Cryo-EM structure of human Pol κ bound to DNA and mono-ubiquitylated PCNA

Y-family DNA polymerase κ (Pol κ) can replicate damaged DNA templates to rescue stalled replication forks. Access of Pol κ to DNA damage sites is facilitated by its interaction with the processivity clamp PCNA and is regulated by PCNA mono-ubiquitylation. Here, we present cryo-EM reconstructions of human Pol κ bound to DNA, an incoming nucleotide, and wild type or mono-ubiquitylated PCNA (Ub-PCNA). In both reconstructions, the internal PIP-box adjacent to the Pol κ Polymerase-Associated Domain (PAD) docks the catalytic core to one PCNA protomer in an angled orientation, bending the DNA exiting the Pol κ active site through PCNA, while Pol κ C-terminal domain containing two Ubiquitin Binding Zinc Fingers (UBZs) is invisible, in agreement with disorder predictions. The ubiquitin moieties are partly flexible and extend radially away from PCNA, with the ubiquitin at the Pol κ-bound protomer appearing more rigid. Activity assays suggest that, when the internal PIP-box interaction is lost, Pol κ is retained on DNA by a secondary interaction between the UBZs and the ubiquitins flexibly conjugated to PCNA. Our data provide a structural basis for the recruitment of a Y-family TLS polymerase to sites of DNA damage.This research was supported by King Abdullah University of Science and Technology through core funding (to S.M.H.) and the Competitive Research Award Grant CRG8 URF/1/4036‐01‐01 (to S.M.H. and A.D.B.), and by the Wellcome Trust (to A.D.B.). R.C. acknowledges funding from the MINECO (CTQ2016-78636-P) and to AGAUR, (2017 SGR 324). The MD project has been carried out using CSUC resources. We acknowledge The Midlands Regional Cryo-EM Facility at the Leicester Institute of Structural and Chemical Biology (LISCB), major funding from MRC (MC_PC_17136). We thank Christos Savva (LISCB, University of Leicester) for his help in cryo-EM data collection and advice on data processing.Peer reviewe

A glutamine-based single α-helix scaffold to target globular proteins

The binding of intrinsically disordered proteins to globular ones can require the folding of motifs into α-helices. These interactions offer opportunities for therapeutic intervention but their modulation with small molecules is challenging because they bury large surfaces. Linear peptides that display the residues that are key for binding can be targeted to globular proteins when they form stable helices, which in most cases requires their chemical modification. Here we present rules to design peptides that fold into single α-helices by instead concatenating glutamine side chain to main chain hydrogen bonds recently discovered in polyglutamine helices. The resulting peptides are uncharged, contain only natural amino acids, and their sequences can be optimized to interact with specific targets. Our results provide design rules to obtain single α-helices for a wide range of applications in protein engineering and drug design.We thank Luis Serrano for help with the Agadir predictions and helpful discussions, Ben Lehner and Ernest Giralt for helpful discussions and the ICTS NMR facility, managed by the scientific and technological centers of the University of Barcelona (CCiT UB), for their help in NMR. B.M. acknowledges funding from the Asociación Española contra el Cáncer (FCAECC project #POSTD211371MATE). C.G. acknowledges a graduate fellowship from MINECO (PRE2018-084684). M.S.-N. acknowledges funding from MINECO (PID2020-119810RB-I00). M.S.-N. holds a Ramón y Cajal contract (RYC2018-024759-I) from the Spanish Ministry of Science, Innovation, and Universities. X.S. acknowledges funding from AGAUR (2017 SGR 324), MINECO (BIO2015-70092-R and PID2019-110198RB-I00), and the European Research Council (CONCERT, contract number 648201). B.B.K acknowledges funding from the Novo Nordisk Foundation (#NNF18OC0033926). M.O. acknowledges funding from the Instituto Nacional de Bioinformática, The EU BioExcel Centre of Excellence for HPC and the Spanish Ministry of Science (PID2021-122478NB-I00) and the Instituto de Salud Carlos III–Instituto Nacional de Bioinformatica (ISCIII PT 17/0009/0007 co-funded by the Fondo Europeo de Desarrollo Regional). M.O. is an ICREA Academy scholar and J.A. is a Juan de la Cierva fellow. M.C. was supported by institutional funds of the Max Planck Society. This project has been carried out using the resources of CSUC. IRB Barcelona is the recipient of a Severo Ochoa Award of Excellence from MINECO (Government of Spain).Peer reviewe

Mg2+-dependent conformational equilibria in CorA and an integrated view on transport regulation

