| 1. |
- Forouzanfar, Mohammad H, et al.
(författare)
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Global, regional, and national comparative risk assessment of 79 behavioural, environmental and occupational, and metabolic risks or clusters of risks in 188 countries, 1990-2013 : a systematic analysis for the Global Burden of Disease Study 2013.
- 2015
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Ingår i: The Lancet. - 0140-6736 .- 1474-547X. ; 386:10010, s. 2287-2323
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The Global Burden of Disease, Injuries, and Risk Factor study 2013 (GBD 2013) is the first of a series of annual updates of the GBD. Risk factor quantification, particularly of modifiable risk factors, can help to identify emerging threats to population health and opportunities for prevention. The GBD 2013 provides a timely opportunity to update the comparative risk assessment with new data for exposure, relative risks, and evidence on the appropriate counterfactual risk distribution.METHODS: Attributable deaths, years of life lost, years lived with disability, and disability-adjusted life-years (DALYs) have been estimated for 79 risks or clusters of risks using the GBD 2010 methods. Risk-outcome pairs meeting explicit evidence criteria were assessed for 188 countries for the period 1990-2013 by age and sex using three inputs: risk exposure, relative risks, and the theoretical minimum risk exposure level (TMREL). Risks are organised into a hierarchy with blocks of behavioural, environmental and occupational, and metabolic risks at the first level of the hierarchy. The next level in the hierarchy includes nine clusters of related risks and two individual risks, with more detail provided at levels 3 and 4 of the hierarchy. Compared with GBD 2010, six new risk factors have been added: handwashing practices, occupational exposure to trichloroethylene, childhood wasting, childhood stunting, unsafe sex, and low glomerular filtration rate. For most risks, data for exposure were synthesised with a Bayesian meta-regression method, DisMod-MR 2.0, or spatial-temporal Gaussian process regression. Relative risks were based on meta-regressions of published cohort and intervention studies. Attributable burden for clusters of risks and all risks combined took into account evidence on the mediation of some risks such as high body-mass index (BMI) through other risks such as high systolic blood pressure and high cholesterol.FINDINGS: All risks combined account for 57·2% (95% uncertainty interval [UI] 55·8-58·5) of deaths and 41·6% (40·1-43·0) of DALYs. Risks quantified account for 87·9% (86·5-89·3) of cardiovascular disease DALYs, ranging to a low of 0% for neonatal disorders and neglected tropical diseases and malaria. In terms of global DALYs in 2013, six risks or clusters of risks each caused more than 5% of DALYs: dietary risks accounting for 11·3 million deaths and 241·4 million DALYs, high systolic blood pressure for 10·4 million deaths and 208·1 million DALYs, child and maternal malnutrition for 1·7 million deaths and 176·9 million DALYs, tobacco smoke for 6·1 million deaths and 143·5 million DALYs, air pollution for 5·5 million deaths and 141·5 million DALYs, and high BMI for 4·4 million deaths and 134·0 million DALYs. Risk factor patterns vary across regions and countries and with time. In sub-Saharan Africa, the leading risk factors are child and maternal malnutrition, unsafe sex, and unsafe water, sanitation, and handwashing. In women, in nearly all countries in the Americas, north Africa, and the Middle East, and in many other high-income countries, high BMI is the leading risk factor, with high systolic blood pressure as the leading risk in most of Central and Eastern Europe and south and east Asia. For men, high systolic blood pressure or tobacco use are the leading risks in nearly all high-income countries, in north Africa and the Middle East, Europe, and Asia. For men and women, unsafe sex is the leading risk in a corridor from Kenya to South Africa.INTERPRETATION: Behavioural, environmental and occupational, and metabolic risks can explain half of global mortality and more than one-third of global DALYs providing many opportunities for prevention. Of the larger risks, the attributable burden of high BMI has increased in the past 23 years. In view of the prominence of behavioural risk factors, behavioural and social science research on interventions for these risks should be strengthened. Many prevention and primary care policy options are available now to act on key risks.FUNDING: Bill & Melinda Gates Foundation.
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| 2. |
- Vos, Theo, et al.
