17 research outputs found
NMR, EM and functional studies on TBsmr, a small multidrug transporter from M. tuberculosis
Antibiotic resistance of pathogenic bacteria is a major worldwide problem. Bacteria can resist antibiotics by active efflux due to multidrug efflux pumps. The focus of this study has been the mycobacterial multidrug transporter TBsmr because it belongs to the small multidrug resistance (SMR) family whose members are a paradigm to study multidrug efflux due to their small size. SMR proteins are typically 11-12 kDa in size and have a four-transmembrane helix topology. They bind cationic, lipophilic antibiotics such as ethidium bromide (EtBr) and TPP+, and transport them across the membrane in exchange for protons. To understand the molecular mechanism of multidrug resistance, we have to gain information about the structure and function of these proteins. The research described in this thesis aimed to deduce details about the topology, transport cycle and key residues of TBsmr using biophysical techniques. Solid-state NMR (ssNMR) can provide detailed insight into structural organization and dynamical properties of these systems. However, a major bottleneck is the preparation of mg amounts of isotope labeled protein. In case of proteoliposomes, the problem is compounded by the presence of lipids which have to fit into the small active volume of the ssNMR rotor. In Chapter 3, an enhanced protein preparation is described which yields large amounts of TBsmr reconstituted in a native lipid environment suitable for further functional and structual studies. The achieved high protein-to-lipid ratios made a further characterization by ssNMR feasible. The transport activity and oligomeric state of the reconstituted protein in different types of lipid was studied as shown in Chapter 4. The exact oligomeric state of native SMR proteins is still uncertain but a number of biochemical and biophysical studies in detergent suggest that the minimal functional unit capable of binding substrate is a dimer. However, binding assays are not ideal since a protein may bind substrate without completing the transport cycle which can only be shown for reconstituted protein in transport assays.By combining functional data of a TPP+ transport assay with information about theoligomeric state of reconstituted TBsmr obtained by freeze-fracture electron microscopy, it could be shown that lipids affect the function and the oligomeric state of the protein, and that the TBsmr dimer is the minimal functional unit necessary for transport. The transport cycle must involve various conformational states of the protein needed for substrate binding, translocation and release. A fluorescent substrate will therefore experience a significant change of environment while being transported, which influences its fluorescence properties. Thus the substrate itself can report intermediate states that form during the transport cycle. In Chapter 5, the existence of such a substrate-transporter complex for the TBsmr and its substrate EtBr could be shown. The pH gradient needed for antiport has been generated by co-reconstituting TBsmr with bacteriorhodopsin. The measurements have shown the formation of a pH-dependant, transient substrate-protein complex between binding and release of EtBr. This state was further characterized by determining the Kd, by inhibiting EtBr transport through titration with non-fluorescent substrate and by fluorescence anisotropy measurements. The findings support a model with a single occluded intermediate state in which the substrate is highly immobile. Liquid-state NMR is a useful tool to monitor protein-ligand interactions by chemical shift mapping and thus identify and characterize important residues in the protein which are involved in substrate binding. In agreement with previous studies (Krueger-Koplin et al., 2004), the detergent LPPG was found to be highly suitable for liquid-state NMR studies of the membrane protein TBsmr and 42% of the residues could be assigned, as reported in Chapter 6. However, no specific interactions with EtBr were found. This observation was confirmed by LILBID mass spectrometry which showed that TBsmr was predominantly in the non-functional monomeric state. Functional protein was prepared in proteoliposomes which can be investigated by solidstate NMR (Chapter 7). Besides the essential E13, the aromatic residues W63, Y40, and Y60 have been shown to be directly involved in drug binding and transport. Different isotope labeling strategies were evaluated to improve the quality of the NMR spectra to identify and characterize these key residues. In a single tryptophan mutant of reconstituted TBsmr W30A, the binding of ethidium bromide could be detected by 13C solid-state NMR. The measurements have revealed two populations of the conserved W63 residue with distinct backbone structures in the presence of substrate. There is a controversy about the parallel or anti-parallel arrangement of the protomers in the EmrE dimer (Schuldiner, 2007) but this structural asymmetry is consistent with both a parallel and anti-parallel topology.Die Antibiotikaresistenz pathogener Bakterien ist ein Problem für die Gesundheit von Menschen