Natural Mutations Research Articles

Mutations in AMBRA1 aggravate β-thalassemia by impairing autophagy-mediated clearance of free α-globin.

Accumulation of free α-globin is a critical factor in the pathogenesis of β-thalassemia. Autophagy plays a crucial role in clearing toxic free α-globin, thereby reducing disease severity. However, the impact of natural mutations in autophagy-related genes (ATGs) on the phenotypic variability of β-thalassemia remains unclear. In this study, we systematically investigated the relationship between variants in ATGs and disease phenotypes in a cohort of 1,022 patients with β-thalassemia, identifying four missense mutations in the autophagy and beclin 1 regulator 1 (AMBRA1) gene. Disruption of the Ambra1 gene in β-thalassemic mice was found to reduce autophagic clearance of α-globin in red blood cell precursors, exacerbating disease phenotypes. Functional characterization of the AMBRA1 gene and these mutations in patient-derived CD34+ cells, edited HUDEP-2 cells, and engineered HUDEP-2 β-thalassemic cells confirmed that AMBRA1 facilitates the autophagic clearance of free α-globin in human erythroid cells. Functional studies demonstrated that AMBRA1 missense mutants destabilize ULK1 protein, inhibit LC3 lipidation, and subsequently hinder autophagic flux, leading to increased α-globin deposition. Additionally, these mutations were associated with erythrotoxic effects in vitro, including increased intracellular reactive oxygen species levels, higher apoptosis rates, and impaired erythroid differentiation and maturation. This study sheds light on the molecular association between mutations in ATGs and the exacerbation of β-thalassemia, highlighting the potential role of the AMBRA1 gene as a promising diagnostic and therapeutic target for β-hemoglobinopathies.

Molecular basis for loss of virulence in Magnaporthe oryzae strain AM16

The rapid virulence variation of Magnaporthe oryzae (M. oryzae) to rice is a big challenge for rice blast control. Even though many studies have been done by scientists all over the world, the mechanism of virulence variation in M. oryzae remains elusive. AM16, an avirulent M. oryzae strain reported in our previous study, provides an excellent entry point to explore the mechanism of virulence variation in M. oryzae. In this study, we found that the Pmk1 and Mac1 had specific mutations in strain AM16. The AM16 strains overexpressing Pmk1Guy11 or (and) Mac1Guy11 allele from strain Guy11 displayed significantly increasing conidiation, functional appressorium formation, and restoring pathogenicity to rice. Moreover, we observed that the strains overexpressing Mac1Guy11 had stronger conidia forming capacity than that of the strains overexpressing Pmk1Guy11, while the appressorium formation rate of strains overexpressing Pmk1Guy11 was similar to that of strains overexpressing Pmk1Guy11-Mac1Guy11, much higher than that of the strains overexpressing Mac1Guy11. Taken together, our results reveal that the natural mutation of Pmk1 and Mac1 genes are important, but not the sole cause, for the loss of virulence in strain AM16. The functional difference between Pmk1 and Mac1 in the growth and development of M. oryzae was first discovered, providing new insight into the pathogenic mechanism of M. oryzae.

Natural mutation in Stay-Green (OsSGR) confers enhanced resistance to rice sheath blight through elevating cytokinin content.

Sheath blight (ShB), caused by Rhizoctonia solani, is a highly destructive disease in many crops worldwide and no major resistance genes are available. Here, we identified a sbr1 (sheath blight resistance 1) rice mutant, which shows enhanced ShB resistance and maintains wildtype agronomic traits including yield, but carries an undesired stay-green phenotype. Through map-based cloning and transgenic validation, we found that an insertion disrupting the Stay-Green (OsSGR) gene is responsible for sbr1 phenotypes. Mechanistically, the sbr1/Ossgr mutants reduce the expression of most OsCKX genes, which function in cytokinin (CK) degradation, to accumulate CK leading to ShB resistance. Importantly, knockout of OsCKX7, predominantly expressed in the leaf sheath and highly induced by R. solani, significantly enhances ShB resistance without stay-green phenotype nor yield penalty, showing high application potential. Thus, our study reveals novel insights that OsSGR and cytokinin play key roles in rice-R. solani interaction and generates a valuable ShB-resistant germplasm.

