Single Amino Acid Variants Research Articles

Assessment of Data-Independent Acquisition Mass Spectrometry (DIA-MS) for the Identification of Single Amino Acid Variants

Proteogenomics integrates genomic and proteomic data to elucidate cellular processes by identifying variant peptides, including single amino acid variants (SAAVs). In this study, we assessed the capability of data-independent acquisition mass spectrometry (DIA-MS) to identify SAAV peptides in HeLa cells using various search engine pipelines. We developed a customised sequence database (DB) incorporating SAAV sequences from the HeLa genome and conducted searches using DIA-NN, Spectronaut, and Fragpipe-MSFragger. Our evaluation focused on identifying true positive SAAV peptides and false positives through entrapment DBs. This study revealed that DIA-MS provides reproducible and comprehensive coverage of the proteome, identifying a substantial proportion of SAAV peptides. Notably, the DIA-MS searches maintained consistent identification of SAAV peptides despite varying sizes of the entrapment DB. A comparative analysis showed that Fragpipe-MSFragger (FP-DIA) demonstrated the most conservative and effective performance, exhibiting the lowest false discovery match ratio (FDMR). Additionally, integrating DIA and data-dependent acquisition (DDA) MS data search outputs enhanced SAAV peptide identification, with a lower false discovery rate (FDR) observed in DDA searches. The validation using stable isotope dilution and parallel reaction monitoring (SID-PRM) confirmed the SAAV peptides identified by DIA-MS and DDA-MS searches, highlighting the reliability of our approach. Our findings underscore the effectiveness of DIA-MS in proteogenomic workflows for identifying SAAV peptides, offering insights into optimising search engine pipelines and DB construction for accurate proteomics analysis. These methodologies advance the understanding of proteome variability, contributing to cancer research and the identification of novel proteoform therapeutic targets.

Selective transformation of crocin-1 to crocetin-glucosyl esters by β-glucosidase (Lf18920) from Leifsonia sp. ZF2019: Insights from molecular docking and point mutations

Crocetin di/mono-glucosyl esters (crocin-4 and crocin-5) are rarely distributed in nature, limiting their potential applications in the food and pharmaceutical industries. In the present study, a novel GH3 family β-glucosidase Lf18920 was identified from Leifsonia sp. ZF2019, which selectively hydrolyzed crocin-1 (crocetin di-gentiobiosyl ester) to crocin-5 and crocin-4, but not to its aglycone, crocetin. Under the optimal condition of 40 °C and pH 6.0 for 120 min, Lf18920 almost completely hydrolyzed crocin-1, yielding 73.50±5.66 % crocin-4 and 16.19±1.38 % crocin-5. Molecular docking and point mutation studies revealed that Lf18920 formed a narrow binding channel that facilitated crocin-1 binding. Five single amino acid variants (D50A, D53A, W274A, G420A, and Q421A) were constructed, all of which showed reduced hydrolytic activity. Mutations at D50 and D53, located distal to the active site, increased binding energy and decreased hydrolytic activity, while mutations at W274, G420, and Q421, proximal to the active site, disrupted hydrolytic function. These findings suggest that the narrow binding channel and specific enzyme-substrate interactions are crucial for Lf18920’s selective hydrolytic activity. Overall, this study is the first to report a β-glucosidase capable of selectively transforming crocin-1 to crocetin di/mono-glucosyl esters, offering potential for synthesizing crocin-4 and crocin-5.

Deep mutational scanning of CYP2C19 in human cells reveals a substrate specificity-abundance tradeoff.

The Cytochrome P450s (CYPs) enzyme family metabolizes ∼80% of small molecule drugs. Variants in CYPs can substantially alter drug metabolism, leading to improper dosing and severe adverse drug reactions. Due to low sequence conservation, predicting variant effects across CYPs is challenging. Even closely related CYPs like CYP2C9 and CYP2C19, which share 92% amino acid sequence identity, display distinct phenotypic properties. Using Variant Abundance by Massively Parallel sequencing (VAMP-seq), we measured the steady-state protein abundance of 7,660 single amino acid variants in CYP2C19 expressed in cultured human cells. Our findings confirmed critical positions and structural features essential for CYP function and revealed how variants at conserved positions influence abundance. We jointly analyzed 4,670 variants whose abundance was measured in both CYP2C19 and CYP2C9, finding that the homologs have different variant abundances in substrate recognition sites within the hydrophobic core. We also measured the abundance of all single and some multiple WT amino acid exchanges between CYP2C19 and CYP2C9. While most exchanges had no effect, substitutions in substrate recognition site 4 (SRS4) reduced abundance in CYP2C19. Double and triple mutants showed distinct interactions, highlighting a region that points to differing thermodynamic properties between the two homologs. These positions are known contributors to substrate specificity, suggesting an evolutionary tradeoff between stability and enzymatic function. Finally, we analyzed 368 previously unannotated human variants, finding that 43% had decreased abundance. By comparing variant effects between these homologs, we uncovered regions underlying their functional differences, advancing our understanding of this versatile family of enzymes.

