Search

11 pags., 5 figs., Understanding early amyloidogenesis is key to rationally develop therapeutic strategies. Tau protein forms well-characterized pathological deposits but its aggregation mechanism is still poorly understood. Using single-molecule force spectroscopy based on a mechanical protection strategy, we studied the conformational landscape of the monomeric tau repeat domain (tauRD244-368). We found two sets of conformational states, whose frequency is influenced by mutations and the chemical context. While pathological mutations Δ280K and P301L and a pro-amyloidogenic milieu favored expanded conformations and destabilized local structures, an anti-amyloidogenic environment promoted a compact ensemble, including a conformer whose topology might mask two amyloidogenic segments. Our results reveal that to initiate aggregation, monomeric tau-RD244-368 decreases its polymorphism adopting expanded conformations. This could account for the distinct structures found in vitro and across tauopathies., Spanish Agency for Research (AEI). Grant Numbers: SAF2016-76678-C2-1-R, SAF2016-76678-C2-2-R, BFU2015-70072-R

12 pags., 6 figs., Bacteriophage-derived endolysins and bacterial autolysins (hereinafter lysins) represent a completely new class of efficient antibacterials. They prevent the development of bacterial resistance and help protect commensal microbiota, producing cell wall lysis. Here we have investigated whether the acquisition of enzymatic active domains (EADs) and cell wall binding domains (CWBDs) of balancing efficiencies could be a way of tuning natural lysin activity. The concept was applied to produce a chimeric lysin of superior antibacterial capacity using the endolysin Skl and the major pneumococcal autolysin LytA. Combination of the Skl EAD and the cell wall choline-binding domain (CBD) of LytA in the chimera QSLA increased the bacterial killing by 2 logs or more compared to parental enzymes at an equal concentration and extended the substrate range to resistant and emergent pneumococci and other pathogens of the mitis group. Contrarily, QLAS, containing LytA EAD and Skl CBD, was inactive against all tested strains, although domain structures were preserved and hydrolysis of purified cell walls maintained in both chimeras. As a whole, our study provides a novel clue to design superior lysins to fight multidrug-resistant pathogens based on domain selection, and a powerful in-vivo active lysin (QSLA) with promising therapeutic perspectives., This work was supported by grants from the Ministry of Economy and Competitiveness (BFU2015-70072-R) and the Ministry of Science, Innovation and Universities (RTI2018-099985-B-I00/AEI/10.13039/501100011033) to M. Menéndez, and by a grant from the Ministry of Economy and Competitiveness (MINECO-FEDER, SAF2017-88664-R) to P. García. Additional funding was provided by the “CIBER de Enfermedades Respiratorias” (CIBERES), an initiative of the Instituto de Salud Carlos III (ISCIII), to M. Menéndez and P. García.

Fungi are among the most abundant and diverse organisms on Earth and play pivotal roles in global carbon processing, nutrient cycling and food webs. Despite their abundant and functional importance, little is known about the patterns and mechanisms governing their community composition in intermittent rivers and ephemeral streams, which are the most common fluvial ecosystems globally. Thus far, it is known that aquatic fungi have evolved various life-history strategies and functional adaptations to cope with drying. Nevertheless, some of these adaptations have a metabolic cost and trade-offs between growth, reproduction and dispersion that may affect ecosystem functioning. Thus, understanding their ecological strategies along a gradient of drying is crucial to assess how species will respond to global change and to identify meaningful taxa to maintain ecosystem functions. By combining in situ hydrological information with a niche-based approach, we analysed the role of drying in explaining the spatial segregation of fungal species, and we determined their specialization and affinity over a gradient of drying. In addition, we estimated whether species niches are good predictors of two key ecosystem processes: organic matter decomposition and fungal biomass accrual. Overall, we found that annual drying duration and frequency were the most influential variables upon species niche differentiation across the 15 studied streams. Our cluster analysis identified four drying niche-based groups with contrasting distributions and responses over the drying gradient: drying-sensitive, partly tolerant to drying, generalist, and drying-resistant specialist. In addition, we found that species belonging to the drying specialist group showed a weak contribution to both ecosystem processes, suggesting trade-offs between drying resistance strategies and the energy invested in growth. Taken together, our results suggest that increased water scarcity may jeopardise the capacity of aquatic fungi to guarantee ecosystem functioning and to maintain biogeochemical cycles despite their ability to cope with drying.