The CorA family of proteins regulates the homeostasis of divalent metal ions in many bacteria, archaea, and eukaryotic mitochondria, making it an important target in the investigation of the mechanisms of transport and its functional regulation. Although numerous structures of open and closed channels are now available for the CorA family, the mechanism of the transport regulation remains elusive. Here, we investigated the conformational distribution and associated dynamic behaviour of the pentameric Mg2+ channel CorA at room temperature using small-angle neutron scattering (SANS) in combination with molecular dynamics (MD) simulations and solid-state nuclear magnetic resonance spectroscopy (NMR). We find that neither the Mg2+-bound closed structure nor the Mg2+-free open forms are sufficient to explain the average conformation of CorA. Our data support the presence of conformational equilibria between multiple states, and we further find a variation in the behaviour of the backbone dynamics with and without Mg2+. We propose that CorA must be in a dynamic equilibrium between different non-conducting states, both symmetric and asymmetric, regardless of bound Mg2+ but that conducting states become more populated in Mg2+-free conditions. These properties are regulated by backbone dynamics and are key to understanding the functional regulation of CorA.Peer reviewe

Dietary inflammatory index and anthropometric measures of obesity in a population sample at high cardiovascular risk from the PREDIMED trial

The dietary inflammatory index (DII) is a new tool to assess the inflammatory potential of diet. We aimed to determine the association between the DII and body mass index (BMI), waist circumference and waist to height ratio (WHtR). We conducted a cross-sectional study of 7,236 participants recruited into the PREDIMED trial (PREvención con DIeta MEDiterránea). Information from a validated 137-item food frequency questionnaire was used to calculate energy, foods and nutrients. A 14-item dietary screener was used to assess adherence to the Mediterranean diet (MeDiet). Sex-specific multivariable linear regression models were fitted to estimate differences (and 95% confidence intervals) in BMI, waist circumference and WHtR across quintiles of the DII. All nutrient intakes, healthy foods and adherence to the MeDiet were higher in the quintile with lowest DII score (more anti-inflammatory values) except for animal protein, saturated and monounsaturated fat. Though an inverse association between DII and total energy was apparent, the DII was associated with higher average BMI, waist circumference and WHtR after adjusting for known risk factors. The adjusted difference in WHtR for women and men between the highest and lowest quintile of DII was 1.60% (95% CI 0.87-2.33) and 1.04% (95% CI 0.35-1.74), respectively. Pro-inflammatory scores remained associated with obesity after controlling for the effect that adherence to a MeDiet had on inflammation. In conclusion, this study shows a direct association between the DII and indices of obesity and supports the hypothesis that diet may have a role in the development of obesity through inflammatory modulation mechanisms

Dietary α‐Linolenic Acid, Marine ω‐3 Fatty Acids, and Mortality in a Population With High Fish Consumption: Findings From the PREvención con DIeta MEDiterránea (PREDIMED) Study

Background: Epidemiological evidence suggests a cardioprotective role of α‐linolenic acid (ALA), a plant‐derived ω‐3 fatty acid. It is unclear whether ALA is beneficial in a background of high marine ω‐3 fatty acids (long‐chain n‐3 polyunsaturated fatty acids) intake. In persons at high cardiovascular risk from Spain, a country in which fish consumption is customarily high, we investigated whether meeting the International Society for the Study of Fatty Acids and Lipids recommendation for dietary ALA (0.7% of total energy) at baseline was related to all‐cause and cardiovascular disease mortality. We also examined the effect of meeting the society's recommendation for long‐chain n‐3 polyunsaturated fatty acids (≥500 mg/day). Methods and Results: We longitudinally evaluated 7202 participants in the PREvención con DIeta MEDiterránea (PREDIMED) trial. Multivariable‐adjusted Cox regression models were fitted to estimate hazard ratios. ALA intake correlated to walnut consumption (r=0.94). During a 5.9‐y follow‐up, 431 deaths occurred (104 cardiovascular disease, 55 coronary heart disease, 32 sudden cardiac death, 25 stroke). The hazard ratios for meeting ALA recommendation (n=1615, 22.4%) were 0.72 (95% CI 0.56–0.92) for all‐cause mortality and 0.95 (95% CI 0.58–1.57) for fatal cardiovascular disease. The hazard ratios for meeting the recommendation for long‐chain n‐3 polyunsaturated fatty acids (n=5452, 75.7%) were 0.84 (95% CI 0.67–1.05) for all‐cause mortality, 0.61 (95% CI 0.39–0.96) for fatal cardiovascular disease, 0.54 (95% CI 0.29–0.99) for fatal coronary heart disease, and 0.49 (95% CI 0.22–1.01) for sudden cardiac death. The highest reduction in all‐cause mortality occurred in participants meeting both recommendations (hazard ratio 0.63 [95% CI 0.45–0.87]). Conclusions: In participants without prior cardiovascular disease and high fish consumption, dietary ALA, supplied mainly by walnuts and olive oil, relates inversely to all‐cause mortality, whereas protection from cardiac mortality is limited to fish‐derived long‐chain n‐3 polyunsaturated fatty acids. Clinical Trial Registration URL: http://www.Controlled-trials.com/. Unique identifier: ISRCTN35739639

