The C‐degron pathway eliminates mislocalized proteins and products of deubiquitinating enzymes

Protein termini are determinants of protein stability. Proteins bearing degradation signals, or degrons, at their amino‐ or carboxyl‐termini are eliminated by the N‐ or C‐degron pathways, respectively. We aimed to elucidate the function of C‐degron pathways and to unveil how normal proteomes are exempt from C‐degron pathway‐mediated destruction. Our data reveal that C‐degron pathways remove mislocalized cellular proteins and cleavage products of deubiquitinating enzymes. Furthermore, the C‐degron and N‐degron pathways cooperate in protein removal. Proteome analysis revealed a shortfall in normal proteins targeted by C‐degron pathways, but not of defective proteins, suggesting proteolysis‐based immunity as a constraint for protein evolution/selection. Our work highlights the importance of protein termini for protein quality surveillance, and the relationship between the functional proteome and protein degradation pathways.     

An ultra‐high affinity protein–protein interface displaying sequence‐robustness

Protein–protein interactions are crucial in biology and play roles in for example, the immune system, signaling pathways, and enzyme regulation. Ultra‐high affinity interactions (K _d <0.1 nM) occur in these systems, however, structures and energetics behind stability of ultra‐high affinity protein–protein complexes are not well understood. Regulation of the starch debranching barley limit dextrinase (LD) and its endogenous cereal type inhibitor (LDI) exemplifies an ultra‐high affinity complex (K _d of 42 pM). In this study the LD–LDI complex is investigated to unveil how robust the ultra‐high affinity is to LDI sequence variation at the protein–protein interface and whether alternative sequences can retain the ultra‐high binding affinity. The interface of LD–LDI was engineered using computational protein redesign aiming at identifying LDI variants predicted to retain ultra‐high binding affinity. These variants present a very diverse set of mutations going beyond conservative and alanine substitutions typically used to probe interfaces. Surface plasmon resonance analysis of the LDI variants revealed that high affinity of LD–LDI requires interactions of several residues at the rim of the protein interface, unlike the classical hotspot arrangement where key residues are found at the center of the interface. Notably, substitution of interface residues in LDI, including amino acids with functional groups different from the wild‐type, could occur without loss of affinity. This demonstrates that ultra‐high binding affinity can be conferred without hotspot residues, thus making complexes more robust to mutational drift in evolution. The present mutational analysis also demonstrates how energetic coupling can emerge between residues at large distances at the interface.     

Gillespie eco‐evolutionary models (GEMs) reveal the role of heritable trait variation in eco‐evolutionary dynamics

Heritable trait variation is a central and necessary ingredient of evolution. Trait variation also directly affects ecological processes, generating a clear link between evolutionary and ecological dynamics. Despite the changes in variation that occur through selection, drift, mutation, and recombination, current eco‐evolutionary models usually fail to track how variation changes through time. Moreover, eco‐evolutionary models assume fitness functions for each trait and each ecological context, which often do not have empirical validation. We introduce a new type of model, Gillespie eco‐evolutionary models (GEMs), that resolves these concerns by tracking distributions of traits through time as eco‐evolutionary dynamics progress. This is done by allowing change to be driven by the direct fitness consequences of model parameters within the context of the underlying ecological model, without having to assume a particular fitness function. GEMs work by adding a trait distribution component to the standard Gillespie algorithm – an approach that models stochastic systems in nature that are typically approximated through ordinary differential equations. We illustrate GEMs with the Rosenzweig–MacArthur consumer–resource model. We show not only how heritable trait variation fuels trait evolution and influences eco‐evolutionary dynamics, but also how the erosion of variation through time may hinder eco‐evolutionary dynamics in the long run. GEMs can be developed for any parameter in any ordinary differential equation model and, furthermore, can enable modeling of multiple interacting traits at the same time. We expect GEMs will open the door to a new direction in eco‐evolutionary and evolutionary modeling by removing long‐standing modeling barriers, simplifying the link between traits, fitness, and dynamics, and expanding eco‐evolutionary treatment of a greater diversity of ecological interactions. These factors make GEMs much more than a modeling advance, but an important conceptual advance that bridges ecology and evolution through the central concept of heritable trait variation.     