Lipid nanocarriers, such as niosomes, are considered attractive candidates for non-viral gene delivery due to their suitable biocompatibility and high versatility. In this work, we studied the influence of incorporating chloroquine in niosomes biophysical performance, as well as the effect of non-ionic surfactant composition and protocol of incorporation in their biophysical performance. An exhaustive comparative evaluation of three niosome formulations differing in these parameters was performed, which included the analysis of their thermal stability, rheological behavior, mean particle size, dispersity, zeta potential, morphology, membrane packing capacity, affinity to bind DNA, ability to release and protect the genetic material, buffering capacity and ability to escape from artificially synthesized lysosomes. Finally, in vitro biological studies were, also, performed in order to determine the compatibility of the formulations with biological systems, their transfection efficiency and transgene expression. Results revealed that the incorporation of chloroquine in niosome formulations improved their biophysical properties and the transfection efficiency, while the substitution of one of the non-ionic surfactants and the phase of addition resulted in less biophysical variations. Of note, the present work provides several biophysical parameters and characterization strategies that could be used as gold standard for gene therapy nanosystems evaluation., Authors wish to thank: ICTS “NANBIOSIS”, specifically the Drug Formulation Unit (U10) of the CIBER in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN) for the intellectual and technical assistance; SGIker (UPV/EHU) for technical and human support; the Department of Education, University and Research of the Basque Country Government (Consolidated Groups, IT907-16); the Spanish Ministry of Science and Innovation (Grants PID2019-106199RB-C21 and RTI2018-099985-B-I00); also the support of CIBER of Respiratory Diseases (CIBERES-ISCIII) and the Cystic Fibrosis Association of the Basque Country. M.S.-R. thanks the University of the Basque Country (UPV/EHU) for the granted pre-doctoral fellowship (PIF17/79). I.V.-B. thanks the University of the Basque Country (UPV/EHU) for the granted postdoctoral fellowship (call for the Specialization of Doctor Researcher Personnel of the UPV/EHU, grant reference: ESPDOC19/47).

The effects of mixing different leaf litter species on litter decomposition in streams have received considerable attention in recent years. However, contrasting results have been reported and the mechanisms behind the effects of litter diversity have been poorly examined. We compared the decomposition rates and associated fungi for two contrasting litter species, when incubated individually and in mixture, at two different current velocities. Coarse-mesh bags with alder litter individually, oak litter individually and with a mixture of both were incubated in a forest headwater stream over 32 days, under fast or slow current velocities. We determined litter decomposition rates, microbial oxygen consumption rates, and aquatic hyphomycete sporulation rates, species richness and community composition; litter species in the mixture were processed individually. Our results provided weak evidences for diversity effects on leaf litter decomposition. Generally, litter decomposition was unaffected by mixing contrasting litter species, with litter species in the mixture decomposing at the same rate as when incubated individually at both current velocities. The same pattern was observed for microbial variables. Decomposition rates and microbial colonization and activity depended primarily on the traits of the target litter species and were not affected by those of the companion species. However, litter-mixing effects were detected on oak litter at late decomposition stages under fast current velocity conditions, suggesting that both current velocity and the incubation time might influence diversity effects on litter decomposition in streams. This finding contributes to explain the lack of litter-mixing effects reported previously by many studies.

14 pags., 4 figs., Background: Amyloids are ordered, insoluble protein aggregates, characterized by a cross-β sheet quaternary structure in which molecules in a β-strand conformation are stacked along the filament axis via intermolecular interactions. While amyloids are typically associated with pathological conditions, functional amyloids have also been identified and are present in a wide variety of organisms ranging from bacteria to humans. The cytoplasmic polyadenylation element-binding (CPEB) prion-like protein is an mRNA-binding translation regulator, whose neuronal isoforms undergo activity-dependent aggregation, a process that has emerged as a plausible biochemical substrate for memory maintenance. CPEB aggregation is driven by prion-like domains (PLD) that are divergent in sequence across species, and it remains unknown whether such divergent PLDs follow a similar aggregating assembly pathway. Here, we describe the amyloid-like features of the neuronal Aplysia CPEB (ApCPEB) PLD and compare them to those of the Drosophila ortholog, Orb2 PLD. Results: Using in vitro single-molecule and bulk biophysical methods, we find transient oligomers and mature amyloid-like filaments that suggest similarities in the late stages of the assembly pathway for both ApCPEB and Orb2 PLDs. However, while prior to aggregation the Orb2 PLD monomer remains mainly as a random coil in solution, ApCPEB PLD adopts a diversity of conformations comprising α-helical structures that evolve to coiled-coil species, indicating structural differences at the beginning of their amyloid assembly pathways. Conclusion: Our results indicate that divergent PLDs of CPEB proteins from different species retain the ability to form a generic amyloid-like fold through different assembly mechanisms., The work was funded by two joint grants from the Ministry of Economy and Competitiveness to MCV (SAF2013-49179-C2-1-R and SAF2016-76678-C2-1-R) and DVL (SAF2013-49179-C2-2-R and SAF2016-76678-C2-2-R) and grants of the Ministry of Economy and Science (BFU2015-70072-R) and the CIBER de Enfermedades Respiratorias (CIBERES; ISCIII) to MM.