Identification of two point mutations associated with inherited antithrombin deficiency

BackgroundAntithrombin (AT) is a serine protease inhibitor which exerts its anticoagulant effect through binding to serine residues in the active centers of procoagulant serine proteases. Its deficiency is associated with increased risk of venous thrombosis. We aim to investigate the pathogenic mechanism of two natural mutants (W221C and M284R) in inherited AT deficiency.MethodsWe analyzed 9 unrelated patients with inherited AT deficiency by extracting peripheral blood DNA and sequencing the SERPINC1 gene after amplification by polymerase chain reaction. Enzyme-linked immunosorbent assay and heparin affinity chromatography were used to assess AT secretion and purification efficiency. The mutant AT models were evaluated via computational simulations.ResultsAmong the 9 patients with inherited AT deficiency, 8 patients had type I AT deficiency, and one patient had type II AT deficiency with subtype of reactive site mutation. Seven of them experienced venous thrombotic events and all patients were found genetic mutations including missense (n = 6), deletion (n = 2) and insertion (n = 1). Two point mutations, W221C and M284R, were identified and were hypothesized to affect AT by destabilizing the central β-sheet. Based on immunoassays and heparin purification, the W221C mutant may impair AT secretion, whereas M284R mutant decreased the total AT production (696.8 ± 151.6 ng/ml versus 3833.72 ± 315.4 ng/ml, p = 0.029). Both mutants delayed the peak of AT release in heparin affinity chromatography.ConclusionsOur study demonstrates that two mutations in SERPINC1 gene altered the production and structure of AT by in vitro protein expression and functional studies, including protein secretion and production. These findings enhance our understanding of the genetic basis of AT deficiency and its possible clinical implications.

Identification of key antigenic sites in hemagglutinin of H10N3 avian influenza virus

The H10 avian influenza viruses (AIV) have been detected in both birds and mammals. Recently, the cases of human infection with H10N8 and H10N3 in China pose high risk to public health. However, the antigenic sites in hemagglutinin (HA) of H10 are poorly understood. In this study, 3 monoclonal antibodies (MAb), designated as 1F4, 6B3 and 6G12, against the HA protein of the H10N3 strain A/chicken/Taizhou/498/2021(H10N3) (TZ498), were first generated. All of these MAb could effectively inhibit TZ498 in haemagglutination inhibition assay and microneutralization assay. Four novel antigenic sites at positions 135, 208, 227, and 266 (H10 numbering) were identified in the HA of TZ498 through escape mutants selected by these 3 MAb. Moreover, natural mutations at positions 135 and 227 were found in the H10 field strains. All these not only provide novel insights into the molecular markers for monitoring the antigenic variation of H10 but also be helpful for developing efficient control strategies against H10.

Sequencing and Annotation of the Chloroplast Genome of <i>Triticum militinae</i> – a “Natural Mutant” of Tetraploid Wheat <i>Triticum timopheevii</i> Zhuk.

Triticum militinae Zhuk. et Migusch. – tetraploid wheat with the GAA genome, considered a natural naked mutant of T. timopheevii Zhuk. Previously, experiments were conducted for this wheat to study the karyotype and crossing characteristics. To clarify its origin and relationship with other representatives of the wheat family, analysis of the chloroplast genome of T. militinae, which has previously remained unstudied, is of great interest. The sucrose gradient method was used to isolate chloroplast DNA from leaves. Sequencing was performed using the FASTASeq 300 Sequencing Kit V1.0 100 M reads/flow cell on a Genolab M sequencer (GeneMind, China). For the first time, we sequenced and annotated the complete chloroplast genome of T. militinae with a size of 135898 bp. In the structure of the plastid genome there are a pair of inverted repeats with a size of 21552 bp each, the region of a small single copy (SSC) – 12791 bp and the region of a large single copy (LSC) – 80003 bp. As part of the chloroplast genome of T. militinae 132 structural genes were found, of which 85 protein-coding genes, 31 tRNAs and 4 rRNA genes.

