molecular understanding of porin-based drug-resistance in Gram-negative bacteria.
Asunto(s)
Proteínas Bacterianas/química , Neisseria meningitidis/metabolismo , Porinas/química , Ampicilina/química , Ampicilina/metabolismo , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Farmacorresistencia Bacteriana/efectos de los fármacos , Liposomas/química , Liposomas/metabolismo , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Permeabilidad/efectos de los fármacos , Porinas/genética , Porinas/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Mutations in the Trypanosoma brucei aquaporin AQP2 are associated with resistance to pentamidine and melarsoprol. We show that TbAQP2 but not TbAQP3 was positively selected for increased pore size from a common ancestor aquaporin. We demonstrate that TbAQP2's unique architecture permits pentamidine permeation through its central pore and show how specific mutations in highly conserved motifs affect drug permeation. Introduction of key TbAQP2 amino acids into TbAQP3 renders the latter permeable to pentamidine. Molecular dynamics demonstrates that permeation by dicationic pentamidine is energetically favourable in TbAQP2, driven by the membrane potential, although aquaporins are normally strictly impermeable for ionic species. We also identify the structural determinants that make pentamidine a permeant although most other diamidine drugs are excluded. Our results have wide-ranging implications for optimising antitrypanosomal drugs and averting cross-resistance. Moreover, these new insights in aquaporin permeation may allow the pharmacological exploitation of other members of this ubiquitous gene family.
African sleeping sickness is a potentially deadly illness caused by the parasite Trypanosoma brucei. The disease is treatable, but many of the current treatments are old and are becoming increasingly ineffective. For instance, resistance is growing against pentamidine, a drug used in the early stages in the disease, as well as against melarsoprol, which is deployed when the infection has progressed to the brain. Usually, cases resistant to pentamidine are also resistant to melarsoprol, but it is still unclear why, as the drugs are chemically unrelated. Studies have shown that changes in a water channel called aquaglyceroporin 2 (TbAQP2) contribute to drug resistance in African sleeping sickness; this suggests that it plays a role in allowing drugs to kill the parasite. This molecular 'drain pipe' extends through the surface of T. brucei, and should allow only water and a molecule called glycerol in and out of the cell. In particular, the channel should be too narrow to allow pentamidine or melarsoprol to pass through. One possibility is that, in T. brucei, the TbAQP2 channel is abnormally wide compared to other members of its family. Alternatively, pentamidine and melarsoprol may only bind to TbAQP2, and then 'hitch a ride' when the protein is taken into the parasite as part of the natural cycle of surface protein replacement. Alghamdi et al. aimed to tease out these hypotheses. Computer models of the structure of the protein were paired with engineered changes in the key areas of the channel to show that, in T. brucei, TbAQP2 provides a much broader gateway into the cell than observed for similar proteins. In addition, genetic analysis showed that this version of TbAQP2 has been actively selected for during the evolution process of T. brucei. This suggests that the parasite somehow benefits from this wider aquaglyceroporin variant. This is a new resistance mechanism, and it is possible that aquaglyceroporins are also larger than expected in other infectious microbes. The work by Alghamdi et al. therefore provides insight into how other germs may become resistant to drugs.
Asunto(s)
Acuaporina 2 , Pentamidina/farmacología , Trypanosoma brucei brucei , Animales , Acuaporina 2/química , Acuaporina 2/genética , Acuaporina 2/metabolismo , Acuaporinas/química , Acuaporinas/genética , Acuaporinas/metabolismo , Resistencia a Medicamentos/efectos de los fármacos , Resistencia a Medicamentos/genética , Melarsoprol/farmacología , Mutación , Tripanocidas/farmacología , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Tripanosomiasis Africana/tratamiento farmacológicoRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
G-protein-coupled receptors (GPCRs) are involved in numerous physiological processes and are the most frequent targets of approved drugs. The explosion in the number of new three-dimensional (3D) molecular structures of GPCRs (3D-GPCRome) over the last decade has greatly advanced the mechanistic understanding and drug design opportunities for this protein family. Molecular dynamics (MD) simulations have become a widely established technique for exploring the conformational landscape of proteins at an atomic level. However, the analysis and visualization of MD simulations require efficient storage resources and specialized software. Here we present GPCRmd (http://gpcrmd.org/), an online platform that incorporates web-based visualization capabilities as well as a comprehensive and user-friendly analysis toolbox that allows scientists from different disciplines to visualize, analyze and share GPCR MD data. GPCRmd originates from a community-driven effort to create an open, interactive and standardized database of GPCR MD simulations.