Polyamine oxidase induces flower formation by promoting spermidine and ABA accumulation in cherry (Cerasus pseudocerasus Lindl.)

Polyamines are important small molecules that play a crucial in flower formation in plants. Polyamine oxidases (PAOs) have a significant function in regulating flower development by influencing polyamine homeostasis. Treatment with exogenous Spd resulted in a significant increase in the Spd content in cherry flower buds, which was accompanied by an increase in abscisic acid (ABA) levels and a decrease in gibberellin (GA) levels, as well as a notable rise in the expression of genes associated with flower formation. Polyamine oxidases are essential for maintaining endogenous polyamine homeostasis. Based on the sweet cherry genome and the ‘Manaohong’ cherry transcriptome data, four polyamine metabolism enzyme genes (CpPAO1/2/3/4) were identified. Sequence comparisons of PAO proteins revealed a high degree of sequence similarity between ‘Manaohong’ cherry and sweet cherry, with only a single amino acid variation. Phylogenetic analysis classified CpPAOs into three subclasses. The expression patterns of CpPAOs were significantly tissue-specific, exhibiting higher levels of expression in flowers and young leaves. Exogenous Spd significantly increased the expression abundance of CpPAOs, as well as ABA-related and flower-forming genes. The function of CpPAO2 was subsequently verified through genetic transformation, which demonstrated that the expression levels of ABA synthesis, signal transduction genes, and flower formation genes were substantially elevated in CpPAO2 transgenic lines. These transgenic lines exhibited earlier flowering and a greater number of inflorescences compared to wild type (WT). Yeast single-hybrid and dual-luciferase assays revealed that CpABF5 binds to the promoter of CpSOC1, promoting its expression. Collectively, these results indicated that exogenous Spd increases the content of Spd and Spm while enhancing ABA levels by promoting the transcript level of CpNCEDs in flower buds, thereby facilitating flower formation through the CpABF5-CpSOC1 pathway.

RBPs: an RNA editor's choice.

RNA-binding proteins (RBPs) play a key role in gene expression and post-transcriptional RNA regulation. As integral components of ribonucleoprotein complexes, RBPs are susceptible to genomic and RNA Editing derived amino acid substitutions, impacting functional interactions. This article explores the prevalent RNA Editing of RBPs, unravelling the complex interplay between RBPs and RNA Editing events. Emphasis is placed on their influence on single amino acid variants (SAAVs) and implications for disease development. The role of Proteogenomics in identifying SAAVs is briefly discussed, offering insights into the RBP landscape. RNA Editing within RBPs emerges as a promising target for precision medicine, reshaping our understanding of genetic and epigenetic variations in health and disease.

Impact of subtype C-specific amino acid variants on HIV-1 Tat-TAR interaction: insights from molecular modelling and dynamics