No Genetic Tradeoffs between Hygienic Behaviour and Individual Innate Immunity in the Honey Bee,Apis mellifera

Many animals have individual and social mechanisms for combating pathogens. Animals may exhibit short-term physiological tradeoffs between social and individual immunity because the latter is often energetically costly. Genetic tradeoffs between these two traits can also occur if mutations that enhance social immunity diminish individual immunity, or vice versa. Physiological tradeoffs between individual and social immunity have been previously documented in insects, but there has been no study of genetic tradeoffs involving these traits. There is strong evidence that some genes influence both innate immunity and behaviour in social insects – a prerequisite for genetic tradeoffs. Quantifying genetic tradeoffs is critical for understanding the evolution of immunity in social insects and for devising effective strategies for breeding disease-resistant pollinator populations. We conducted two experiments to test the hypothesis of a genetic tradeoff between social and individual immunity in the honey bee, Apis mellifera. First, we estimated the relative contribution of genetics to individual variation in innate immunity of honey bee workers, as only heritable traits can experience genetic tradeoffs. Second, we examined if worker bees with hygienic sisters have reduced individual innate immune response. We genotyped several hundred workers from two colonies and found that patriline genotype does not significantly influence the antimicrobial activity of a worker’s hemolymph. Further, we did not find a negative correlation between hygienic behaviour and the average antimicrobial activity of a worker’s hemolymph across 30 honey bee colonies. Taken together, our work indicates no genetic tradeoffs between hygienic behaviour and innate immunity in honey bees. Our work suggests that using artificial selection to increase hygienic behaviour of honey bee colonies is not expected to concurrently compromise individual innate immunity of worker bees.     

Effects of Imidacloprid and Varroa destructor on survival and health of European honey bees,Apis mellifera

There has been growing concern over declines in populations of honey bees and other pollinators which are a vital part to our food security. It is imperative to identify factors responsible for accelerated declines in bee populations and develop solutions for reversing bee losses. While exact causes of colony losses remain elusive, risk factors thought to play key roles are ectoparasitic mites Varroa destructor and neonicotinoid pesticides. The present study aims to investigate effects of a neonicotinoid pesticide Imidacloprid and Varroa mites individually on survivorship, growth, physiology, virus dynamics and immunity of honey bee workers. Our study provides clear evidence that the exposure to sublethal doses of Imidacloprid could exert a significantly negative effect on health and survival of honey bees. We observed a significant reduction in the titer of vitellogenin (Vg), an egg yolk precursor that regulates the honey bees development and behavior and often are linked to energy homeostasis, in bees exposed to Imidacloprid. This result indicates that sublethal exposure to neonicotinoid could lead to increased energy usage in honey bees as detoxification is a energy‐consuming metabolic process and suggests that Vg could be a useful biomarker for measuring levels of energy stress and sublethal effects of pesticides on honey bees. Measurement of the quantitative effects of different levels of Varroa mite infestation on the replication dynamic of Deformed wing virus (DWV), an RNA virus associated with Varroa infestation, and expression level of immune genes yields unique insights into how honey bees respond to stressors under laboratory conditions.     

Drivers of Hsp104 potentiation revealed by scanning mutagenesis of the middle domain

Hsp104, a yeast protein disaggregase, can be potentiated via numerous missense mutations at disparate locations throughout the coiled‐coil middle domain (MD). Potentiated Hsp104 variants can counter the toxicity and misfolding of TDP‐43, FUS, and α‐synuclein, proteins which are implicated in neurodegenerative disorders. However, potentiated MD variants typically exhibit off‐target toxicity. Further, it has remained confounding how numerous degenerate mutations confer potentiation, hampering engineering of therapeutic Hsp104 variants. Here, we sought to comprehensively define the key drivers of Hsp104 potentiation. Using scanning mutagenesis, we iteratively studied the effects of modulation at each position in the Hsp104 MD. Screening this library to identify enhanced variants reveals that missense mutations at 26% of positions in the MD yield variants that counter FUS toxicity. Modulation of the helix 2–helix 3/4 MD interface potentiates Hsp104, whereas mutations in the analogous helix 1–2 interface do not. Surprisingly, we find that there is a higher likelihood of enhancing Hsp104 activity against human disease substrates than impairing Hsp104 native function. We find that single mutations can broadly destabilize the MD structure and lead to functional potentiation, suggesting this may be a common mechanism conferring Hsp104 potentiation. Using this approach, we have demonstrated that modulation of the MD can yield engineered variants with decreased off‐target effects.     