14 p.-6 fig.-1 tab., Endolysins are bacteriophage-encoded peptidoglycan hydrolases targeting the cell wall of host bacteria via their cell wall-binding domains (CBDs). The molecular basis for selective recognition of surface carbohydrate ligands by CBDs remains elusive. Here, we describe, in atomic detail, the interaction between the Listeria phage endolysin domain CBD500 and its cell wall teichoic acid (WTA) ligands. We show that 30O-acetylated GlcNAc residues integrated into the WTA polymer chain are the key epitope recognized by a CBD binding cavity located at the interface of tandem copies of beta-barrel, pseudosymmetric SH3b-like repeats. This cavity consists of multiple aromatic residues making extensive interactions with two GlcNAc acetyl groups via hydrogen bonds and van der Waals contacts, while permitting the docking of the diastereomorphic ligands. Our multidisciplinary approach tackled an extremely challenging protein–glycopolymer complex and delineated a previously unknown recognition mechanism by which a phage endolysin specifically recognizes and targets WTA, suggesting an adaptable model for regulation of endolysin specificity., E. T. S. has been supported by the Swiss National Science Foundation (SNF) grant 310030_156947/1. F. J. C.and M. M. have been supported by the Spanish Ministry of Science Innovation and Universities and FEDER funding (grants RTI2018-094751-B-C22 and BFU2015-70072R) and CIBERES, an initiative from the Spanish Institute of Health Carlos III.

Flow interruption in intermittent rivers (IRs) generates a mosaic of terrestrial and aquatic habitats across the river network affecting ecosystem processes, as organic matter (OM) decomposition. Water use for farming in arid and semi-arid climates intensifies the dry conditions and affects local river characteristics. In that way, flow intermittence and the distribution of land uses may affect the OM processing along the river. To understand the role of IRs in global OM dynamics and how global change affecting water flow regimes determines these dynamics, it is important to estimate OM-processing rates at a basin scale. The aim of this study was to evaluate the effect of the intensity of flow intermittence on OM processing, and how this effect was modulated by local environmental factors related to land uses across a Mediterranean river basin. To do this, wood decomposition (mass loss and fungal biomass) was selected as a functional indicator. Drying duration and frequency were measured to characterize flow intermittence in different reaches along the river, as well as local environmental factors. Linear models stablished the role of factors on decomposition. The results showed that differences in decomposition rates across the river network were negatively related to the duration of flow interruption. Dissolved inorganic nitrogen associated with agriculture counteracted the negative effect of intermittence on mass loss (increasing by up to three times); but with a higher duration of dry conditions, its effect was insignificant. An increase of 20% of canopy (higher in natural areas) resulted in increases of up to 5% of mass loss. Overall, our study is relevant to understanding the interaction between flow intermittence and land uses on OM processing, especially considering the intensification of flow intermittence and its increased distribution to other regions, which is expected to be a consequence of climate warming and human activities.