Substrate specificity and protein stability drive the divergence of plant-specific DNA methyltransferases.

DNA methylation is an important epigenetic mechanism essential for transposon silencing and genome integrity. Across evolution, the substrates of DNA methylation have diversified between kingdoms. In plants, chromomethylase3 (CMT3) and CMT2 mediate CHG and CHH methylation, respectively. However, how these two methyltransferases diverge on substrate specificities during evolution remains unknown. Here, we reveal that CMT2 originates from a duplication of an evolutionarily ancient CMT3 in flowering plants. Lacking a key arginine residue recognizing CHG in CMT2 impairs its CHG methylation activity in most flowering plants. An engineered V1200R mutation empowers CMT2 to restore CHG and CHH methylations in Arabidopsis cmt2cmt3 mutant, testifying a loss-of-function effect for CMT2 during evolution. CMT2 has evolved a long and unstructured amino terminus critical for protein stability, especially under heat stress, and is plastic to tolerate various natural mutations. Together, this study reveals the mechanism of chromomethylase divergence for context-specific DNA methylation in plants and sheds important lights on DNA methylation evolution and function.

Reducing amylose content in wheat (Triticum aestivum L.) using a novel Wx-D1 null allele generated by chemical mutagenesis.

Amylose has a major influence over starch properties and end-use quality in wheat. The granule-bound starch synthase I, encoded by Wx-1, is the single enzyme responsible for amylose synthesis. Natural null mutants of Wx-1 appear at extremely low frequencies, particularly in the Wx-D1 locus, where only four spontaneous null variants have been identified, with different geographic origins. The current study identified an induced Wx-D1 null mutant (M4-9484) from the M4 generation of an ethyl methanesulfonate-mutagenized population of wheat cv. 'SM126'. The sequencing showed that the complete Wx-D1 ORF sequences of 'SM126' and M4-9484 were 2862 bp long and that there was one SNP mutation between them. The mutation was located at the RNA splice site within the junction of exon 8 and intron 8, which led to abnormal transcription of Wx-D1, with five different aberrant transcripts being identified in the mutant. The Wx-D1 null allele resulted in amylose and total starch content being decreased in M4-9484 in comparison with the wild-type 'SM126', with higher swelling capacity and being fully pasted at higher temperatures than the wild-type parent. The mutation of the Wx-D1 null gene affects the formation of amylose directly, resulting in significantly altered starch properties. This discovery offers valuable insights for enhancing wheat starch quality and contributes to the diversification of starch characteristics. It also deepens our understanding of the genetic and molecular mechanisms underlying amylose synthesis, thereby supporting breeding programs. © 2024 Society of Chemical Industry.

Generative AI in the Advancement of Viral Therapeutics for Predicting and Targeting Immune-Evasive SARS-CoV-2 Mutations.

The emergence of immune-evasive mutations in the SARS-CoV-2 spike protein is consistently challenging existing vaccines and therapies, making precise prediction of their escape potential a critical imperative. Artificial Intelligence(AI) holds great promise for deciphering the intricate language of protein. Here, we employed a Generative Adversarial Network to decipher the hidden escape pathways within the spike protein by generating spikes that closely resemble natural ones. Through comprehensive analysis, we demonstrated that generated sequences capture natural escape characteristics. Moreover, incorporating these sequences into an AI-based escape prediction model significantly enhanced its performance, achieving a 7% increase in detecting natural escape mutations on the experimentally validated Greaney dataset. Similar improvements were observed on other datasets, demonstrating the model's generalizability. Precisely predicting immune-evasive spikes not only enables the design of strategically targeted therapies but also has the potential to expedite future viral therapeutics. This breakthrough carries profound implications for shaping a more resilient future against viral threats.