Evidence for a synergistic effect of post‐translational modifications and genomic composition of eEF‐1α on the adaptation of Phytophthora infestans

Genetic variation plays a fundamental role in pathogen''s adaptation to environmental stresses. Pathogens with low genetic variation tend to survive and proliferate more poorly due to their lack of genotypic/phenotypic polymorphisms in responding to fluctuating environments. Evolutionary theory hypothesizes that the adaptive disadvantage of genes with low genomic variation can be compensated for structural diversity of proteins through post‐translation modification (PTM) but this theory is rarely tested experimentally and its implication to sustainable disease management is hardly discussed. In this study, we analyzed nucleotide characteristics of eukaryotic translation elongation factor‐1α (eEF‐lα) gene from 165 Phytophthora infestans isolates and the physical and chemical properties of its derived proteins. We found a low sequence variation of eEF‐lα protein, possibly attributable to purifying selection and a lack of intra‐genic recombination rather than reduced mutation. In the only two isoforms detected by the study, the major one accounted for >95% of the pathogen collection and displayed a significantly higher fitness than the minor one. High lysine representation enhances the opportunity of the eEF‐1α protein to be methylated and the absence of disulfide bonds is consistent with the structural prediction showing that many disordered regions are existed in the protein. Methylation, structural disordering, and possibly other PTMs ensure the ability of the protein to modify its functions during biological, cellular and biochemical processes, and compensate for its adaptive disadvantage caused by sequence conservation. Our results indicate that PTMs may function synergistically with nucleotide codes to regulate the adaptive landscape of eEF‐1α, possibly as well as other housekeeping genes, in P. infestans. Compensatory evolution between pre‐ and post‐translational phase in eEF‐1α could enable pathogens quickly adapting to disease management strategies while efficiently maintaining critical roles of the protein playing in biological, cellular, and biochemical activities. Implications of these results to sustainable plant disease management are discussed.     

Interactomes of SARS‐CoV‐2 and human coronaviruses reveal host factors potentially affecting pathogenesis

Host–virus protein–protein interactions play key roles in the life cycle of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). We conducted a comprehensive interactome study between the virus and host cells using tandem affinity purification and proximity‐labeling strategies and identified 437 human proteins as the high‐confidence interacting proteins. Further characterization of these interactions and comparison to other large‐scale study of cellular responses to SARS‐CoV‐2 infection elucidated how distinct SARS‐CoV‐2 viral proteins participate in its life cycle. With these data mining, we discovered potential drug targets for the treatment of COVID‐19. The interactomes of two key SARS‐CoV‐2‐encoded viral proteins, NSP1 and N, were compared with the interactomes of their counterparts in other human coronaviruses. These comparisons not only revealed common host pathways these viruses manipulate for their survival, but also showed divergent protein–protein interactions that may explain differences in disease pathology. This comprehensive interactome of SARS‐CoV‐2 provides valuable resources for the understanding and treating of this disease.     