(författare)
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Global, regional, and national incidence, prevalence, and years lived with disability for 301 acute and chronic diseases and injuries in 188 countries, 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013
- 2015
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Ingår i: The Lancet. - 1474-547X .- 0140-6736. ; 386:9995, s. 743-800
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Tidskriftsartikel (refereegranskat)abstract
- Background Up-to-date evidence about levels and trends in disease and injury incidence, prevalence, and years lived with disability (YLDs) is an essential input into global, regional, and national health policies. In the Global Burden of Disease Study 2013 (GBD 2013), we estimated these quantities for acute and chronic diseases and injuries for 188 countries between 1990 and 2013. Methods Estimates were calculated for disease and injury incidence, prevalence, and YLDs using GBD 2010 methods with some important refinements. Results for incidence of acute disorders and prevalence of chronic disorders are new additions to the analysis. Key improvements include expansion to the cause and sequelae list, updated systematic reviews, use of detailed injury codes, improvements to the Bayesian meta-regression method (DisMod-MR), and use of severity splits for various causes. An index of data representativeness, showing data availability, was calculated for each cause and impairment during three periods globally and at the country level for 2013. In total, 35 620 distinct sources of data were used and documented to calculated estimates for 301 diseases and injuries and 2337 sequelae. The comorbidity simulation provides estimates for the number of sequelae, concurrently, by individuals by country, year, age, and sex. Disability weights were updated with the addition of new population-based survey data from four countries. Findings Disease and injury were highly prevalent; only a small fraction of individuals had no sequelae. Comorbidity rose substantially with age and in absolute terms from 1990 to 2013. Incidence of acute sequelae were predominantly infectious diseases and short-term injuries, with over 2 billion cases of upper respiratory infections and diarrhoeal disease episodes in 2013, with the notable exception of tooth pain due to permanent caries with more than 200 million incident cases in 2013. Conversely, leading chronic sequelae were largely attributable to non-communicable diseases, with prevalence estimates for asymptomatic permanent caries and tension-type headache of 2.4 billion and 1.6 billion, respectively. The distribution of the number of sequelae in populations varied widely across regions, with an expected relation between age and disease prevalence. YLDs for both sexes increased from 537.6 million in 1990 to 764.8 million in 2013 due to population growth and ageing, whereas the age-standardised rate decreased little from 114.87 per 1000 people to 110.31 per 1000 people between 1990 and 2013. Leading causes of YLDs included low back pain and major depressive disorder among the top ten causes of YLDs in every country. YLD rates per person, by major cause groups, indicated the main drivers of increases were due to musculoskeletal, mental, and substance use disorders, neurological disorders, and chronic respiratory diseases; however HIV/AIDS was a notable driver of increasing YLDs in sub-Saharan Africa. Also, the proportion of disability-adjusted life years due to YLDs increased globally from 21.1% in 1990 to 31.2% in 2013. Interpretation Ageing of the world's population is leading to a substantial increase in the numbers of individuals with sequelae of diseases and injuries. Rates of YLDs are declining much more slowly than mortality rates. The non-fatal dimensions of disease and injury will require more and more attention from health systems. The transition to non-fatal outcomes as the dominant source of burden of disease is occurring rapidly outside of sub-Saharan Africa. Our results can guide future health initiatives through examination of epidemiological trends and a better understanding of variation across countries.
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| 3. |
- Naghavi, Mohsen, et al.
(författare)
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Global, regional, and national age-sex specific all-cause and cause-specific mortality for 240 causes of death, 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013
- 2015
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Ingår i: The Lancet. - 1474-547X .- 0140-6736. ; 385:9963, s. 117-171
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Tidskriftsartikel (refereegranskat)abstract
- Background Up-to-date evidence on levels and trends for age-sex-specifi c all-cause and cause-specifi c mortality is essential for the formation of global, regional, and national health policies. In the Global Burden of Disease Study 2013 (GBD 2013) we estimated yearly deaths for 188 countries between 1990, and 2013. We used the results to assess whether there is epidemiological convergence across countries. Methods We estimated age-sex-specifi c all-cause mortality using the GBD 2010 methods with some refinements to improve accuracy applied to an updated database of vital registration, survey, and census data. We generally estimated cause of death as in the GBD 2010. Key improvements included the addition of more recent vital registration data for 72 countries, an updated verbal autopsy literature review, two new and detailed data systems for China, and more detail for Mexico, UK, Turkey, and Russia. We improved statistical models for garbage code redistribution. We used six different modelling strategies across the 240 causes; cause of death ensemble modelling (CODEm) was the dominant strategy for causes with sufficient information. Trends for Alzheimer's disease and other dementias were informed by meta-regression of prevalence studies. For pathogen-specifi c causes of diarrhoea and lower respiratory infections we used a counterfactual approach. We computed two measures of convergence (inequality) across countries: the average relative difference across all pairs of countries (Gini coefficient) and the average absolute difference across countries. To summarise broad findings, we used multiple decrement life-tables to decompose probabilities of death from birth to exact age 15 years, from exact age 15 years to exact age 50 years, and from exact age 50 years to exact age 75 years, and life expectancy at birth into major causes. For all quantities reported, we computed 95% uncertainty intervals (UIs). We constrained cause-specific fractions within each age-sex-country-year group to sum to all-cause mortality based on draws from the uncertainty distributions. Findings Global life expectancy for both sexes increased from 65.3 years (UI 65.0-65.6) in 1990, to 71.5 years (UI 71.0-71.9) in 2013, while the number of deaths increased from 47.5 million (UI 46.8-48.2) to 54.9 million (UI 53.6-56.3) over the same interval. Global progress masked variation by age and sex: for children, average absolute diff erences between countries decreased but relative diff erences increased. For women aged 25-39 years and older than 75 years and for men aged 20-49 years and 65 years and older, both absolute and relative diff erences increased. Decomposition of global and regional life expectancy showed the prominent role of reductions in age-standardised death rates for cardiovascular diseases and cancers in high-income regions, and reductions in child deaths from diarrhoea, lower respiratory infections, and neonatal causes in low-income regions. HIV/AIDS reduced life expectancy in southern sub-Saharan Africa. For most communicable causes of death both numbers of deaths and age-standardised death rates fell whereas for most non-communicable causes, demographic shifts have increased numbers of deaths but decreased age-standardised death rates. Global deaths from injury increased by 10.7%, from 4.3 million deaths in 1990 to 4.8 million in 2013; but age-standardised rates declined over the same period by 21%. For some causes of more than 100 000 deaths per year in 2013, age-standardised death rates increased between 1990 and 2013, including HIV/AIDS, pancreatic cancer, atrial fibrillation and flutter, drug use disorders, diabetes, chronic kidney disease, and sickle-cell anaemias. Diarrhoeal diseases, lower respiratory infections, neonatal causes, and malaria are still in the top five causes of death in children younger than 5 years. The most important pathogens are rotavirus for diarrhoea and pneumococcus for lower respiratory infections. Country-specific probabilities of death over three phases of life were substantially varied between and within regions. Interpretation For most countries, the general pattern of reductions in age-sex specifi c mortality has been associated with a progressive shift towards a larger share of the remaining deaths caused by non-communicable disease and injuries. Assessing epidemiological convergence across countries depends on whether an absolute or relative measure of inequality is used. Nevertheless, age-standardised death rates for seven substantial causes are increasing, suggesting the potential for reversals in some countries. Important gaps exist in the empirical data for cause of death estimates for some countries; for example, no national data for India are available for the past decade.