Predicting the impacts of global change on highly dynamic ecosystems requires a better understanding of how communities respond to disturbance duration, frequency and timing. Intermittent rivers and ephemeral streams are dynamic ecosystems that are recognized as the most common fluvial ecosystem globally. The complexity of the drying process can give rise to different annual and antecedent hydrological conditions, but their effect on aquatic communities remains unclear. Here, using aquatic invertebrates from 33 streams across a flow-intermittence gradient, we assessed how annual (drying duration and frequency) and recent drying characteristics (duration of the last dry period and flowing duration since the last rewetting) affect the density and diversity metrics of communities and trophic groups while controlling for other key abiotic factors (dissolved oxygen and altitude). We characterized invertebrate communities using taxonomy and functional traits to capture biological features that increase vulnerability to drying. In addition, using structural equation modelling (SEM), we evaluated pathways by which drying characteristics directly impact invertebrate density and whether diversity indirectly mediates such relationships. We show that drying frequency drove reductions in diversity at the community level and within trophic groups, whereas both the drying duration and frequency had a negative influence on density metrics. Reductions in taxonomic richness were linked to increased annual drying duration, whereas functional diversity declined in response to annual drying frequency. Filterer, predator and shredder trophic groups exhibited the strongest negative responses to drying. Recent drying characteristics had a minor effect on density and diversity metrics. Our SEM results demonstrated that diversity mediates the negative impacts of annual drying duration and frequency on invertebrate density through reductions in their taxonomic richness and functional diversity. Our results underscore the importance of considering multiple drying characteristics together with the interdependence of density and diversity to better anticipate drying responses in freshwater ecosystems. Keywords: biodiversity-ecosystem functioning, drying, flow intermittence, functional traits, intermittent rivers and ephemeral streams, Mediterranean streams

20 pags, 9 figs, 2 tabs. -- The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2021.740914/full#supplementary-material, We have structurally and functionally characterized Skl and Pal endolysins, the latter being the first endolysin shown to kill effectively Streptococcus pneumoniae, a leading cause of deathly diseases. We have proved that Skl and Pal are cysteine-amidases whose catalytic domains, from CHAP and Amidase_5 families, respectively, share an α3β6-fold with papain-like topology. Catalytic triads are identified (for the first time in Amidase_5 family), and residues relevant for substrate binding and catalysis inferred from in silico models, including a calcium-binding site accounting for Skl dependence on this cation for activity. Both endolysins contain a choline-binding domain (CBD) with a β-solenoid fold (homology modeled) and six conserved choline-binding loci whose saturation induced dimerization. Remarkably, Pal and Skl dimers display a common overall architecture, preserved in choline-bound dimers of pneumococcal lysins with other catalytic domains and bond specificities, as disclosed using small angle X-ray scattering (SAXS). Additionally, Skl is proved to be an efficient anti-pneumococcal agent that kills multi-resistant strains and clinical emergent-serotype isolates. Interestingly, Skl and Pal time-courses of pneumococcal lysis were sigmoidal, which might denote a limited access of both endolysins to target bonds at first stages of lysis. Furthermore, their DTT-mediated activation, of relevance for other cysteine-peptidases, cannot be solely ascribed to reversal of catalytic-cysteine oxidation., This work was supported by grants from the Ministry of Economy and Competitiveness (BFU2015-70072-R) and the Ministry of Science, Innovation and Universities (RTI2018-099985-B-I00/AEI/10.13039/501100011033) to MM, and by a grant from the Ministry of Economy and Competitiveness (MINECO-FEDER, SAF2017-88664-R) to PG. Additional funding was provided by the Centro de Investigacion Biomedica en Red de Enfermedades Respiratorias (CIBERES), an initiative of the Instituto de Salud Carlos III (ISCIII), to MM and PG.