Row crop fields provide mid‐summer forage for honey bees

Honey bees provide invaluable economic and ecological services while simultaneously facing stressors that may compromise their health. For example, agricultural landscapes, such as a row crop system, are necessary for our food production, but they may cause poor nutrition in bees from a lack of available nectar and pollen. Here, we investigated the foraging dynamics of honey bees in a row crop environment. We decoded, mapped, and analyzed 3459 waggle dances, which communicate the location of where bees collected food, for two full foraging seasons (April–October, 2018–2019). We found that bees recruited nestmates mostly locally (<2 km) throughout the season. The shortest communicated median distances (0.474 and 0.310 km), indicating abundant food availability, occurred in July in both years, which was when our row crops were in full bloom. We determined, by plotting and analyzing the communicated locations, that almost half of the mid‐summer recruitment was to row crops, with 37% (2018) and 50% (2019) of honey bee dances indicating these fields. Peanut was the most attractive in July, followed by corn and cotton but not soybean. Overall, row crop fields are indicated by a surprisingly large proportion of recruitment dances, suggesting that similar agricultural landscapes may also provide mid‐summer foraging opportunities for honey bees.     

Reproductive trait differences drive offspring production in urban cavity‐nesting bees and wasps

The contrasting and idiosyncratic changes in biodiversity that have been documented across urbanization gradients call for a more mechanistic understanding of urban community assembly. The reproductive success of organisms in cities should underpin their population persistence and the maintenance of biodiversity in urban landscapes. We propose that exploring individual‐level reproductive traits and environmental drivers of reproductive success could provide the necessary links between environmental conditions, offspring production, and biodiversity in urban areas. For 3 years, we studied cavity‐nesting solitary bees and wasps in four urban green space types across Toronto, Canada. We measured three reproductive traits of each nest: the total number of brood cells, the proportion of parasite‐free cells, and the proportion of non‐emerged brood cells that were parasite‐free. We determined (a) how reproductive traits, trait diversity and offspring production respond to multiple environmental variables and (b) how well reproductive trait variation explains the offspring production of single nests, by reflecting the different ways organisms navigate trade‐offs between gathering of resources and exposure to parasites. Our results showed that environmental variables were poor predictors of mean reproductive trait values, trait diversity, and offspring production. However, offspring production was highly positively correlated with reproductive trait evenness and negatively correlated with trait richness and divergence. This suggests that a narrow range of reproductive traits are optimal for reproduction, and the even distribution of individual reproductive traits across those optimal phenotypes is consistent with the idea that selection could favor diverse reproductive strategies to reduce competition. This study is novel in its exploration of individual‐level reproductive traits and its consideration of multiple axes of urbanization. Reproductive trait variation did not follow previously reported biodiversity‐urbanization patterns; the insensitivity to urbanization gradients raise questions about the role of the spatial mosaic of habitats in cities and the disconnections between different metrics of biodiversity.     

Reproduction of ectoparasitic mites in a coevolved system: Varroa spp.—Eastern honey bees,Apis cerana

Parasite host shifts can impose a high selective pressure on novel hosts. Even though the coevolved systems can reveal fundamental aspects of host–parasite interactions, research often focuses on the new host–parasite relationships. This holds true for two ectoparasitic mite species, Varroa destructor and Varroa jacobsonii, which have shifted hosts from Eastern honey bees, Apis cerana, to Western honey bees, Apis mellifera, generating colony losses of these pollinators globally. Here, we study infestation rates and reproduction of V. destructor and V. jacobsonii haplotypes in 185 A. cerana colonies of six populations in China and Thailand to investigate how coevolution shaped these features. Reproductive success was mostly similar and low, indicating constraints imposed by hosts and/or mite physiology. Infestation rates varied between mite haplotypes, suggesting distinct local co‐evolutionary scenarios. The differences in infestation rates and reproductive output between haplotypes did not correlate with the virulence of the respective host‐shifted lineages suggesting distinct selection scenarios in novel and original host. The occasional worker brood infestation was significantly lower than that of drone brood, except for the V. destructor haplotype (Korea) from which the invasive lineage derived. Whether mites infesting and reproducing in atypical intraspecific hosts (i.e., workers and queens) actually predisposes for and may govern the impact of host shifts on novel hosts should be determined by identifying the underlying mechanisms. In general, the apparent gaps in our knowledge of this coevolved system need to be further addressed to foster the adequate protection of wild and managed honey bees from these mites globally.     