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| 4. |
- Wilson, L. F. L., et al.
(författare)
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The structure of EXTL3 helps to explain the different roles of bi-domain exostosins in heparan sulfate synthesis
- 2022
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 13:3314
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Tidskriftsartikel (refereegranskat)abstract
- Heparan sulfate is a highly modified O-linked glycan that performs diverse physiological roles in animal tissues. Though quickly modified, it is initially synthesised as a polysaccharide of alternating β-d-glucuronosyl and N-acetyl-α-d-glucosaminyl residues by exostosins. These enzymes generally possess two glycosyltransferase domains (GT47 and GT64)—each thought to add one type of monosaccharide unit to the backbone. Although previous structures of murine exostosin-like 2 (EXTL2) provide insight into the GT64 domain, the rest of the bi-domain architecture is yet to be characterised; hence, how the two domains co-operate is unknown. Here, we report the structure of human exostosin-like 3 (EXTL3) in apo and UDP-bound forms. We explain the ineffectiveness of EXTL3’s GT47 domain to transfer β-d-glucuronosyl units, and we observe that, in general, the bi-domain architecture would preclude a processive mechanism of backbone extension. We therefore propose that heparan sulfate backbone polymerisation occurs by a simple dissociative mechanism.
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| 5. |
- Awad, Wael, et al.
(författare)
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Improvements in the order, isotropy and electron density of glypican-1 crystals by controlled dehydration.
- 2013
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Ingår i: Acta Crystallographica. Section D: Biological Crystallography. - 1399-0047. ; 69:Pt 12, s. 2524-2533
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Tidskriftsartikel (refereegranskat)abstract
- The use of controlled dehydration for improvement of protein crystal diffraction quality is increasing in popularity, although there are still relatively few documented examples of success. A study has been carried out to establish whether controlled dehydration could be used to improve the anisotropy of crystals of the core protein of the human proteoglycan glypican-1. Crystals were subjected to controlled dehydration using the HC1 device. The optimal protocol for dehydration was developed by careful investigation of the following parameters: dehydration rate, final relative humidity and total incubation time Tinc. Of these, the most important was shown to be Tinc. After dehydration using the optimal protocol the crystals showed significantly reduced anisotropy and improved electron density, allowing the building of previously disordered parts of the structure.
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| 6. |
- Awad, Wael, et al.
(författare)
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Structural and Biophysical Characterization of Human EXTL3 : Domain Organization, Glycosylation, and Solution Structure
- 2018
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 57:7, s. 1166-1177
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Tidskriftsartikel (refereegranskat)abstract
- Heparan sulfate proteoglycans are proteins substituted with one or more heparan sulfate (HS) polysaccharides, found in abundance at cell surfaces. HS chains influence the activity of many biologically important molecules involved in cellular communication and signaling. The exostosin (EXT) proteins are glycosyltransferases in the Golgi apparatus that assemble HS chains on HSPGs. The EXTL3 enzyme mainly works as an initiator in HS biosynthesis. In this work, human lumenal N-glycosylated EXTL3 (EXTL3ΔN) was cloned, expressed in human embryonic kidney cells, and purified. Various biophysical and biochemical approaches were then employed to elucidate the N-glycosylation sites and the function of their attached N-glycans. Furthermore, the stability and conformation of the purified EXTL3ΔN protein in solution have been analyzed. Our data show that EXTL3ΔN has N-glycans at least at two positions, Asn290 and Asn592, which seem to be critical for proper protein folding and/or release. EXTL3ΔN is quite stable, as high temperature (∼59 °C) was required for denaturation. Deconvolution of the EXTL3ΔN far-UV CD spectrum revealed a substantial fraction of β sheets (25%) with a minor proportion of α-helices (14%) in the secondary structure. Solution small-angle X-ray scattering and dynamic light scattering revealed an extended structure suggestive of a dimeric arrangement and consisting of two distinct regions, narrow and broad, respectively. This is consistent with bioinformatics analyses suggesting a 3-domain structure with two glycosyltransferase domains and a coiled-coil domain.