9 pags, 6 figs. -- Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.actbio.2021.08.019., The adenovirus (AdV) icosahedral capsid encloses a nucleoprotein core formed by the dsDNA genome bound to numerous copies of virus-encoded, positively charged proteins. For an efficient delivery of its genome, AdV must undergo a cascade of dismantling events from the plasma membrane to the nuclear pore. Throughout this uncoating process, the virion moves across potentially disruptive environments whose influence in particle stability is poorly understood. In this work we analyze the effect of acidic conditions on AdV particles by exploring their mechanical properties, genome accessibility and capsid disruption. Our results show that under short term acidification the AdV virion becomes softer and its genome less accessible to an intercalating dye, even in the presence of capsid openings. The AFM tip penetrates deeper in virions at neutral pH, and mechanical properties of genome-less particles are not altered upon acidification. Altogether, these results indicate that the main effect of acidification is the compaction of the nucleoproteic core, revealing a previously unknown role for chemical cues in AdV uncoating. STATEMENT OF SIGNIFICANCE: Studying the behavior of virus particles under changing environmental conditions is key to understand cell entry and propagation. One such change is the acidification undergone in certain cell compartments, which is thought to play a role in the programmed uncoating of virus genomes. Mild acidification in the early endosome has been proposed as a trigger signal for human AdV uncoating. However, the actual effect of low pH in AdV stability and entry is not well defined. Understanding the consequences of acidification in AdV structure and stability is also relevant to define storage conditions for therapeutic vectors, or design AdV variants resistant to intestinal conditions for oral administration of vaccines., We thank M. G. Mateu (CBMSO-CSIC-UAM) for careful read-ing and insightful comments on early drafts, M. Castellanos and L. A. Campos (CNB-CSIC) for advice with fluorescence measurements and analyses, and M.I. Laguna (CNB-CSIC) for expert technical help. This work was supported by grants from the Spanish Ministry of Economy, Industry and Competitiveness (FIS2017-89549-R ; "Maria de Maeztu" Program for Units of Excellence in R&D MDM-2014-0377; and FIS2017-90701-REDT) and from the Human Frontiers Science Program (HFSPO RGP0012/2018) to P.J.P.; as well as grants PID2019-104098GB-I00/AEI/10.13039/501100011033 and BFU2016-74868-P, co-funded by the Spanish State Research Agency and the European Regional Development Fund , and 2019AEP045 from the Agencia Estatal CSIC to C.S.M. The CNB-CSIC is further supported by a Severo Ochoa Excellence grant (SEV 2017-0712) . MM was funded by grant RTI2018-099985-B-I0 0, (MICINN/FEDER, UE) and the Ciber of Respiratory Diseases (CIBERES) , an initiative from the Spanish Institute of Health Carlos III (ISCIII) . M.H.-P. was a recipient of a Juan de la Cierva Incorporation postdoctoral contract funded by the Spanish State Research Agency. M.P.-I. held a predoctoral contract from La Caixa Foundation (ID 10 0 010434, under agreement LCF/BQ/SO16/52270 032) . J. G. is a re-cipient of a FPI predoctoral contract (BES-2017-079868) funded by the Spanish State Research Agency.

20 pags., 7 figs., 2 tabs., Genetic variants arising from within-patient evolution shed light on bacterial adaptation during chronic infection. Contingency loci generate high levels of genetic variation in bacterial genomes, enabling adaptation to the stringent selective pressures exerted by the host. A significant gap in our understanding of phase-variable contingency loci is the extent of their contribution to natural infections. The human-adapted pathogen nontypeable Haemophilus influenzae (NTHi) causes persistent infections, which contribute to underlying disease progression. The phase-variable high-molecular-weight (HMW) adhesins located on the NTHi surface mediate adherence to respiratory epithelial cells and, depending on the allelic variant, can also confer high epithelial invasiveness or hyperinvasion. In this study, we characterize the dynamics of HMW-mediated hyperinvasion in living cells and identify a specific HMW binding domain shared by hyperinvasive NTHi isolates of distinct pathological origins. Moreover, we observed that HMW expression decreased over time by using a longitudinal set of persistent NTHi strains collected from chronic obstructive pulmonary disease (COPD) patients, resulting from increased numbers of simple-sequence repeats (SSRs) downstream of the functional P2hmw1A promoter, which is the one primarily driving HMW expression. Notably, the increased SSR numbers at the hmw1 promoter region also control a phenotypic switch toward lower bacterial intracellular invasion and higher biofilm formation, likely conferring adaptive advantages during chronic airway infection by NTHi. Overall, we reveal novel molecular mechanisms of NTHi pathoadaptation based on within-patient lifestyle switching controlled by phase variation. IMPORTANCE Human-adapted bacterial pathogens have evolved specific mechanisms to colonize their host niche. Phase variation is a contingency strategy to allow adaptation to changing conditions, as phase-variable bacterial loci rapidly and reversibly switch their expression. Several NTHi adhesins are phase variable. These adhesins are required for colonization but also immunogenic, in such a way that bacteria with lower adhesin levels are better equipped to survive an immune response, making their contribution to natural infections unclear. We show here that the major NTHi adhesin HMW1A displays allelic variation, which can drive a phase-variable epithelial hyperinvasion phenotype. Over time, hmw1A phase variation lowers adhesin expression, which controls an NTHi lifestyle switch from high epithelial invasiveness to lower invasion and higher biofilm formation. This reversible loss of function aligns with the previously stated notion that epithelial infection is essential for NTHi infection establishment, but once established, persistence favors gene inactivation, in this case facilitating biofilm growth., This work has been funded by grants SAF2015-66520-R and RTI2018-096369-B-I00 from MINECO, 875/2019 from SEPAR, and PC150-151-152 from the Gobierno de Navarra to J.G. and RTI2018-099985-B-I00 to M.M. CIBER is an initiative from the Instituto de Salud Carlos III (ISCIII), Madrid, Spain. A.T.-A. is funded by a Ministry of Science and Innovation grant (PID2019-105216GB-I00)