BackgroundHIV-1 produces Tat, a crucial protein for transcription, viral replication, and CNS neurotoxicity. Tat interacts with TAR, enhancing HIV reverse transcription. Subtype C Tat variants (C31S, R57S, Q63E) are associated with reduced transactivation and neurovirulence compared to subtype B. However, their precise impact on Tat-TAR binding is unclear. This study investigates how these substitutions affect Tat-TAR interaction.MethodsWe utilized molecular modelling techniques, including MODELLER, to produce precise three-dimensional structures of HIV-1 Tat protein variants. We utilized Tat subtype B as the reference or wild type, and generated Tat variants to mirror those amino acid variants found in Tat subtype C. Subtype C-specific amino acid substitutions were selected based on their role in the neuropathogenesis of HIV-1. Subsequently, we conducted molecular docking of each Tat protein variant to TAR using HDOCK, followed by molecular dynamic simulations.ResultsMolecular docking results indicated that Tat subtype B (TatWt) showed the highest affinity for the TAR element (-262.07), followed by TatC31S (-261.61), TatQ63E (-256.43), TatC31S/R57S/Q63E (-238.92), and TatR57S (-222.24). However, binding free energy analysis showed higher affinities for single variants TatQ63E (-349.2 ± 10.4 kcal/mol) and TatR57S (-290.0 ± 9.6 kcal/mol) compared to TatWt (-247.9 ± 27.7 kcal/mol), while TatC31S and TatC31S/R57SQ/63E showed lower values. Interactions over the protein trajectory were also higher for TatQ63E and TatR57S compared to TatWt, TatC31S, and TatC31S/R57SQ/63E, suggesting that modifying amino acids within the Arginine/Glutamine-rich region notably affects TAR interaction. Single amino acid mutations TatR57S and TatQ63E had a significant impact, while TatC31S had minimal effect. Introducing single amino acid variants from TatWt to a more representative Tat subtype C (TatC31S/R57SQ/63E) resulted in lower predicted binding affinity, consistent with previous findings.ConclusionsThese identified amino acid positions likely contribute significantly to Tat-TAR interaction and the differential pathogenesis and neuropathogenesis observed between subtype B and subtype C. Additional experimental investigations should prioritize exploring the influence of these amino acid signatures on TAR binding to gain a comprehensive understanding of their impact on viral transactivation, potentially identifying them as therapeutic targets.

Deep mutational scanning quantifies DNA binding and predicts clinical outcomes of PAX6 variants.

Nonsense and missense mutations in the transcription factor PAX6 cause a wide range of eye development defects, including aniridia, microphthalmia and coloboma. To understand how changes of PAX6:DNA binding cause these phenotypes, we combined saturation mutagenesis of the paired domain of PAX6 with a yeast one-hybrid (Y1H) assay in which expression of a PAX6-GAL4 fusion gene drives antibiotic resistance. We quantified binding of more than 2700 single amino-acid variants to two DNA sequence elements. Mutations in DNA-facing residues of the N-terminal subdomain and linker region were most detrimental, as were mutations to prolines and to negatively charged residues. Many variants caused sequence-specific molecular gain-of-function effects, including variants in position 71 that increased binding to the LE9 enhancer but decreased binding to a SELEX-derived binding site. In the absence of antibiotic selection, variants that retained DNA binding slowed yeast growth, likely because such variants perturbed the yeast transcriptome. Benchmarking against known patient variants and applying ACMG/AMP guidelines to variant classification, we obtained supporting-to-moderate evidence that 977 variants are likely pathogenic and 1306 are likely benign. Our analysis shows that most pathogenic mutations in the paired domain of PAX6 can be explained simply by the effects of these mutations on PAX6:DNA association, and establishes Y1H as a generalisable assay for the interpretation of variant effects in transcription factors.

Switch-2 determines Mg2+ADP-release kinetics and fine-tunes the duty ratio of Dictyostelium class-1 myosins.

Though myosins share a structurally conserved motor domain, single amino acid variations of active site elements, including the P-loop, switch-1 and switch-2, which act as nucleotide sensors, can substantially determine the kinetic signature of a myosin, i.e., to either perform fast movement or enable long-range transport and tension generation. Switch-2 essentially contributes to the ATP hydrolysis reaction and determines product release. With few exceptions, class-1 myosin harbor a tyrosine in the switch-2 consensus sequence DIYGFE, at a position where class-2 myosins and a selection of myosins from other classes have a substitution. Here, we addressed the role of the tyrosine in switch-2 of class-1 myosins as potential determinant of the duty ratio. We generated constitutively active motor domain constructs of two class-1 myosins from the social amoeba Dictyostelium discoideum, namely, Myo1E, a high duty ratio myosin and Myo1B, a low duty ratio myosin. In Myo1E we introduced mutation Y388F and in Myo1B mutation F387Y. The detailed functional characterization by steady-state and transient kinetic experiments, combined with in vitro motility and landing assays revealed an almost reciprocal relationship of a number of critical kinetic parameters and equilibrium constants between wild-type and mutants that dictate the lifetime of the strongly actin-attached states of myosin. The Y-to-F mutation increased the duty ratio of Moy1B by almost one order of magnitude, while the introduction of the phenylalanine in switch-2 of Myo1E transformed the myosin into a low duty ratio motor. These data together with structural considerations propose a role of switch-2 in fine-tuning ADP release through a mechanism, where the class-specific tyrosine together with surrounding residues contributes to the coordination of Mg2+ and ADP. Our results highlight the importance of conserved switch-2 residues in class-1 myosins for efficient chemo-mechanical coupling, revealing that switch-2 is important to adjust the duty ratio of the amoeboid class-1 myosins for performing movement, transport or gating functions.