Asunto(s)
Simulación de Dinámica Molecular , Receptores Acoplados a Proteínas G/química , Programas Informáticos , Metaboloma , Modelos Moleculares , Conformación ProteicaRESUMEN
The transport of charged molecules across biological membranes faces the dual problem of accommodating charges in a highly hydrophobic environment while maintaining selective substrate translocation. This has been the subject of a particular controversy for the exchange of ammonium across cellular membranes, an essential process in all domains of life. Ammonium transport is mediated by the ubiquitous Amt/Mep/Rh transporters that includes the human Rhesus factors. Here, using a combination of electrophysiology, yeast functional complementation and extended molecular dynamics simulations, we reveal a unique two-lane pathway for electrogenic NH4+ transport in two archetypal members of the family, the transporters AmtB from Escherichia coli and Rh50 from Nitrosomonas europaea. The pathway underpins a mechanism by which charged H+ and neutral NH3 are carried separately across the membrane after NH4+ deprotonation. This mechanism defines a new principle of achieving transport selectivity against competing ions in a biological transport process.
Asunto(s)
Amoníaco/metabolismo , Compuestos de Amonio/metabolismo , Escherichia coli/metabolismo , Transporte Iónico , Nitrosomonas europaea/metabolismoRESUMEN
Bacteria use a number of mechanisms to defend themselves from antimicrobial drugs. One important defense strategy is the ability to export drugs by multidrug transporters. One class of multidrug transporter, the so-called multidrug and toxic compound extrusion (MATE) transporters, extrude a variety of antibiotic compounds from the bacterial cytoplasm. These MATE transporters are driven by a Na+, H+, or combined Na+/H+ gradient, and act as antiporters to drive a conformational change in the transporter from the outward to the inward-facing conformation. In the inward-facing conformation, a chemical compound (drug) binds to the protein, resulting in a switch to the opposite conformation, thereby extruding the drug. Using molecular dynamics simulations, we now report the structural basis for Na+ and H+ binding in the dual ion coupled MATE transporter ClbM from Escherichia coli, which is connected to colibactin-induced genotoxicity, yielding novel insights into the ion/drug translocation mechanism of this bacterial transporter.
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Proteínas de Escherichia coli/química , Simulación de Dinámica Molecular , Proteínas de Transporte de Catión Orgánico/química , Antibacterianos/química , Sitios de Unión , Proteínas de Escherichia coli/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Unión Proteica , Protones , Sodio/metabolismoRESUMEN
Tetratricopeptide repeat (TPR) proteins belong to the class of α-solenoid proteins, in which repetitive units of α-helical hairpin motifs stack to form superhelical, often highly flexible structures. TPR domains occur in a wide variety of proteins, and perform key functional roles including protein folding, protein trafficking, cell cycle control and post-translational modification. Here, we look at the TPR domain of the enzyme O-linked GlcNAc-transferase (OGT), which catalyses O-GlcNAcylation of a broad range of substrate proteins. A number of single-point mutations in the TPR domain of human OGT have been associated with the disease Intellectual Disability (ID). By extended steered and equilibrium atomistic simulations, we show that the OGT-TPR domain acts as an elastic nanospring, and that each of the ID-related local mutations substantially affect the global dynamics of the TPR domain. Since the nanospring character of the OGT-TPR domain is key to its function in binding and releasing OGT substrates, these changes of its biomechanics likely lead to defective substrate interaction. We find that neutral mutations in the human population, selected by analysis of the gnomAD database, do not incur these changes. Our findings may not only help to explain the ID phenotype of the mutants, but also aid the design of TPR proteins with tailored biomechanical properties.
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Discapacidad Intelectual/genética , N-Acetilglucosaminiltransferasas/química , N-Acetilglucosaminiltransferasas/genética , Mutación Puntual , Humanos , Simulación de Dinámica Molecular , N-Acetilglucosaminiltransferasas/metabolismo , Conformación Proteica , Dominios Proteicos , Repeticiones de TetratricopéptidosRESUMEN
The permeation of most antibiotics through the outer membrane of Gram-negative bacteria occurs through porin channels. To design drugs with increased activity against Gram-negative bacteria in the face of the antibiotic resistance crisis, the strict constraints on the physicochemical properties of the permeants imposed by these channels must be better understood. Here we show that a combination of high-resolution electrophysiology, new noise-filtering analysis protocols and atomistic biomolecular simulations reveals weak binding events between the ß-lactam antibiotic ampicillin and the porin PorB from the pathogenic bacterium Neisseria meningitidis. In particular, an asymmetry often seen in the electrophysiological characteristics of ligand-bound channels is utilised to characterise the binding site and molecular interactions in detail, based on the principles of electro-osmotic flow through the channel. Our results provide a rationale for the determinants that govern the binding and permeation of zwitterionic antibiotics in porin channels.