Investigating the influence of biodiversity on ecosystem functioning over environmental gradients is needed to anticipate ecosystem responses to global change. However, our understanding of the functional role of freshwater biodiversity, especially for microbes, is mainly based on manipulative experiments, where biodiversity and environmental variability are minimized. Here, we combined observational and manipulative experiments to analyse how fungal biodiversity responds to and mediates the impacts of drying on two key ecosystem processes: organic matter decomposition and fungal biomass accrual. Our observational data set consists of fungal biodiversity and ecosystem processes from 15 streams spanning a natural gradient of flow intermittence. Our manipulative design evaluates the responses of ecosystem processes to two fungal richness levels crossed with three levels of drying. For the observational experiment, we found that increasing the duration of drying reduced fungal species richness and caused compositional changes. Changes in species composition were driven by species turnover, suggesting resistance mechanisms to cope with drying. We also found that fungal richness had a positive effect on organic matter decomposition and fungal biomass accrual. Positive effects of fungal biodiversity were consistent when controlling for the effects of drying duration on richness by means of structural equation modelling. In addition, our results for the manipulative experiment showed that the positive effects of higher richness on both ecosystem processes were evident even when exposed to short or long simulated drying. Overall, our study suggests that maintaining high levels of biodiversity is crucial for maintaining functional freshwater ecosystems in response to ongoing and future environmental changes.

Knowledge on the molecular bases of early amyloid assembly is fundamental to understand its structure-dysfunction relationship during disease progression. Tauopathies, a well-defined set of neurodegenerative disorders that includes Alzheimer’s disease, are characterized by the pathological amyloid aggregation of tau. However, the underlying molecular mechanisms that trigger tau aggregation and toxicity are poorly understood. Here, using a single-molecule approach, AFM-based single molecule-force spectroscopy (AFM-SMFS), combined with a protein-engineering mechanical protection strategy, we have analyzed the fluctuations of the conformational space of tau during the start of its pathological amyloid assembly. Specifically, we have analyzed the region that includes the four tau microtubule-binding repeats, known to play a key role on tau aggregation. We find that, unlike other amyloid-forming proteins, tau aggregation is accompanied by a decrease of conformational polymorphism, which is driven by amyloid-promoting factors, such as the Δ280K and P301L mutations, linked to Frontotemporal Dementia-17, or by specific chemical conditions. Such perturbations have distinct effects and lead to different tau (aggregate) structures. In addition to providing insight into how tau aggregates in a context dependent manner, these findings may help delve into how protein aggregation-based diseases, like Alzheimer’s, might be treated using monomer fluctuations as a pharmacological target.Abstract Figure