ATP regulates RNA‐driven cold inducible RNA binding protein phase separation

Intrinsically disordered proteins and proteins containing intrinsically disordered regions are highly abundant in the proteome of eukaryotes and are extensively involved in essential biological functions. More recently, their role in the organization of biomolecular condensates has become evident and along with their misregulation in several neurologic disorders. Currently, most studies involving these proteins are carried out in vitro and using purified proteins. Given that in cells, condensate‐forming proteins are exposed to high, millimolar concentrations of cellular metabolites, we aimed to reveal the interactions of cellular metabolites and a representative condensate‐forming protein. Here, using the arginine–glycine/arginine–glycine–glycine (RG/RGG)‐rich cold inducible RNA binding protein (CIRBP) as paradigm, we studied binding of the cellular metabolome to CIRBP. We found that most of the highly abundant cellular metabolites, except nucleotides, do not directly bind to CIRBP. ATP, ADP, and AMP as well as NAD⁺, NADH, NADP⁺, and NADPH directly interact with CIRBP, involving both the folded RNA‐recognition motif and the disordered RG/RGG region. ATP binding inhibited RNA‐driven phase separation of CIRBP. Thus, it might be beneficial to include cellular metabolites in in vitro liquid–liquid phase separation studies of RG/RGG and other condensate‐forming proteins in order to better mimic the cellular environment in the future.     

A jacalin‐like lectin domain‐containing protein of Sclerospora graminicola acts as an apoplastic virulence effector in plant–oomycete interactions

The plant extracellular space, including the apoplast and plasma membrane, is the initial site of plant–pathogen interactions. Pathogens deliver numerous secreted proteins, called effectors, into this region to suppress plant immunity and establish infection. Downy mildew caused by the oomycete pathogen Sclerospora graminicola (Sg) is an economically important disease of Poaceae crops including foxtail millet (Setaria italica). We previously reported the genome sequence of Sg and showed that the jacalin‐related lectin (JRL) gene family has significantly expanded in this lineage. However, the biological functions of JRL proteins remained unknown. Here, we show that JRL from Sg (SgJRL) functions as an apoplastic virulence effector. We identified eight SgJRLs by protein mass spectrometry analysis of extracellular fluid from Sg‐inoculated foxtail millet leaves. SgJRLs consist of a jacalin‐like lectin domain and an N‐terminal putative secretion signal; SgJRL expression is induced by Sg infection. Heterologous expression of three SgJRLs with N‐terminal secretion signal peptides in Nicotiana benthamiana enhanced the virulence of the pathogen Phytophthora palmivora inoculated onto the same leaves. Of the three SgJRLs, SG06536 fused with green fluorescent protein (GFP) localized to the apoplastic space in N. benthamiana leaves. INF1‐mediated induction of defence‐related genes was suppressed by co‐expression of SG06536‐GFP. These findings suggest that JRLs are novel apoplastic effectors that contribute to pathogenicity by suppressing plant defence responses.     

Decomposing Spatial β‐Diversity in the temperate forests of Northeastern China

β‐Diversity, which describes the extent of change in species composition in a given region, has become a core issue in ecology in recent years. However, it is hard to understand the underlying mechanisms of β‐diversity by using indices that yield identical values under species replacement and nestedness pattern. Partitioning β‐diversity into turnover (caused by species replacement among plots) and nestedness components (caused by species loss or gain among plots) may provide improved understanding of the variation in species composition. Here, we collected presence–absence data of 456 one‐tenth ha circular plots in the temperate forests of Northeastern China spanning a latitudinal range of 12° (41–53°N). We decomposed β‐diversity to assess the relative contribution of the turnover and nestedness components across latitudinal gradients. We used regression analysis to assess the relationship between spatial distance and β‐diversity. We applied variation partitioning to evaluate the importance of the measured environmental and spatial variables in explaining β‐diversity. We used the Tukey honest significant difference test to test the differences of β‐diversity along latitudinal gradients. Pearson correlations (r) and significance (p‐value) were computed using the Mantel tests to verify the relationship between distance and β‐diversity. The ANOVA test was used to verify whether the variation of β‐diversity explained by the environment and distance was significant. Our results showed that (1) β‐diversity and the turnover component were higher at low latitudes (zones A and B) than at high latitudes (zones C and D), while there was no relationship between the nestedness component and latitude. (2) The turnover component was dominant. (3) The spatial distance explained more variation of β‐diversity than the measured environmental factors. Therefore, we conclude that β‐diversity is mainly a product of species turnover in our temperate forests, suggesting that different localities harbor different species. We find that decomposing β‐diversity into the turnover and nestedness components is a useful approach to explore the variation of community composition and their causes.     