weltweit und damit ein wichtiges Forschungsziel. Ein Mechanismus zur Resistenz gegen Antibiotika ist der aktive Transport von Antibiotika aus der Zelle heraus durch so genannte Multidrug-Transportproteine. Zusätzlich sind diese Transporter als Modellsysteme auch von großem generellem Interesse, denn ihre erstaunliche Fähigkeit, eine Vielzahl sehr diverser Wirkstoffe spezifisch zu binden, scheint den verbreiteten Ansichten über Substrat-Protein-Wechselwirkung zu widersprechen. Zur Untersuchung des Multidrug-Transports befasst sich die vorliegende Arbeit mit der SMR-Familie (small multidrug resistance), deren Mitglieder sich auf Grund ihrer kleinen Größe hervorragend als Modellsysteme eignen. Die SMR-Proteine sind typischerweise 11-12 kDa schwer und bestehen aus vier transmembranen ? - Helizes. Sie transportiere eine Reihe unterschiedlicher aromatischer und positiv geladener Substrate wie zum Beispiel Ethidium Bromid im Austausch gegen Protonen durch die Membran. Um offene strukturelle und mechanistische Fragen zu beantworten, wurde TBsmr von M. tuberculosis, als ein typisches SMR Protein, ausgewählt. Ziel dieser Arbeit war es, ein besseres Verständnis von der Oligomerisierung, dem Transportzyklus und wichtigen, konservierten Aminosäuren der SMR-Familie durch Untersuchungen mit biophysikalischen Methoden zu bekommen. Die Festkörper-NMR-Spektroskopie eignet sich, um Informationen über die Struktur und Dynamik dieser Systeme zu erhalten. Vorteilhaft sind Messungen an Proteoliposomen, da sich das Protein dann in seiner nativen Membranumgebung befindet. Die Notwendigkeit von mg-Mengen isotopenmarkierter Proteine ist aber ein großer Engpass bei der Umsetzung. Der Nachteil dieser Proben ist darüber hinaus das zusätzliche Volumen durch Lipide, da die Festkörper-NMR-Rotoren nur ein geringes Probenvolumen fassen. Durch eine verbesserte Probenpräparation ließen sich große Mengen isotopenmarkiertes TBsmr gewinnen und mit einem hohen Protein-zu-Lipid Verhältnis in Liposomen rekonstituieren. Diese Verbesserungen ermöglichten die nachfolgenden Messungen zur Charakterisierung von TBsmr. Der Oligomerisierungszustand der nativen SMR-Proteine ist noch nicht sicher bestimmt, aber die meisten biochemischen und biophysikalischen Untersuchungen in Detergenz deuten darauf hin, dass ein Dimer die minimale funktionelle Einheit ist, die Substrat binden kann. Ligand-Bindungs-Experimente haben aber den Nachteil, dass eventuell Substrat an das Protein bindet, es aber trotzdem nicht transportiert werden kann. Daher wurde der Transport des Substrats Tetraphenylphosphonium (TPP+) in verschiedenen Lipiden untersucht. Die Messungen erfolgten mit Hilfe von pH-Sprüngen und einer TPP+ -sensitiven Elektrode. Dabei wurde ein Einfluss der Lipidsorte auf die Aktivität und den Oligomerisierungszustand von TBsmr festgestellt. Durch Kombination mit Informationen über den Oligomerisierungsgrad des rekonstitutierten Proteins aus Gefrierbruch-Elektronenmikroskopie-Untersuchungen, konnte die Existenz von funktionalen Dimeren, die Substrat transportieren, in POPC nachgewiesen werden. In Übereinstimmung mit bisherigen Untersuchungen, die auf die Existenz höherer Oligomere hindeuten, wurden in E. coli Lipiden Tetramere gefunden. Der Transportzyklus muss eine Reihe von verschiedenen Konformationen für die Bindung, den Transport und die Freisetzung des Substrats enthalten. Eine fluoreszierende Substanz wird sich beim Transport durch die starken Änderungen der Umgebung in ihren Fluoreszenzeigenschaften verändern. Deshalb könnte das Substrat selbst als Reporter für Zwischenzustände im Transportzyklus benutzt werden. Es konnte die Existenz eines solchen Substrat-Transporter-Komplexes für TBsmr und das Substrat Ethidium Bromid mit einem neu entwickelten Assay nachgewiesen werden. Die Möglichkeit eines stabilen pH-Gradienten wurde durch die Korekonstitution von TBsmr mit Bakteriorhodopsin geschaffen. Die beobachtete Fluoreszenzänderung wurde durch einen pH-abhängigen, transienten Substrat-Protein-Komplex zwischen der Bindung und der Freisetzung von Ethidium verursacht. Zusätzlich wurde dieser Zustand durch die Bestimmung eines Kds, der Hemmung des Ethidiumtransports durch die Titration mit einem nicht fluoreszierenden Substrat und durch Fluoreszenzanisotropie-Messungen genauer charakterisiert. Die Ergebnisse deuten auf einen einzelnen verdeckten Übergangszustand hin, in dem das Substrat unbeweglich ist. Lösungs-NMR-Experimente wurden durchgeführt, um zu evaluieren, ob Substrat-Protein-Interaktionen gemessen werden können. Dabei eignete sich besonders das Detergenz LPPG und es war möglich, 42 Prozent aller Resonanzen des Protein-Rückgrates vorläufig zuzuordnen. Es konnten aber keine spezifische Interaktion von TBsmr mit Ethidium Bromid nachgewiesen werden. Die Beobachtungen wurden durch LILBID-Massenspektrometrie-Messungen (Laser Induced Liquid Beam Ionization/Desorption) unterstützt, welche nicht-funktionale TBsmr Monomere in LPPG nachwiesen. Das Problem einer funktionalen Präparation ließ sich durch Aktivität in Lipidmembranen lösen, wo Membranproteine sehr gut mittels Festkörper-NMR untersucht werden können. Neben dem essentiellen Glutamat 13 sind einige weitere, aromatische Aminosäuren (Y40, Y60, W63) für den Transportzyklus wichtig. Es wurden Festkörper-NMR-Messungen an vollständig und selektiv markierten Proben durchgeführt, um durch verschiedene Isotopenmarkierungs-Schemata die Qualität der NMR-Spektren soweit zu verbessern, dass einzelne Aminosäuren identifiziert und charakterisiert werden können. Mit der Mutante TBsmr W30A, in der ein einzelnes Tryptophan vollständig mit 13C Isotopen markiert war, konnte die Bindung von Ethidium Bromid detektiert werden. Die Messungen offenbarten zwei Populationen der konservierten Aminosäure W63 mit verschiedenen Konformationen des Peptidrückgrats in Gegenwart von Substrat. Die beobachtete strukturelle Asymmetrie von W63 ist sowohl in einer parallelen, wie auch anti-parallelen Topologie der Dimere möglich