Stabilization of alpha-helices by side-chain to main hydrogen bonds. Can current force fields describe the hydrophobic shielding?

Poly-glutamine (polyQ) rich tracts are common in transcription factors and their length determines several health disorders. The polyQ tract of the androgen receptor shows a surprisingly high helical content. We previously showed that this is caused by stabilizing side-chain to main-chain hydrogen bonds [1]. Here we use NMR and Molecular Dynamics to understand how the polyQ flanking residues a¿ect these hydrogen bonds. We also explore to which extent current force fields can describe the hydrophobic shielding that is the source of the stabilization

Crowding Induces Differences in the Diffusion of Thermophilic and Mesophilic Proteins: A New Look at Neutron Scattering Results

The dynamical basis underlying the increased thermal stability of thermophilic proteins remains uncertain. Here, we challenge the new paradigm established by neutron scattering experiments in solution, in which the adaptation of thermophilic proteins to high temperatures lies in the lower sensitivity of their flexibility to temperature changes. By means of a combination of molecular dynamics and Brownian dynamics simulations, we report a reinterpretation of those experiments and show evidence that under crowding conditions, such as in vivo, thermophilic and homolog mesophilic proteins have diffusional properties with different thermal behavior

SS-map: Visualizing cooperative secondary structure elements in protein ensembles

We present SS-map, a tool to visualize the secondary structure content of ensembles of proteins. When generating ensembles of intrinsically disordered proteins, we lose the understanding a single native structure gives for folded proteins. It then becomes difficult to visualize the composition of the ensembles or to detect transient helices such as MoRFs. Conformational propensities for single residues also hide the nature of cooperative structures. Here we show how SS-map describes folded and unfolded ensembles of some peptides and gives a new view of the ensembles used to describe intrinsically disordered proteins with residual structure in computational and NMR experiments. This tool is implemented in an open-source python code located at code.google.com/p/ss-mapWe acknowledge financial support from the Ministerio de Innovación y Competitividad (CTQ2012-33324) and the Generalitat de Catalunya (2009SGR01472).Peer reviewe

Can the MaxFlux algorithm describe bifurcating paths?

7 pages, 6 figures.-- Printed version published in Oct 2007.Temperature-dependent reaction paths have been calculated with theMaxFlux algorithm for a bifurcating region of the potential energy surface of the blocked alanine dipeptide. The resulting paths agree with an ensemble of trajectories generated with Transition Path sampling, which has also been used to determine product distributions. The Valley Ridge Inflection point has been shown to lie close to the point of maximum curvature of the MaxFlux paths.R.C. would like to thank the Ramón y Cajal programme and A.J. the CSIC and the European Social Fund for her I3P fellowship. We also acknowledge financial support from the Spanish Drección General de Investigación Científica y Técnica (Grant CTQ2006-01345/BQU) and by the Generalitat de Catalunya (Grant 2005SGR00111). Some of the calculations were performed at the CESCA, which we also acknowledge.Peer reviewe

Can the MaxFlux algorithm describe bifurcating paths?

7 pages, 6 figures.-- Printed version published in Oct 2007.Temperature-dependent reaction paths have been calculated with theMaxFlux algorithm for a bifurcating region of the potential energy surface of the blocked alanine dipeptide. The resulting paths agree with an ensemble of trajectories generated with Transition Path sampling, which has also been used to determine product distributions. The Valley Ridge Inflection point has been shown to lie close to the point of maximum curvature of the MaxFlux paths.R.C. would like to thank the Ramón y Cajal programme and A.J. the CSIC and the European Social Fund for her I3P fellowship. We also acknowledge financial support from the Spanish Drección General de Investigación Científica y Técnica (Grant CTQ2006-01345/BQU) and by the Generalitat de Catalunya (Grant 2005SGR00111). Some of the calculations were performed at the CESCA, which we also acknowledge.Peer reviewe