Meta-Analysis of Rice Phosphoproteomics Data to Understand Variation in Cell Signaling Across the Rice Pan-Genome.

Phosphorylation is the most studied post-translational modification, and has multiple biological functions. In this study, we have reanalyzed publicly available mass spectrometry proteomics data sets enriched for phosphopeptides from Asian rice (Oryza sativa). In total we identified 15,565 phosphosites on serine, threonine, and tyrosine residues on rice proteins. We identified sequence motifs for phosphosites, and link motifs to enrichment of different biological processes, indicating different downstream regulation likely caused by different kinase groups. We cross-referenced phosphosites against the rice 3,000 genomes, to identify single amino acid variations (SAAVs) within or proximal to phosphosites that could cause loss of a site in a given rice variety and clustered the data to identify groups of sites with similar patterns across rice family groups. The data has been loaded into UniProt Knowledge-Base─enabling researchers to visualize sites alongside other data on rice proteins, e.g., structural models from AlphaFold2, PeptideAtlas, and the PRIDE database─enabling visualization of source evidence, including scores and supporting mass spectra.

Dissecting the causal polymorphism of the Lr67res multipathogen resistance gene.

Partial resistance to multiple biotrophic fungal pathogens in wheat (Triticum aestivum L.) is conferred by a variant of the Lr67 gene, which encodes a hexose-proton symporter. Two mutations (G144R and V387L) differentiate the resistant and susceptible protein variants (Lr67res and Lr67sus). Lr67res lacks sugar transport capability and was associated with anion transporter-like properties when expressed in Xenopus laevis oocytes. Here, we extended this functional characterization to include yeast and in planta studies. The Lr67res allele, but not Lr67sus, induced sensitivity to ions in yeast (including NaCl, LiCl, and KI), which is consistent with our previous observations that Lr67res expression in oocytes induces novel ion fluxes. We demonstrate that another naturally occurring single amino acid variant in wheat, containing only the Lr67G144R mutation, confers rust resistance. Transgenic barley plants expressing the orthologous HvSTP13 gene carrying the G144R and V387L mutations were also more resistant to Puccinia hordei infection. NaCl treatment of pot-grown adult wheat plants with the Lr67res allele induced leaf tip necrosis and partial leaf rust resistance. An Lr67res-like function can be introduced into orthologous plant hexose transporters via single amino acid mutation, highlighting the strong possibility of generating disease resistance in other crops, especially with gene editing.

Structural and Dynamic Features of Acinetobacter baumannii OXA-66 β-Lactamase Explain Its Stability and Evolution of Novel Variants

OXA-66 is a member of the OXA-51 subfamily of class D β-lactamases native to the Acinetobacter genus that includes Acinetobacter baumannii, one of the ESKAPE pathogens and a major cause of drug-resistant nosocomial infections. Although both wild type OXA-66 and OXA-51 have low catalytic activity, they are ubiquitous in the Acinetobacter genomes. OXA-51 is also remarkably thermostable. In addition, newly emerging, single and double amino acid variants show increased activity against carbapenems, indicating that the OXA-51 subfamily is growing and gaining clinical significance. In this study, we used molecular dynamics simulations, X-ray crystallography, and thermal denaturation data to examine and compare the dynamics of OXA-66 wt and its gain-of-function variants: I129L (OXA-83), L167V (OXA-82), P130Q (OXA-109), P130A, and W222L (OXA-234). Our data indicate that OXA-66 wt also has a high melting temperature, and its remarkable stability is due to an extensive and rigid hydrophobic bridge formed by a number of residues around the active site and harbored by the three loops, P, Ω, and β5-β6. Compared to the WT enzyme, the mutants exhibit higher flexibility only in the loop regions, and are more stable than other robust carbapenemases, such as OXA-23 and OXA-24/40. All the mutants show increased rotational flexibility of residues I129 and W222, which allows carbapenems to bind. Overall, our data support the hypothesis that structural features in OXA-51 and OXA-66 promote evolution of multiple highly stable variants with increased clinical relevance in A. baumannii.