9 pags., 7 figs., 1 tab., Adenovirus minor coat protein VI contains a membrane-disrupting peptide that is inactive when VI is bound to hexon trimers. Protein VI must be released during entry to ensure endosome escape. Hexon:VI stoichiometry has been uncertain, and only fragments of VI have been identified in the virion structure. Recent findings suggest an unexpected relationship between VI and the major core protein, VII. According to the high-resolution structure of the mature virion, VI and VII may compete for the same binding site in hexon; and noninfectious human adenovirus type 5 particles assembled in the absence of VII (Ad5-VII-) are deficient in proteolytic maturation of protein VI and endosome escape. Here we show that Ad5-VII- particles are trapped in the endosome because they fail to increase VI exposure during entry. This failure was not due to increased particle stability, because capsid disruption happened at lower thermal or mechanical stress in Ad5-VII- compared to wild-type (Ad5-wt) particles. Cryoelectron microscopy difference maps indicated that VII can occupy the same binding pocket as VI in all hexon monomers, strongly arguing for binding competition. In the Ad5-VII- map, density corresponding to the immature amino-terminal region of VI indicates that in the absence of VII the lytic peptide is trapped inside the hexon cavity, and clarifies the hexon:VI stoichiometry conundrum. We propose a model where dynamic competition between proteins VI and VII for hexon binding facilitates the complete maturation of VI, and is responsible for releasing the lytic protein from the hexon cavity during entry and stepwise uncoating, We thank the Centro Nacional de Biotecnología and the Centro de Investigaciones Biológicas cryoelectron microscopy facility for data acquisition.This work was supported by Grant BFU2016-74868-P, cofunded by the Spanish State Research Agency and the European Regional Development Fund (to C.S.M.); as well as by grants from the Spanish Ministry of Economy, Industry and Competitiveness BIO2015-68990-REDT (the Spanish Adenovirus Network, AdenoNet) (to C.S.M.), FIS2017-89549-R, “María de Maeztu” Program for Units of Excellence in R&D (MDM-2014-0377), and FIS2017-90701-REDT (to P.J.d.P.). P.H. was funded by National Institutes of Health Grants CA122677 and AI102577. U.F.G. and M.S. were funded by the Swiss National Science Foundation (SNSF 31003A_179256/1) and the Swiss National Science Foundation program Sinergia (CRSII5_170929/1). M.M. was funded by Grant BFU2015-70052R (Spanish Ministry of Economy, Industry and Competitiveness and European Regional Development Fund) and the Centro de Investigación Biomédica en Red de Enfermedades Respiratorias, an initiative from the Spanish Institute of Health Carlos III. M.H.-P. is a recipient of a Juan de la Cierva postdoctoral contract funded by the Spanish State Research Agency. M.P.-I. holds a predoctoral contract from La Caixa Foundation.

Endolysins are bacteriophage-encoded peptidoglycan hydrolases targeting the cell wall of host bacteria via their cell wall-binding domains (CBDs). The molecular basis for selective recognition of surface carbohydrate ligands by CBDs remains elusive. Here, we describe, in atomic detail, the interaction between the Listeria phage endolysin domain CBD500 and its cell wall teichoic acid (WTA) ligands. We show that 3’ O-acetylated GlcNAc residues integrated into the WTA polymer chain are the key epitope recognized by a CBD binding cavity located at the interface of tandem copies of beta-barrel, pseudo-symmetric SH3b-like repeats. This cavity consists of multiple aromatic residues making extensive interactions with two GlcNAc acetyl groups via hydrogen bonds and van der Waals contacts, while permitting the docking of the diastereomorphic ligands. The multidisciplinary approach described here delineates a previously unknown recognition mechanism by which a phage endolysin specifically recognizes and targets WTA, suggesting an adaptable model for regulation of endolysin specificity.

6 figs., 2 tabs., Molecular recognition is a fundamental phenomenon underpinning almost all aspects of biological processes. Associated energy changes result in release or absorption of heat that can be measured, providing valuable information about the interaction specific features. Isothermal titration calorimetry (ITC) is a label-free binding assay which measures the affinity, stoichiometry, and thermodynamics of molecular interactions from the reaction heat. It is considered the gold-standard technique owing to its unique capacity to provide a complete thermodynamic and even kinetic profile of the interaction in a single experiment, thus giving a detailed understanding of driving forces and underlying processes. Moreover, ITC can be used to characterise complex systems hardly amenable using other techniques and also enzyme-catalysed reactions. Consequently, ITC is being used to study a wide range of interactions mediated by proteins, nucleic acids, lipids, oligosaccharides and other natural or synthetic molecules, and has become a key component of drug discovery platforms.