Enhancing Cellular and Enzymatic Properties Through In Vivo Continuous Evolution.

Directed evolution seeks to evolve target genes at a rate far exceeding the natural mutation rate, thereby endowing cellular and enzymatic properties with desired traits. In vivo continuous directed evolution achieves these purposes by generating libraries within living cells, enabling a continuous cycle of mutant generation and selection, enhancing the exploration of gene variants. Continuous evolution has become powerful tools for unraveling evolution mechanism and improving cellular and enzymatic properties. This review categorizes current continuous evolution into three distinct classes: non-targeted chromosomal, targeted chromosomal, and extra-chromosomal hypermutation approaches. It also compares various continuous evolution strategies based on different principles, providing a reference for selecting suitable methods for specific evolutionary goals. Furthermore, this review discusses the two primary limitations for further widespread application of in vivo continuous evolution, which are lack of general applicability and insufficient mutagenic capability. We envision that developing generally applicable mutagenic components and methods to enhance mutation rates for in vivo continuous evolution are promising future directions for wide range applications of continuous evolution.

Apolipoprotein-L1 (APOL1): From Sleeping Sickness to Kidney Disease.

Apolipoprotein-L1 (APOL1) is a membrane-interacting protein induced by inflammation, which confers human resistance to infection by African trypanosomes. APOL1 kills Trypanosoma brucei through induction of apoptotic-like parasite death, but two T. brucei clones acquired resistance to APOL1, allowing them to cause sleeping sickness. An APOL1 C-terminal sequence alteration, such as occurs in natural West African variants G1 and G2, restored human resistance to these clones. However, APOL1 unfolding induced by G1 or G2 mutations enhances protein hydrophobicity, resulting in kidney podocyte dysfunctions affecting renal filtration. The mechanism involved in these dysfunctions is debated. The ability of APOL1 to generate ion pores in trypanosome intracellular membranes or in synthetic membranes was provided as an explanation. However, transmembrane insertion of APOL1 strictly depends on acidic conditions, and podocyte cytopathology mainly results from secreted APOL1 activity on the plasma membrane, which occurs under non-acidic conditions. In this review, I argue that besides inactivation of APOL3 functions in membrane dynamics (fission and fusion), APOL1 variants induce inflammation-linked podocyte toxicity not through pore formation, but through plasma membrane disturbance resulting from increased interaction with cholesterol, which enhances cation channels activity. A natural mutation in the membrane-interacting domain (N264K) abrogates variant APOL1 toxicity at the expense of slightly increased sensitivity to trypanosomes, further illustrating the continuous mutual adaptation between host and parasite.

New insight into acid-resistant enzymes from natural mutations of Escherichia coli Nissle 1917

The probiotic Escherichia coli Nissle 1917 (EcN), known for its superior acid resistance (AR), serves as a promising chassis for live therapeutics due to the effective colonization capabilities. However, the enzymatic activity regarding AR in EcN remains poorly understood. First, we investigated the AR systems of EcN by measuring cell growth under acidic stress and exploring the relationship of mutations to their corresponding enzymatic activities. As a result, the catalytic activity of inducible decarboxylases of GadB, AdiA and CadA, responsible for metabolizing glutamate, arginine, and lysine, exhibited an average 2-fold increase in EcN compared to the reference strain MG1655. Furthermore, we discovered that the glutamate-dependent AR2 system in EcN was meticulously regulated by specific regulons such as GadW. This study not only revealed the physiology of EcN under acidic conditions, but also highlighted that the mutated core enzymes in the AR system of EcN exhibit improved activities.

Differentially heterogeneous hydration environment of the familial mutants of α-synuclein.