Asunto(s)
Ampicilina/metabolismo , Antibacterianos/metabolismo , Neisseria meningitidis/metabolismo , Porinas/metabolismo , Ampicilina/farmacocinética , Antibacterianos/farmacocinética , Humanos , Meningitis Meningocócica/tratamiento farmacológico , Meningitis Meningocócica/microbiología , Modelos Moleculares , Neisseria meningitidis/efectos de los fármacos , Permeabilidad , beta-Lactamas/metabolismo , beta-Lactamas/farmacocinéticaRESUMEN
The movement of ammonium across biologic membranes is a fundamental process in all living organisms and is mediated by the ubiquitous ammonium transporter/methylammonium permease/rhesus protein (Amt/Mep/Rh) family of transporters. Recent structural analysis and coupled mass spectrometry studies have shown that the Escherichia coli ammonium transporter AmtB specifically binds 1-palmitoyl-2-oleoyl phosphatidylglycerol (POPG). Upon POPG binding, several residues of AmtB undergo a small conformational change, which stabilizes the protein against unfolding. However, no studies have so far been conducted, to our knowledge, to explore whether POPG binding to AmtB has functional consequences. Here, we used an in vitro experimental assay with purified components, together with molecular dynamics simulations, to characterize the relation between POPG binding and AmtB activity. We show that the AmtB activity is electrogenic. Our results indicate that the activity, at the molecular level, of Amt in archaebacteria and eubacteria may differ. We also show that POPG is an important cofactor for AmtB activity and that, in the absence of POPG, AmtB cannot complete the full translocation cycle. Furthermore, our simulations reveal previously undiscovered POPG binding sites on the intracellular side of the lipid bilayer between the AmtB subunits. Possible molecular mechanisms explaining the functional role of POPG are discussed.-Mirandela, G. D., Tamburrino, G., Hoskisson, P. A., Zachariae, U., Javelle, A. The lipid environment determines the activity of the Escherichia coli ammonium transporter AmtB.
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Proteínas de Transporte de Catión/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Simulación de Dinámica Molecular , Fosfatidilgliceroles/química , Sitios de Unión , Proteínas de Transporte de Catión/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Fosfatidilgliceroles/genéticaRESUMEN
The seeming contradiction that K+ channels conduct K+ ions at maximal throughput rates while not permeating slightly smaller Na+ ions has perplexed scientists for decades. Although numerous models have addressed selective permeation in K+ channels, the combination of conduction efficiency and ion selectivity has not yet been linked through a unified functional model. Here, we investigate the mechanism of ion selectivity through atomistic simulations totalling more than 400 µs in length, which include over 7,000 permeation events. Together with free-energy calculations, our simulations show that both rapid permeation of K+ and ion selectivity are ultimately based on a single principle: the direct knock-on of completely desolvated ions in the channels' selectivity filter. Herein, the strong interactions between multiple 'naked' ions in the four filter binding sites give rise to a natural exclusion of any competing ions. Our results are in excellent agreement with experimental selectivity data, measured ion interaction energies and recent two-dimensional infrared spectra of filter ion configurations.
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Cationes Monovalentes/química , Cationes Monovalentes/metabolismo , Canales de Potasio/metabolismo , Potasio/química , Potasio/metabolismo , Transporte Iónico , Canales de Potasio/química , Sodio/química , Sodio/metabolismo , Espectrofotometría Infrarroja , Especificidad por SustratoRESUMEN
In-solution small-angle X-ray and neutron scattering (SAXS/SANS) have become popular methods to characterize the structure of membrane proteins, solubilized by either detergents or nanodiscs. SANS studies of protein-detergent complexes usually require deuterium-labeled proteins or detergents, which in turn often lead to problems in their expression or purification. Here, we report an approach whose novelty is the combined analysis of SAXS and SANS data from an unlabeled membrane protein complex in solution in two complementary ways. First, an explicit atomic analysis, including both protein and detergent molecules, using the program WAXSiS, which has been adapted to predict SANS data. Second, the use of MONSA which allows one to discriminate between detergent head- and tail-groups in an ab initio approach. Our approach is readily applicable to any detergent-solubilized protein and provides more detailed structural information on protein-detergent complexes from unlabeled samples than SAXS or SANS alone.
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Técnicas de Química Analítica/métodos , Detergentes/química , Proteínas de la Membrana/química , Difracción de Neutrones , Difracción de Rayos X , Simulación de Dinámica Molecular , SolubilidadRESUMEN
O-linked ß-N-acetyl-D-glucosamine (O-GlcNAc) transferase (OGT) regulates protein O-GlcNAcylation, an essential post-translational modification that is abundant in the brain. Recently, OGT mutations have been associated with intellectual disability, although it is not understood how they affect OGT structure and function. Using a multi-disciplinary approach we show that the L254F OGT mutation leads to conformational changes of the tetratricopeptide repeats and reduced activity, revealing the molecular mechanisms contributing to pathogenesis.