Red mason bees cannot compete with honey bees for floral resources in a cage experiment

Intensive beekeeping to mitigate crop pollination deficits and habitat loss may cause interspecific competition between bees. Studies show negative correlations between flower visitation of honey bees (Apis mellifera) and wild bees, but effects on the reproduction of wild bees were not proven. Likely reasons are that honey bees can hardly be excluded from controls and wild bee nests are generally difficult to detect in field experiments. The goal of this study was to investigate whether red mason bees (Osmia bicornis) compete with honey bees in cages in order to compare the reproduction of red mason bees under different honey bee densities. Three treatments were applied, each replicated in four cages of 18 m³ with 38 red mason bees in all treatments and 0, 100, and 300 honey bees per treatment with 10–20% being foragers. Within the cages, the flower visitation and interspecific displacements from flowers were observed. Niche breadths and resource overlaps of both bee species were calculated, and the reproduction of red mason bees was measured. Red mason bees visited fewer flowers when honey bees were present. Niche breadth of red mason bees decreased with increasing honey bee density while resource overlaps remained constant. The reproduction of red mason bees decreased in cages with honey bees. In conclusion, our experimental results show that in small and isolated flower patches, wild bees can temporarily suffer from competition with honey bees. Further research should aim to test for competition on small and isolated flower patches in real landscapes.     

Systematic discovery of linear binding motifs targeting an ancient protein interaction surface on MAP kinases

Mitogen‐activated protein kinases (MAPK) are broadly used regulators of cellular signaling. However, how these enzymes can be involved in such a broad spectrum of physiological functions is not understood. Systematic discovery of MAPK networks both experimentally and in silico has been hindered because MAPKs bind to other proteins with low affinity and mostly in less‐characterized disordered regions. We used a structurally consistent model on kinase‐docking motif interactions to facilitate the discovery of short functional sites in the structurally flexible and functionally under‐explored part of the human proteome and applied experimental tools specifically tailored to detect low‐affinity protein–protein interactions for their validation in vitro and in cell‐based assays. The combined computational and experimental approach enabled the identification of many novel MAPK‐docking motifs that were elusive for other large‐scale protein–protein interaction screens. The analysis produced an extensive list of independently evolved linear binding motifs from a functionally diverse set of proteins. These all target, with characteristic binding specificity, an ancient protein interaction surface on evolutionarily related but physiologically clearly distinct three MAPKs (JNK, ERK, and p38). This inventory of human protein kinase binding sites was compared with that of other organisms to examine how kinase‐mediated partnerships evolved over time. The analysis suggests that most human MAPK‐binding motifs are surprisingly new evolutionarily inventions and newly found links highlight (previously hidden) roles of MAPKs. We propose that short MAPK‐binding stretches are created in disordered protein segments through a variety of ways and they represent a major resource for ancient signaling enzymes to acquire new regulatory roles.     

Six years of wild bee monitoring shows changes in biodiversity within and across years and declines in abundance