The behavior of hydration water around familial Parkinson's disease linked mutants of α-synuclein may be linked to the early-onset of Parkinson's disease. For the first time, this study compares the local structure and dynamics of hydration water around different segments of some of the natural mutants of α-synuclein, i.e., E46K, G51D, A30P, and A53E, with that of the wild-type protein through explicit water MD simulations. The results show that the C-terminal segments of the fast aggregating mutants such as E46K and A30P are less exposed to water, while those of the slow aggregating ones such as A53E and G51D are more exposed to water relative to that of the wild-type protein. In addition, the water molecules are found to be more ordered around the C-terminal segment of the A53E and G51D mutants as compared to the wild-type protein. This is due to an increase in the overall charge of α-syn upon A53E and G51D mutations. The translational and rotational motions of water molecules in the hydration shell of the C-terminal segment of A53E and G51D mutants are found to be faster relative to that of the wild-type protein. This study validates the differential hydration environment around the C-terminal segment for the causative and protective mutants of α-synuclein.

OsKANADI1 and OsYABBY5 regulate rice plant height by targeting GIBERELLIN 2-OXIDASE6.

Plant height is an important agronomic characteristic of rice (Oryza sativa L.). Map-based cloning analyses of a natural semi-dwarf rice mutant with inwardly curled leaves found in the field revealed that the defects were due to a mutation of a SHAQKYF-class MYB family transcription factor, OsKANADI1 (OsKAN1). OsKAN1 directly bound to the OsYABBY5 (OsYAB5) promoter to repress its expression and interacted with OsYAB5 to form a functional OsKAN1-OsYAB5 complex. GIBERELLIN 2-OXIDASE6 (OsGA2ox6), encoding an enzyme in the gibberellin (GA) catabolic pathway, was activated by OsYAB5. Furthermore, the OsKAN1-OsYAB5 complex suppressed the inhibitory effect of OsKAN1 toward OsYAB5 and inhibited OsYAB5-induced OsGA2ox6 expression. The proOsKAN1:OsYAB5 transgenic plants were taller than wild-type plants, whereas oskan1 proOsKAN1:OsYAB5 plants exhibited a severe dwarf phenotype due to the absence of the OsKAN1-OsYAB5 complex. The OsKAN1-OsYAB5 complex modulated OsGA2ox6 expression, thereby regulating the levels of bioactive gibberellins and, consequently, plant height. This study elucidated the mechanism underlying the effect of the OsKAN1-OsYAB5-OsGA2ox6 regulatory pathway on plant height at different positions in rice stems and provided insights on stem development and candidate genes for the aerial architecture improvement of crop plants.

Mathematical Modelling of Spatially Inhomogeneous Non-Stationary Interaction of Pests with Transgenic and Non-Modified Crops Considering Taxis

Introduction. This paper addresses a unified spatially inhomogeneous, non-stationary model of interaction between genetically modified crop resources (corn) and the corn borer pest, which is also present on a relatively small section of non-modified corn. The model assumes that insect pests influence both types of crops and are capable of independent movement (taxis) towards the gradient of plant resources. It also considers diffusion processes in the dynamics of all components of the unified model, biomass growth, genetic characteristics of both types of plant resources, processes of crop consumption, phenomena of growth and degradation, diffusion, and mutation of pests. The model allows for predictive calculations aimed at reducing crop losses and increasing the resistance of transgenic crops to pests by slowing down the natural mutation rate of the pest. Materials and Methods. The mathematical model is an extension of Kostitsin’s model and is formulated as an initial-boundary value problem for a nonlinear system of convection-diffusion equations. These equations describe the spatiotemporal dynamics of biomass density changes in two types of crops — transgenic and non-modified — as well as the specific populations (densities) of three genotypes of pests (the corn borer) resulting from mutations. The authors linearized the convection-diffusion equations by applying a time-lag method on the time grid, with nonlinear terms from eachequation taken from the previous time layer. The terms describing taxis are presented in a symmetric form, ensuring the skew-symmetry of the corresponding continuous operator and, in the case of spatial grid approximation, the finite-difference operator. Results. A stable monotonic finite-difference scheme is developed, approximating the original problem with second-order accuracy on a uniform 2D spatial grid. Numerical solutions of model problems are provided, qualitatively corresponding to observed processes. Solutions are obtained for various ratios of modified and non-modified sections of the field. Discussion and Conclusion. The obtained results regarding pest behavior, depending on the type of taxis, could significantly extend the time for pests to acquire Bt resistance. The concentration dynamics of pests moving in the direction of the food gradient differs markedly from the concentration of pests moving towards a mate for reproduction.