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| 7. |
- Awad, Wael, et al.
(författare)
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Structural Basis for YjbH Adaptor-Mediated Recognition of Transcription Factor Spx
- 2019
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Ingår i: Structure. - : Elsevier BV. - 0969-2126. ; 27:6, s. 6-936
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Tidskriftsartikel (refereegranskat)abstract
- YjbH is a bacterial adaptor protein required for efficient proteolysis of the RNA polymerase-binding transcription factor Spx by the ClpXP protease. We report the structure of YjbH in complex with Spx. YjbH comprises a DsbA-like thioredoxin domain connected via a linker to a C-terminal domain reminiscent of the winged helix-turn-helix fold. The interaction between YjbH and Spx involves a large surface area. Binding to YjbH stabilizes the C-terminal ClpX recognition region of Spx. We show that mutation of critical YjbH contact residues abrogates Spx recognition. Small-angle X-ray scattering and hydrogen-deuterium exchange mass spectrometry analyses determined the existence of a stable heterodimeric complex in solution and provide evidence that binding of Spx to YjbH reduces the overall conformational flexibility of Spx. Our findings provide insights into the molecular basis for Spx recognition and suggest a model for how YjbH stabilizes Spx and displays the C terminus of Spx for engagement by ClpXP. Awad et al. determined the crystal structure of the ClpXP adaptor protein YjbH in complex with the transcription factor Spx. Structural dynamics of the complex were investigated by hydrogen-deuterium exchange mass spectrometry. The insights provided in this work add molecular details to the recognition of Spx by YjbH.
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| 8. |
- Bimai, Ornella, et al.
(författare)
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Nucleotide binding to the ATP-cone in anaerobic ribonucleotide reductases allosterically regulates activity by modulating substrate binding
- 2024
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Ingår i: eLIFE. - : eLife Sciences Publications Ltd. - 2050-084X. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- A small, nucleotide-binding domain, the ATP-cone, is found at the N- terminus of most ribonucleotide reductase (RNR) catalytic subunits. By binding adenosine triphosphate (ATP) or deoxyadenosine triphosphate (dATP) it regulates the enzyme activity of all classes of RNR. Functional and structural work on aerobic RNRs has revealed a plethora of ways in which dATP inhibits activity by inducing oligomerisation and preventing a productive radical transfer from one subunit to the active site in the other. Anaerobic RNRs, on the other hand, store a stable glycyl radical next to the active site and the basis for their dATP-dependent inhibition is completely unknown. We present biochemical, biophysical, and structural information on the effects of ATP and dATP binding to the anaerobic RNR from Prevotella copri. The enzyme exists in a dimertetramer equilibrium biased towards dimers when two ATP molecules are bound to the ATP- cone and tetramers when two dATP molecules are bound. In the presence of ATP, P. copri NrdD is active and has a fully ordered glycyl radical domain ( GRD) in one monomer of the dimer. Binding of dATP to the ATP-cone results in loss of activity and increased dynamics of the GRD, such that it cannot be detected in the cryo-EM structures. The glycyl radical is formed even in the dATP-bound form, but the substrate does not bind. The structures implicate a complex network of interactions in activity regulation that involve the GRD more than 30 A away from the dATP molecules, the allosteric substrate specificity site and a conserved but previously unseen flap over the active site. Taken together, the results suggest that dATP inhibition in anaerobic RNRs acts by increasing the flexibility of the flap and GRD, thereby preventing both substrate binding and radical mobilisation.
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| 9. |
- Bågenholm, Viktoria, et al.
(författare)
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A surface-exposed GH26 -mannanase from Bacteroides ovatus : Structure, role, and phylogenetic analysis of BoMan26B
- 2019
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Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 294:23, s. 9100-9117
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Tidskriftsartikel (refereegranskat)abstract
- The galactomannan utilization locus (BoManPUL) of the human gut bacterium Bacteroides ovatus encodes BoMan26B, a cell-surface– exposed endomannanase whose functional and structural features have been unclear. Our study now places BoMan26B in context with related enzymes and reveals the structural basis for its specificity. BoMan26B prefers longer substrates and is less restricted by galactose side-groups than the mannanase BoMan26A of the same locus. Using galactomannan, BoMan26B generated a mixture of (galactosyl) manno-oligosaccharides shorter than mannohexaose. Three defined manno-oligosaccharides had affinity for the SusD-like surface–exposed glycan-binding protein, predicted to be implicated in saccharide transport. Co-incubation of BoMan26B and the periplasmic -galactosidase BoGal36A increased the rate of galactose release by about 10-fold compared with the rate without BoMan26B. The results suggested that BoMan26B performs the initial attack on galactomannan, generating oligosaccharides that after transport to the periplasm are processed by BoGal36A. A crystal structure of BoMan26B with galactosyl-mannotetraose bound in subsites 5 to 2 revealed an open and long active-site cleft with Trp-112 in subsite 5 concluded to be involved in mannosyl interaction. Moreover, Lys-149 in the 4 subsite interacted with the galactosyl side-group of the ligand. A phylogenetic tree consisting of GH26 enzymes revealed four strictly conserved GH26 residues and disclosed that BoMan26A and BoMan26B reside on two distinct phylogenetic branches (A and B). The three other branches contain lichenases, xylanases, or enzymes with unknown activities. Lys-149 is conserved in a narrow part of branch B, and Trp-112 is conserved in a wider group within branch B.