A lipid-dependent link between activity and oligomerization state of the M. tuberculosis SMR protein TBsmr
AbstractTBsmr is a secondary active multidrug transporter from Mycobacterium tuberculosis that transports a plethora of compounds including antibiotics and fluorescent dyes. It belongs to the small multidrug resistance (SMR) superfamily and is structurally and functionally related to E. coli EmrE. Of particular importance is the link between protein function, oligomeric state and lipid composition. By freeze fracture EM, we found three different size distributions in three different lipid environments for TBsmr indicating different oligomeric states. The link of these states with protein activity has been probed by fluorescence spectroscopy revealing significant differences. The drug binding site has been probed further by 19F-MAS NMR through chemical labeling of native cysteine residues showing a water accessible environment in agreement with the alternating access model
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Global burden of 288 causes of death and life expectancy decomposition in 204 countries and territories and 811 subnational locations, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021
BACKGROUND Regular, detailed reporting on population health by underlying cause of death is fundamental for public health decision making. Cause-specific estimates of mortality and the subsequent effects on life expectancy worldwide are valuable metrics to gauge progress in reducing mortality rates. These estimates are particularly important following large-scale mortality spikes, such as the COVID-19 pandemic. When systematically analysed, mortality rates and life expectancy allow comparisons of the consequences of causes of death globally and over time, providing a nuanced understanding of the effect of these causes on global populations. METHODS The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2021 cause-of-death analysis estimated mortality and years of life lost (YLLs) from 288 causes of death by age-sex-location-year in 204 countries and territories and 811 subnational locations for each year from 1990 until 2021. The analysis used 56 604 data sources, including data from vital registration and verbal autopsy as well as surveys, censuses, surveillance systems, and cancer registries, among others. As with previous GBD rounds, cause-specific death rates for most causes were estimated using the Cause of Death Ensemble model-a modelling tool developed for GBD to assess the out-of-sample predictive validity of different statistical models and covariate permutations and combine those results to produce cause-specific mortality estimates-with alternative strategies adapted to model causes with insufficient data, substantial changes in reporting over the study period, or unusual epidemiology. YLLs were computed as the product of the number of deaths for each cause-age-sex-location-year and the standard life expectancy at each age. As part of the modelling process, uncertainty intervals (UIs) were generated using the 2·5th and 97·5th percentiles from a 1000-draw distribution for each metric. We decomposed life expectancy by cause of death, location, and year to show cause-specific effects on life expectancy from 1990 to 2021. We also used the coefficient of variation and the fraction of population affected by 90% of deaths to highlight concentrations of mortality. Findings are reported in counts and age-standardised rates. Methodological improvements for cause-of-death estimates in GBD 2021 include the expansion of under-5-years age group to include four new age groups, enhanced methods to account for stochastic variation of sparse data, and the inclusion of COVID-19 and other pandemic-related mortality-which includes excess mortality associated with the pandemic, excluding COVID-19, lower respiratory infections, measles, malaria, and pertussis. For this analysis, 199 new country-years of vital registration cause-of-death data, 5 country-years of surveillance data, 21 country-years of verbal autopsy data, and 94 country-years of other data types were added to those used in previous GBD rounds. FINDINGS The leading causes of age-standardised deaths globally were the same in 2019 as they were in 1990; in descending order, these were, ischaemic heart disease, stroke, chronic obstructive pulmonary disease, and lower respiratory infections. In 2021, however, COVID-19 