TMX5/TXNDC15, a natural trapping mutant of the PDI family is a client of the proteostatic factor ERp44.

The ER is the organelle of nucleated cells that produces lipids, sugars, and proteins. More than 20 ER-resident members of the protein disulfide isomerase (PDI) family regulate formation, isomerization, and disassembly of covalent bonds in newly synthesized polypeptides. The PDI family includes few membrane-bound members. Among these, TMX1, TMX2, TMX3, TMX4, and TMX5 belong to the thioredoxin-related transmembrane (TMX) protein family. TMX5 is the least-known member of the family. Here, we establish that TMX5 covalently engages via its active site cysteine residue at position 220 a subset of secretory proteins, mainly single- and multipass Golgi-resident polypeptides. TMX5 also interacts non-covalently, and covalently, via non-catalytic cysteine residues, with the PDI family members PDI, ERp57, and ERp44. The association between TMX5 and ERp44 requires formation of a mixed disulfide between the catalytic cysteine residue 29 of ERp44 and the non-catalytic cysteine residues 114 and/or 124 of TMX5 and controls the ER localization of TMX5 in pre-Golgi compartments. Thus, TMX5 belongs to the family of proteins including Ero1α, Ero1β, Prx4, ERAP1, and SUMF1 that operate in pre-Golgi compartments but lack localization sequences required to position themselves and rely on ERp44 engagement for proper intercompartmental distribution.

Public health implications of antibiotic resistance in sewage water: an epidemiological perspective

The emergence and rapid spread of antibiotic resistance pose a major threat to global health, attributing to misuse and overuse of antibiotics resulting in antibiotics-resistant bacteria through natural mutation or transfer of resistance genes. A cross-sectional study was carried out, in which a total of 36 samples were systematically collected; of these, 26 were derived from the wastewater efflux and 10 from the receiving waters at several critical junctures along the Sutlej River. Herein, this study elucidated elevated levels of antibiotic resistance among bacterial isolates sourced from urban wastewater. Escherichia coli (E. coli) was the highest at 90% among the isolates, followed by Klebsiella pneumoniae (K. pneumoniae) at 58%, Pseudomonas aeruginosa (P. aeruginosa) at 55%, and Salmonella spp. at 53%. Many antibiotics were found to be more resistant including Ciproflaxacin, Co-Trimaxazole, Ampicillin and Tetracycline. Several antibiotic-resistance genes were found in isolated bacterial spp., such as Aminoglycosides (aadA), Sulfonamides (Sul1, Sul3), Tetracyclines (Tet (A/B/D)) and Cephalosporins (Bla_CTM X) at 41%, 35%, 29% and 12% respectively. Furthermore, the development of innovative wastewater treatment models and surveillance programs are crucial to counteract the dissemination of antibiotic resistance. To investigate the genetic determinants of antibiotic resistance, molecular analysis was performed, including DNA isolation, PCR amplification, and sequence analysis. The study helps investigate a diverse range of ARBs and ARGs in wastewater, which highlights the need of better laws for antibiotic usage and wastewater treatment processes. This investigation also stresses on regular monitoring of ARBs and ARGs in sewage wastewater. Through proactive interventions and sustained scientific inquiry, we can strive toward preserving environmental integrity and public health for successive generations.Graphical

Genomic and Pathological Characterization of Acute Hepatopancreatic Necrosis Disease (AHPND)-Associated Natural Mutant Vibrio parahaemolyticus Isolated from Penaeus vannamei Cultured in Korea.