Wild bees form diverse communities that pollinate plants in both native and agricultural ecosystems making them both ecologically and economically important. The growing evidence of bee declines has sparked increased interest in monitoring bee community and population dynamics using standardized methods. Here, we studied the dynamics of bee biodiversity within and across years by monitoring wild bees adjacent to four apple orchard locations in Southern Pennsylvania, USA. We collected bees using passive Blue Vane traps continuously from April to October for 6 years (2014–2019) amassing over 26,000 bees representing 144 species. We quantified total abundance, richness, diversity, composition, and phylogenetic structure. There were large seasonal changes in all measures of biodiversity with month explaining an average of 72% of the variation in our models. Changes over time were less dramatic with years explaining an average of 44% of the variation in biodiversity metrics. We found declines in all measures of biodiversity especially in the last 3 years, though additional years of sampling are needed to say if changes over time are part of a larger trend. Analyses of population dynamics over time for the 40 most abundant species indicate that about one third of species showed at least some evidence for declines in abundance. Bee family explained variation in species‐level seasonal patterns but we found no consistent family‐level patterns in declines, though bumble bees and sweat bees were groups that declined the most. Overall, our results show that season‐wide standardized sampling across multiple years can reveal nuanced patterns in bee biodiversity, phenological patterns of bees, and population trends over time of many co‐occurring species. These datasets could be used to quantify the relative effects that different aspects of environmental change have on bee communities and to help identify species of conservation concern.     

Adenosine triphosphate energy‐independently controls protein homeostasis with unique structure and diverse mechanisms

Proteins function in the crowded cellular environments with high salt concentrations, thus facing tremendous challenges of misfolding/aggregation which represents a pathological hallmark of aging and an increasing spectrum of human diseases. Recently, intrinsically disordered regions (IDRs) were recognized to drive liquid–liquid phase separation (LLPS), a common principle for organizing cellular membraneless organelles (MLOs). ATP, the universal energy currency for all living cells, mysteriously has concentrations of 2–12 mM, much higher than required for its previously‐known functions. Only recently, ATP was decoded to behave as a biological hydrotrope to inhibit protein LLPS and aggregation at mM. We further revealed that ATP also acts as a bivalent binder, which not only biphasically modulates LLPS driven by IDRs of human and viral proteins, but also bind to the conserved nucleic‐acid‐binding surfaces of the folded proteins. Most unexpectedly, ATP appears to act as a hydration mediator to antagonize the crowding‐induced destabilization as well as to enhance folding of proteins without significant binding. Here, this review focuses on summarizing the results of these biophysical studies and discussing their implications in an evolutionary context. By linking triphosphate with unique hydration property to adenosine, ATP appears to couple the ability for establishing hydrophobic, π‐π, π‐cation and electrostatic interactions to the capacity in mediating hydration of proteins, which is at the heart of folding, dynamics, stability, phase separation and aggregation. Consequently, ATP acquired a category of functions at ~mM to energy‐independently control protein homeostasis with diverse mechanisms, thus implying a link between cellular ATP concentrations and protein‐aggregation diseases.     

Revisiting the phosphotyrosine binding pocket of Fyn SH2 domain led to the identification of novel SH2 superbinders.

Protein engineering through directed evolution is an effective way to obtain proteins with novel functions with the potential applications as tools for diagnosis or therapeutics. Many natural proteins have undergone directed evolution in vitro in the test tubes in the laboratories worldwide, resulting in the numerous protein variants with novel or enhanced functions. we constructed here an SH2 variant library by randomizing 8 variable residues in its phosphotyrosine (pTyr) binding pocket. Selection of this library by a pTyr peptide led to the identification of SH2 variants with enhanced affinities measured by EC50. Fluorescent polarization was then applied to quantify the binding affinities of the newly identified SH2 variants. As a result, three SH2 variants, named V3, V13 and V24, have comparable binding affinities with the previously identified SH2 triple‐mutant superbinder. Biolayer Interferometry assay was employed to disclose the kinetics of the binding of these SH2 superbinders to the phosphotyrosine peptide. The results indicated that all the SH2 superbinders have two‐orders increase of the dissociation rate when binding the pTyr peptide while there was no significant change in their associate rates. Intriguingly, though binding the pTyr peptide with comparable affinity with other SH2 superbinders, the V3 does not bind to the sTyr peptide. However, variant V13 and V24 have cross‐reactivity with both pTyr and sTyr peptides. The newly identified superbinders could be utilized as tools for the identification of pTyr‐containing proteins from tissues under different physiological or pathophysiological conditions and may have the potential in the therapeutics.