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| 10. |
- Caldararu, Octav, et al.
(författare)
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Are crystallographic : B-factors suitable for calculating protein conformational entropy?
- 2019
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Ingår i: Physical Chemistry Chemical Physics. - : Royal Society of Chemistry (RSC). - 1463-9076 .- 1463-9084. ; 21:33, s. 18149-18160
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Tidskriftsartikel (refereegranskat)abstract
- Conformational entropies are of great interest when studying the binding of small ligands to proteins or the interaction of proteins. Unfortunately, there are no experimental methods available to measure conformational entropies of all groups in a protein. Instead, they are normally estimated from molecular dynamics (MD) simulations, although such methods show problems with convergence and correlation of motions, and depend on the accuracy of the underlying potential-energy function. Crystallographic atomic displacement parameters (also known as B-factors) are available in all crystal structures and contain information about the atomic fluctuations, which can be converted to entropies. We have studied whether B-factors can be employed to extract conformational entropies for proteins by comparing such entropies to those measured by NMR relaxation experiments or obtained from MD simulations in solution or in the crystal. Unfortunately, our results show that B-factor entropies are unreliable, because they include the movement and rotation of the entire protein, they exclude correlation of the movements and they include contributions other than the fluctuations, e.g. static disorder, as well as errors in the model and the scattering factors. We have tried to reduce the first problem by employing translation-libration-screw refinement, the second by employing a description of the correlated movement from MD simulations, and the third by studying only the change in entropy when a pair of ligands binds to the same protein, thoroughly re-refining the structures in exactly the same way and using the same set of alternative conformations. However, the experimental B-factors seem to be incompatible with fluctuations from MD simulations and the precision is too poor to give any reliable entropies.
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| 11. |
- Caldararu, Octav, et al.
(författare)
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Exploring ligand dynamics in protein crystal structures with ensemble refinement
- 2021
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Ingår i: Acta Crystallographica Section D: Structural Biology. - 2059-7983. ; 77, s. 1099-1115
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Tidskriftsartikel (refereegranskat)abstract
- Understanding the dynamics of ligands bound to proteins is an important task in medicinal chemistry and drug design. However, the dominant technique for determining protein-ligand structures, X-ray crystallography, does not fully account for dynamics and cannot accurately describe the movements of ligands in protein binding sites. In this article, an alternative method, ensemble refinement, is used on six protein-ligand complexes with the aim of understanding the conformational diversity of ligands in protein crystal structures. The results show that ensemble refinement sometimes indicates that the flexibility of parts of the ligand and some protein side chains is larger than that which can be described by a single conformation and atomic displacement parameters. However, since the electron-density maps are comparable and R free values are slightly increased, the original crystal structure is still a better model from a statistical point of view. On the other hand, it is shown that molecular-dynamics simulations and automatic generation of alternative conformations in crystallographic refinement confirm that the flexibility of these groups is larger than is observed in standard refinement. Moreover, the flexible groups in ensemble refinement coincide with groups that give high atomic displacement parameters or non-unity occupancy if optimized in standard refinement. Therefore, the conformational diversity indicated by ensemble refinement seems to be qualitatively correct, indicating that ensemble refinement can be an important complement to standard crystallographic refinement as a tool to discover which parts of crystal structures may show extensive flexibility and therefore are poorly described by a single conformation. However, the diversity of the ensembles is often exaggerated (probably partly owing to the rather poor force field employed) and the ensembles should not be trusted in detail.
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| 12. |
- Caldararu, Octav, et al.
(författare)
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Refinement of protein structures using a combination of quantum-mechanical calculations with neutron and X-ray crystallographic data
- 2019
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Ingår i: Acta Crystallographica Section D: Structural Biology. - 2059-7983. ; 75, s. 368-380
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Tidskriftsartikel (refereegranskat)abstract
- Neutron crystallography is a powerful method to determine the positions of H atoms in macromolecular structures. However, it is sometimes hard to judge what would constitute a chemically reasonable model, and the geometry of H atoms depends more on the surroundings (for example the formation of hydrogen bonds) than heavy atoms, so that the empirical geometry information for the H atoms used to supplement the experimental data is often less accurate. These problems may be reduced by using quantum-mechanical calculations. A method has therefore been developed to combine quantum-mechanical calculations with joint crystallographic refinement against X-ray and neutron data. A first validation of this method is provided by re-refining the structure of the galectin-3 carbohydrate-recognition domain in complex with lactose. The geometry is improved, in particular for water molecules, for which the method leads to better-resolved hydrogen-bonding interactions. The method has also been applied to the active copper site of lytic polysaccharide monooxygenase and shows that the protonation state of the amino-terminal histidine residue can be determined.