Polyproline II (PPII) helices play vital roles in biochemical recognition events and structures like collagen and form part of the conformational landscapes of intrinsically disordered proteins (IDPs). Nevertheless, this structure is generally hard to detect and quantify. Here, we report the first thorough NMR characterization of a PPII helical bundle protein, the Hypogastrura harveyi ¿snow flea¿ antifreeze protein (sfAFP). J-couplings and nuclear Overhauser enhancement spectroscopy confirm a natively folded structure consisting of six PPII helices. NMR spectral analyses reveal quite distinct H¿2 versus H¿3 chemical shifts for 28 Gly residues as well as 13C¿, 15N, and 1HN conformational chemical shifts (¿¿) unique to PPII helical bundles. The 15N ¿¿ and 1HN ¿¿ values and small negative 1HN temperature coefficients evince hydrogen-bond formation. 1H¿15N relaxation measurements reveal that the backbone structure is generally highly rigid on ps¿ns time scales. NMR relaxation parameters and biophysical characterization reveal that sfAFP is chiefly a dimer. For it, a structural model featuring the packing of long, flat hydrophobic faces at the dimer interface is advanced. The conformational stability, measured by amide H/D exchange to be 6.24 ± 0.2 kcal·mol¿1, is elevated. These are extraordinary findings considering the great entropic cost of fixing Gly residues and, together with the remarkable upfield chemical shifts of 28 Gly 1H¿, evidence significant stabilizing contributions from C¿H¿ ||| O¿C hydrogen bonds. These stabilizing interactions are corroborated by density functional theory calculations and natural bonding orbital analysis. The singular conformational chemical shifts, J-couplings, high hNOE ratios, small negative temperature coefficients, and slowed H/D exchange constitute a unique set of fingerprints to identify PPII helical bundles, which may be formed by hundreds of Gly-rich motifs detected in sequence databases. These results should aid the quantification of PPII helices in IDPs, the development of improved antifreeze proteins, and the incorporation of PPII helices into novel designed proteins.

Adenovirus minor coat protein VI contains a membrane-disrupting peptide which is inactive when VI is bound to hexon trimers. Protein VI must be released during entry to ensure endosome escape. Hexon:VI stoichiometry has been uncertain, and only fragments of VI have been identified in the virion structure. Recent findings suggest an unexpected relationship between VI and the major core protein, VII. According to the high resolution structure of the mature virion, VI and VII may compete for the same binding site in hexon; and non-infectious human adenovirus type 5 particles assembled in the absence of VII (Ad5-VII-) are deficient in proteolytic maturation of protein VI and endosome escape. Here we show that Ad5-VII- particles are trapped in the endosome because they fail to increase VI exposure during entry. This failure was not due to increased particle stability, because capsid disruption happened at lower thermal or mechanical stress in Ad5-VII- compared to wildtype (Ad5-wt) particles. Cryo-EM difference maps indicated that VII can occupy the same binding pocket as VI in all hexon monomers, strongly arguing for binding competition. In the Ad5-VII- map, density corresponding to the immature amino-terminal region of VI indicates that in the absence of VII the lytic peptide is trapped inside the hexon cavity, and clarifies the hexon:VI stoichiometry conundrum. We propose a model where dynamic competition between proteins VI and VII for hexon binding facilitates the complete maturation of VI, and is responsible for releasing the lytic protein from the hexon cavity during entry and stepwise uncoating.Significance StatementCorrect assembly of an adenovirus infectious particle involves the highly regulated interaction of more than ten different proteins as well as the viral genome. Here we examine the interplay between two of these proteins: the major core protein VII, involved in genome condensation, and the multifunctional minor coat protein VI. Protein VI binds to the inner surface of adenovirus hexons (trimers of the major coat protein) and contains a lytic peptide which must be released during entry to ensure endosome rupture. We present data supporting a dynamic competition model between proteins VI and VII for hexon binding during assembly. This competition facilitates the release of the lytic peptide from the hexon cavity and ensures virus escape from the early endosome.

Changes in hydrology are one of the main causes of mangrove degradation; however, the reforestation of mangrove has been the main restoration activity and very little information on how pioneer species can facilitate the colonization and development of the mangrove species is available. After carrying out a water reconnection as the sole restoration action, secondary succession has occurred in the mangrove rehabilitation area of Celestun (Yucatan, SE Mexico). Two pioneer species, Batis maritima and Salicornia virginica were observed in plots with different coverage (0%, 20%, 100%) where the three natural mangrove species were established, with Laguncularia racemosa as the dominant species in density. The greatest interstitial mean salinity (79.9 g/kg) was recorded in the plots with 20% cover, while the lowest salinity (40.7 g/kg) was recorded in the plots with 100% of coverage. At the end of sampling period nutrient content (carbon, nitrogen and phosphorus) and organic matter in the sediment were greatest in the plots with 100% cover, whereas the lowest concentrations were observed in the site devoid of vegetation. The percentage cover of S. virginica and B. maritima changed over time; in the plot that started with 100% cover it decreased until it disappeared at the end of the study period, whereas it increased in the plots with 20% and 0% cover. This study shows that B. maritima and S. virginica has a function as “facilitator species” initially colonizing the bare soil and modifying its conditions (decreasing interstitial salinity and increasing nutrient), which favored colonization and growing of mangrove seedlings.