Acute hepatopancreatic necrosis disease (AHPND) is one of the most important diseases in the global shrimp industry. The emergence of mutant AHPND-associated V. parahaemolyticus (VpAHPND) strains has raised concerns regarding potential misdiagnosis and unforeseen pathogenicity. In this study, we report the first emergence of a type II (pirA-, pirB+) natural mutant, VpAHPND (strain 20-082A3), isolated from cultured Penaeus vannamei in Korea. Phenotypic and genetic analyses revealed a close relationship between the mutant strain 20-082A3 and the virulent Korean VpAHPND strain 19-021-D1, which caused an outbreak in 2019. Detailed sequence analysis of AHPND-associated plasmids showed that plasmid pVp_20-082A3B in strain 20-082A3 was almost identical (>99.9%) to that of strain 19-021-D1. Moreover, strains 20-082A3 and 19-021-D1 exhibited the same multilocus sequence type (ST 413) and serotype (O1:Un-typeable K-serogroup), suggesting that the mutant strain is closely related to and may have originated from the virulent strain 19-021-D1. Similar to previous reports on the natural mutant VpAHPND, strain 20-082A3 did not induce AHPND-related symptoms or cause mortality in the shrimp bioassay. The emergence of a mutant strain which is almost identical to the virulent VpAHPND highlights the need for surveillance of the pathogen prevalent in Korea. Further investigations to elucidate the potential relationship between ST 413 and recent Korean VpAHPND isolates are needed.

Conserved role of spike S2 domain N-glycosylation across beta-coronavirus family.

Besides acting as an immunological shield, the N-glycans of SARS-CoV-2 are also critical for viral life cycle. As the S2 subunit of spike is highly conserved across beta-coronaviruses, we determined the functional significance of the five 'stem N-glycans' located in S2 between N1098-N1194. Studies were performed with 31 Asn-to-Gln mutants, beta-coronavirus virus-like particles and single-cycle viral replicons. Deletions of stem N-glycans enhanced S1 shedding from trimeric spike, reduced ACE2 binding and abolished syncytia formation. When three or more N-glycans were deleted, spike expression on cell surface and incorporation into virions was both reduced. Viral entry function was progressively lost upon deleting the N1098 glycan in combination with additional glycosite modifications. In addition to SARS-CoV-2, deleting stem N-glycans in SARS-CoV and MERS-CoV spike also prevented viral entry into target cells. These data suggest multiple functional roles for the stem N-glycans, and evolutionarily conserved properties for these complex carbohydrates across human beta-coronaviruses.

Structural and functional effects of phosphopriming and scaffolding in the kinase GSK-3β.

Glycogen synthase kinase 3β (GSK-3β) targets specific signaling pathways in response to distinct upstream signals. We used structural and functional studies to dissect how an upstream phosphorylation step primes the Wnt signaling component β-catenin for phosphorylation by GSK-3β and how scaffolding interactions contribute to this reaction. Our crystal structure of GSK-3β bound to a phosphoprimed β-catenin peptide confirmed the expected binding mode of the phosphoprimed residue adjacent to the catalytic site. An aspartate phosphomimic in the priming site of β-catenin adopted an indistinguishable structure but reacted approximately 1000-fold slower than the native phosphoprimed substrate. This result suggests that substrate positioning alone is not sufficient for catalysis and that native phosphopriming interactions are necessary. We also obtained a structure of GSK-3β with an extended peptide from the scaffold protein Axin that bound with greater affinity than that of previously crystallized Axin fragments. This structure neither revealed additional contacts that produce the higher affinity nor explained how substrate interactions in the GSK-3β active site are modulated by remote Axin binding. Together, our findings suggest that phosphopriming and scaffolding produce small conformational changes or allosteric effects, not captured in the crystal structures, that activate GSK-3β and facilitate β-catenin phosphorylation. These results highlight limitations in our ability to predict catalytic activity from structure and have potential implications for the role of natural phosphomimic mutations in kinase regulation and phosphosite evolution.