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| 13. |
- Dahlqvist, Alexander, et al.
(författare)
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3-Substituted 1-Naphthamidomethyl-C-galactosyls Interact with Two Unique Sub-Sites for High-Affinity and High-Selectivity Inhibition of Galectin-3
- 2019
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Ingår i: Molecules. - : MDPI AG. - 1420-3049. ; 24:24
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Tidskriftsartikel (refereegranskat)abstract
- The galectins are a family of galactose-binding proteins playing key roles in inflammatory processes and cancer. However, they are structurally very closely related, and discovery of highly selective inhibitors is challenging. In this work, we report the design of novel inhibitors binding to a subsite unique to galectin-3, which confers both high selectivity and affinity towards galectin-3. Olefin cross metathesis between allyl β-C-galactopyranosyl and 1-vinylnaphthalenes or acylation of aminomethyl β-C-galactopyranosyl with 1-naphthoic acid derivatives gave C-galactopyranosyls carrying 1-naphthamide structural elements that interacted favorably with a galectin-3 unique subsite according to molecular modeling and X-ray structural analysis of two inhibitor-galectin-3 complexes. Affinities were down to sub-µM and selectivities over galectin-1, 2, 4 N-terminal domain, 4 C-terminal domain, 7, 8 N-terminal domain, 9 N-terminal domain, and 9 C-terminal domain were high. These results show that high affinity and selectivity for a single galectin can be achieved by targeting unique subsites, which holds promise for further development of small and selective galectin inhibitors.
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| 14. |
- Franza, Thierry, et al.
(författare)
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NAD+ pool depletion as a signal for the Rex regulon involved in Streptococcus agalactiae virulence
- 2021
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Ingår i: PLoS Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 17:8
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Tidskriftsartikel (refereegranskat)abstract
- In many Gram-positive bacteria, the redox-sensing transcriptional repressor Rex controls central carbon and energy metabolism by sensing the intra cellular balance between the reduced and oxidized forms of nicotinamide adenine dinucleotide; the NADH/NAD+ ratio. Here, we report high-resolution crystal structures and characterization of a Rex ortholog (Gbs1167) in the opportunistic pathogen, Streptococcus agalactiae, also known as group B streptococcus (GBS). We present structures of Rex bound to NAD+ and to a DNA operator which are the first structures of a Rex-family member from a pathogenic bacterium. The structures reveal the molecular basis of DNA binding and the conformation alterations between the free NAD+ complex and DNA-bound form of Rex. Transcriptomic analysis revealed that GBS Rex controls not only central metabolism, but also expression of the monocistronic rex gene as well as virulence gene expression. Rex enhances GBS virulence after disseminated infection in mice. Mechanistically, NAD+ stabilizes Rex as a repressor in the absence of NADH. However, GBS Rex is unique compared to Rex regulators previously characterized because of its sensing mechanism: we show that it primarily responds to NAD+ levels (or growth rate) rather than to the NADH/NAD+ ratio. These results indicate that Rex plays a key role in GBS pathogenicity by modulating virulence factor gene expression and carbon metabolism to harvest nutrients from the host.
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| 15. |
- Grāve, Kristīne, et al.
(författare)
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Redox-induced structural changes in the di-iron and di-manganese forms of Bacillus anthracis ribonucleotide reductase subunit NrdF suggest a mechanism for gating of radical access
- 2019
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Ingår i: Journal of Biological Inorganic Chemistry. - : Springer Science and Business Media LLC. - 0949-8257 .- 1432-1327. ; 24:6, s. 849-861
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Tidskriftsartikel (refereegranskat)abstract
- Class Ib ribonucleotide reductases (RNR) utilize a di-nuclear manganese or iron cofactor for reduction of superoxide or molecular oxygen, respectively. This generates a stable tyrosyl radical (Y center dot) in the R2 subunit (NrdF), which is further used for ribonucleotide reduction in the R1 subunit of RNR. Here, we report high-resolution crystal structures of Bacillus anthracis NrdF in the metal-free form (1.51 angstrom) and in complex with manganese (Mn-II/Mn-II, 1.30 angstrom). We also report three structures of the protein in complex with iron, either prepared anaerobically (Fe-II/Fe-II form, 1.32 angstrom), or prepared aerobically in the photo-reduced Fe-II/Fe-II form (1.63 angstrom) and with the partially oxidized metallo-cofactor (1.46 angstrom). The structures reveal significant conformational dynamics, likely to be associated with the generation, stabilization, and transfer of the radical to the R1 subunit. Based on observed redox-dependent structural changes, we propose that the passage for the superoxide, linking the FMN cofactor of NrdI and the metal site in NrdF, is closed upon metal oxidation, blocking access to the metal and radical sites. In addition, we describe the structural mechanics likely to be involved in this process.
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| 16. |
- Hansson, Sebastian, et al.