replaced stroke as the second-leading age-standardised cause of death, with 94·0 deaths (95% UI 89·2-100·0) per 100 000 population. The COVID-19 pandemic shifted the rankings of the leading five causes, lowering stroke to the third-leading and chronic obstructive pulmonary disease to the fourth-leading position. In 2021, the highest age-standardised death rates from COVID-19 occurred in sub-Saharan Africa (271·0 deaths [250·1-290·7] per 100 000 population) and Latin America and the Caribbean (195·4 deaths [182·1-211·4] per 100 000 population). The lowest age-standardised death rates from COVID-19 were in the high-income super-region (48·1 deaths [47·4-48·8] per 100 000 population) and southeast Asia, east Asia, and Oceania (23·2 deaths [16·3-37·2] per 100 000 population). Globally, life expectancy steadily improved between 1990 and 2019 for 18 of the 22 investigated causes. Decomposition of global and regional life expectancy showed the positive effect that reductions in deaths from enteric infections, lower respiratory infections, stroke, and neonatal deaths, among others have contributed to improved survival over the study period. However, a net reduction of 1·6 years occurred in global life expectancy between 2019 and 2021, primarily due to increased death rates from COVID-19 and other pandemic-related mortality. Life expectancy was highly variable between super-regions over the study period, with southeast Asia, east Asia, and Oceania gaining 8·3 years (6·7-9·9) overall, while having the smallest reduction in life expectancy due to COVID-19 (0·4 years). The largest reduction in life expectancy due to COVID-19 occurred in Latin America and the Caribbean (3·6 years). Additionally, 53 of the 288 causes of death were highly concentrated in locations with less than 50% of the global population as of 2021, and these causes of death became progressively more concentrated since 1990, when only 44 causes showed this pattern. The concentration phenomenon is discussed heuristically with respect to enteric and lower respiratory infections, malaria, HIV/AIDS, neonatal disorders, tuberculosis, and measles. INTERPRETATION Long-standing gains in life expectancy and reductions in many of the leading causes of death have been disrupted by the COVID-19 pandemic, the adverse effects of which were spread unevenly among populations. Despite the pandemic, there has been continued progress in combatting several notable causes of death, leading to improved global life expectancy over the study period. Each of the seven GBD super-regions showed an overall improvement from 1990 and 2021, obscuring the negative effect in the years of the pandemic. Additionally, our findings regarding regional variation in causes of death driving increases in life expectancy hold clear policy utility. Analyses of shifting mortality trends reveal that several causes, once widespread globally, are now increasingly concentrated geographically. These changes in mortality concentration, alongside further investigation of changing risks, interventions, and relevant policy, present an important opportunity to deepen our understanding of mortality-reduction strategies. Examining patterns in mortality concentration might reveal areas where successful public health interventions have been implemented. Translating these successes to locations where certain causes of death remain entrenched can inform policies that work to improve life expectancy for people everywhere. FUNDING Bill & Melinda Gates Foundation
Transport cycle intermediate in small multidrug resistance protein is revealed by substrate fluorescence
Efflux pumps of the small multidrug resistance family bind cationic, lipophilic antibiotics and transport them across the membrane in exchange for protons. The transport cycle must involve various conformational states of the protein needed for substrate binding, translocation, and release. A fluorescent substrate will therefore experience a significant change of environment while being transported, which influences its fluorescence properties. Thus the substrate itself can report intermediate states that form during the transport cycle. We show the existence of such a substrate-transporter complex for the EmrE homologMycobacterium tuberculosisTBsmr and its substrate ethidium bromide. The pH gradient needed for antiport has been generated by co-reconstituting TBsmr with bacteriorhodopsin. Sample illumination generates a ΔpH, which results in enhanced ethidium fluorescence intensity, which is abolished when ΔpH or ΔΨ is collapsed or when the essential residue Glu-13 in TBsmr is exchanged with Ala. This observation shows the formation of a pH-dependent, transient substrate-protein complex between binding and release of ethidium. We have further characterized this state by determining theKd, by inhibiting ethidium transport through titration with nonfluorescent substrate and by fluorescence anisotropy measurements. Our findings support a model with a single occluded intermediate state in which the substrate is highly immobile.—Basting, D., Lorch, M., Lehner, I., Glaubitz, C. Transport cycle intermediate in small multidrug resistance protein is revealed by substrate fluorescence