(författare)
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Crystal structure of a mutant elongation factor G trapped with a GTP analogue
- 2005
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Ingår i: FEBS Letters. - : Wiley. - 1873-3468 .- 0014-5793. ; 579:20, s. 4492-4497
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Tidskriftsartikel (refereegranskat)abstract
- Elongation factor G (EF-G) is a G protein factor that catalyzes the translocation step in protein synthesis on the ribosome. Its GTP conformation in the absence of the ribosome is currently unknown. We present the structure of a mutant EF-G (T84A) in complex with the non-hydrolysable GTP analogue GDPNP. The crystal structure provides a first insight into conformational changes induced in EF-G by GTP. Comparison of this structure with that of EF-G in complex with GDP suggests that the GTP and GDP conformations in solution are very similar and that the major contribution to the active GTPase conformation, which is quite different, therefore comes from its interaction with the ribosome. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
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| 17. |
- Hasan, Mahmudul, et al.
(författare)
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Solution Structure of the dATP-Inactivated Class I Ribonucleotide Reductase From Leeuwenhoekiella blandensis by SAXS and Cryo-Electron Microscopy
- 2021
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media SA. - 2296-889X. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- The essential enzyme ribonucleotide reductase (RNR) is highly regulated both at the level of overall activity and substrate specificity. Studies of class I, aerobic RNRs have shown that overall activity is downregulated by the binding of dATP to a small domain known as the ATP-cone often found at the N-terminus of RNR subunits, causing oligomerization that prevents formation of a necessary alpha(2)beta(2) complex between the catalytic (alpha(2)) and radical generating (beta(2)) subunits. In some relatively rare organisms with RNRs of the subclass NrdAi, the ATP-cone is found at the N-terminus of the beta subunit rather than more commonly the alpha subunit. Binding of dATP to the ATP-cone in beta results in formation of an unusual beta(4) tetramer. However, the structural basis for how the formation of the active complex is hindered by such oligomerization has not been studied. Here we analyse the low-resolution three-dimensional structures of the separate subunits of an RNR from subclass NrdAi, as well as the alpha(4)beta(4) octamer that forms in the presence of dATP. The results reveal a type of oligomer not previously seen for any class of RNR and suggest a mechanism for how binding of dATP to the ATP-cone switches off catalysis by sterically preventing formation of the asymmetrical alpha(2)beta(2) complex.
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| 18. |
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| 19. |
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| 20. |
- Johansson, Renzo, et al.
(författare)
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Structural Mechanism of Allosteric Activity Regulation in a Ribonucleotide Reductase with Double ATP Cones
- 2016
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Ingår i: Structure. - : Elsevier BV. - 0969-2126 .- 1878-4186. ; 24:6, s. 906-917
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Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotide reductases (RNRs) reduce ribonucleotides to deoxyribonucleotides. Their overall activity is stimulated by ATP and downregulated by dATP via a genetically mobile ATP cone domain mediating the formation of oligomeric complexes with varying quaternary structures. The crystal structure and solution X-ray scattering data of a novel dATP-induced homotetramer of the Pseudomonas aeruginosa class I RNR reveal the structural bases for its unique properties, namely one ATP cone that binds two dATP molecules and a second one that is non-functional, binding no nucleotides. Mutations in the observed tetramer interface ablate oligomerization and dATP-induced inhibition but not the ability to bind dATP. Sequence analysis shows that the novel type of ATP cone may be widespread in RNRs. The present study supports a scenario in which diverse mechanisms for allosteric activity regulation are gained and lost through acquisition and evolutionary erosion of different types of ATP cone.
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| 21. |
- Kulkarni, Tejas, et al.
(författare)
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Crystal structure of β-glucosidase 1A from Thermotoga neapolitana and comparison of active site mutants for hydrolysis of flavonoid glucosides
- 2017
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Ingår i: Proteins: Structure, Function and Bioinformatics. - : Wiley. - 1097-0134 .- 0887-3585. ; 85:5, s. 872-884
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Tidskriftsartikel (refereegranskat)abstract
- The β-glucosidase TnBgl1A catalyses hydrolysis of O-linked terminal β-glycosidic bonds at the nonreducing end of glycosides/oligosaccharides. Enzymes with this specificity have potential in lignocellulose conversion (degrading cellobiose to glucose) and conversion of bioactive flavonoids (modification of glycosylation results in modulation of bioavailability). Previous work has shown TnBgl1A to hydrolyse 3, 4′ and 7 glucosylation in flavonoids, and although conversion of 3-glucosylated substrate to aglycone was low, it was improved by mutagenesis of residue N220. To further explore structure-function relationships, the crystal structure of the nucleophile mutant TnBgl1A-E349G was determined at 1.9 Å resolution, and docking studies of flavonoid substrates were made to reveal substrate interacting residues. A series of single amino acid changes were introduced in the aglycone binding region [N220(S/F), N221(S/F), F224(I), F310(L/E), and W322(A)] of the wild type. Activity screening was made on eight glucosylated flavonoids, and kinetic parameters were monitored for the flavonoid quercetin-3-glucoside (Q3), as well as for the model substrate para-nitrophenyl-β-d-glucopyranoside (pNPGlc). Substitution by Ser at N220 or N221 increased the catalytic efficiency on both pNPGlc and Q3. Residue W322 was proven important for substrate accomodation, as mutagenesis to W322A resulted in a large reduction of hydrolytic activity on 3-glucosylated flavonoids. Flavonoid glucoside hydrolysis was unaffected by mutations at positions 224 and 310. The mutations did not significantly affect thermal stability, and the variants kept an apparent unfolding temperature of 101°C. This work pinpoints positions in the aglycone region of TnBgl1A of importance for specificity on flavonoid-3-glucosides, improving the molecular understanding of activity in GH1 enzymes.
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| 22. |
- Kumar, Rohit, et al.
(författare)
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Structure and Energetics of Ligand–Fluorine Interactions with Galectin-3 Backbone and Side-Chain Amides : Insight into Solvation Effects and Multipolar Interactions
- 2019
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Ingår i: ChemMedChem. - : Wiley. - 1860-7179 .- 1860-7187. ; 14:16, s. 1528-1536
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Tidskriftsartikel (refereegranskat)abstract
- Multipolar fluorine–amide interactions with backbone and side-chain amides have been described as important for protein–ligand interactions and have been used to improve the potency of synthetic inhibitors. In this study, fluorine interactions within a well-defined binding pocket on galectin-3 were investigated systematically using phenyltriazolyl-thiogalactosides fluorinated singly or multiply at various positions on the phenyl ring. X-ray structures of the C-terminal domain of galectin-3 in complex with eight of these ligands revealed potential orthogonal fluorine–amide interactions with backbone amides and one with a side-chain amide. The two interactions involving main-chain amides seem to have a strong influence on affinity as determined by fluorescence anisotropy. In contrast, the interaction with the side-chain amide did not influence affinity. Quantum mechanics calculations were used to analyze the relative contributions of these interactions to the binding energies. No clear correlation could be found between the relative energies of the fluorine–main-chain amide interactions and the overall binding energy. Instead, dispersion and desolvation effects play a larger role. The results confirm that the contribution of fluorine–amide interactions to protein–ligand interactions cannot simply be predicted, on geometrical considerations alone, but require careful consideration of the energetic components.
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| 23. |
- Kumar, Rohit, et al.
(författare)
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Substituted polyfluoroaryl interactions with an arginine side chain in galectin-3 are governed by steric-, desolvation and electronic conjugation effects
- 2019
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Ingår i: Organic and Biomolecular Chemistry. - : Royal Society of Chemistry (RSC). - 1477-0520 .- 1477-0539. ; 17:5, s. 1081-1089
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Tidskriftsartikel (refereegranskat)abstract
- In the β-d-galactopyranoside-binding protein galectin-3, synthetic inhibitors substituted at the 3-position of a thiodigalactoside core cause the formation of an aglycone binding pocket through the displacement of an arginine residue (Arg144) from its position in the apoprotein. To examine in detail the role of different molecular interactions in this pocket, we have synthesized a series of nine 3-(4-(2,3,5,6-tetrafluorophenyl)-1,2,3-triazol-1-yl)-thiogalactosides with different para substituents and measured their affinities to galectin-3 using a fluorescence polarization assay. High-resolution crystal structures (<1.3 Å) have been determined for five of the ligands in complex with the C-terminal domain of galectin-3. The binding affinities are rationalised with the help of the three-dimensional structures and quantum-mechanical calculations. Three effects seem to be involved: Firstly, the binding pocket is too small for the largest ligands with ethyl and methyl. Secondly, for the other ligands, the affinity seems to be determined mainly by desolvation effects, disfavouring the polar substituents, but this is partly counteracted by the cation-π interaction with Arg144, which stacks on top of the substituted tetrafluorophenyl group in all complexes. The results provide detailed insight into interactions of fluorinated phenyl moieties with arginine-containing protein binding sites and the complex interplay of different energetic components in defining the binding affinity.
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| 24. |
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| 25. |
- Lima, Gustavo M A, et al.
(författare)
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FragMAX : the fragment-screening platform at the MAX IV Laboratory
- 2020
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Ingår i: Acta Crystallographica Section D: Structural Biology. - 2059-7983. ; 76:Pt 8, s. 771-777
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Tidskriftsartikel (refereegranskat)abstract
- Advances in synchrotron storage rings and beamline automation have pushed data-collection rates to thousands of data sets per week. With this increase in throughput, massive projects such as in-crystal fragment screening have become accessible to a larger number of research groups. The quality of support offered at large-scale facilities allows medicinal chemistry-focused or biochemistry-focused groups to supplement their research with structural biology. Preparing the experiment, analysing multiple data sets and prospecting for interesting complexes of protein and fragments require, for both newcomers and experienced users, efficient management of the project and extensive computational power for data processing and structure refinement. Here, FragMAX, a new complete platform for fragment screening at the BioMAX beamline of the MAX IV Laboratory, is described. The ways in which users are assisted in X-ray-based fragment screenings and in which the fourth-generation storage ring available at the facility is best exploited are also described.
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