Insight into the function of voltage-sensing phosphatase in hindgut-derived pseudoplacenta of a viviparous teleost Xenotoca eiseni.

Nutrient absorption is essential for animal survival and development. Our previous study on zebrafish reported that nutrient absorption in lysosome-rich enterocytes (LREs) is promoted by the voltage-sensing phosphatase (VSP), which regulates phosphoinositide (PIP) homeostasis via electrical signaling in biological membranes. However, it remains unknown whether this VSP function is shared by different absorptive tissues in other species. Here, we focused on the function of VSP in a viviparous teleost Xenotoca eiseni, whose intraovarian embryos absorb nutrients from the maternal ovarian fluid through a specialized hindgut-derived pseudoplacental structure called trophotaenia. Xenotoca eiseni VSP (Xe-VSP) is expressed in trophotaenia epithelium, an absorptive tissue functionally similar to zebrafish LREs. Notably, the apical distribution of Xe-VSP in trophotaenia epithelial cells closely resembles zebrafish VSP (Dr-VSP) distribution in zebrafish LREs, suggesting a shared role for VSP in absorptive tissues between the two species. Electrophysiological analysis using a heterologous expression system revealed that Xe-VSP preserves functional voltage sensors and phosphatase activity with the leftward shifted voltage sensitivity compared with zebrafish VSP (Dr-VSP). We also identified a single amino acid variation in the S4 helix of Xe-VSP as one of the factors contributing to the leftward shifted voltage sensitivity. This study highlights the biological variation and significance of VSP in various animal species, as well as hinting at the potential role of VSP in nutrient absorption in X. eiseni trophotaenia.NEW & NOTEWORTHY We investigate the voltage-sensing phosphatase (VSP) in Xenotoca eiseni, a viviparous fish whose intraovarian embryos utilize trophotaenia for nutrient absorption. Although X. eiseni VSP (Xe-VSP) shares key features with known VSPs, its distinct voltage sensitivity arises from species-specific amino acid variation. Xe-VSP in trophotaenia epithelium suggests its involvement in nutrient absorption, similar to VSP in zebrafish enterocytes and potentially in species with similar absorptive cells. Our findings highlight the potential role of VSP across species.

Mutational scanning pinpoints distinct binding sites of key ATGL regulators in lipolysis

ATGL is a key enzyme in intracellular lipolysis and plays an important role in metabolic and cardiovascular diseases. ATGL is tightly regulated by a known set of protein-protein interaction partners with activating or inhibiting functions in the control of lipolysis. Here, we use deep mutational protein interaction perturbation scanning and generate comprehensive profiles of single amino acid variants that affect the interactions of ATGL with its regulatory partners: CGI-58, G0S2, PLIN1, PLIN5 and CIDEC. Twenty-three ATGL amino acid variants yield a specific interaction perturbation pattern when validated in co-immunoprecipitation experiments in mammalian cells. We identify and characterize eleven highly selective ATGL switch mutations which affect the interaction of one of the five partners without affecting the others. Switch mutations thus provide distinct interaction determinants for ATGL’s key regulatory proteins at an amino acid resolution. When we test triglyceride hydrolase activity in vitro and lipolysis in cells, the activity patterns of the ATGL switch variants trace to their protein interaction profile. In the context of structural data, the integration of variant binding and activity profiles provides insights into the regulation of lipolysis and the impact of mutations in human disease.

Substrate Promiscuity of the Triceptide Maturase XncB Leads to Incorporation of Various Amino Acids and Detection of Oxygenated Products.

Triceptides are cyclophane-containing ribosomally synthesized and post-translationally modified peptides. The characteristic cross-links are formed between an aromatic ring to Cβ on three-residue Ω1X2X3 motifs (Ω1 = aromatic). Here, we explored the promiscuity of the XYE family triceptide maturase, XncB from Xenorhabdus nematophila DSM 3370. Single amino acid variants were coexpressed with XncB in vivo in Escherichia coli, and we show that a variety of amino acids can be incorporated into the Phe-Gly-Asn cyclophane. Aromatic amino acids at the X3 position were accepted by the enzyme but yielded hydroxylated, rather than the typical cyclophane, products. These studies show that oxygen can be inserted but diverges in the final product formed relative to daropeptide maturases. Finally, truncations of the leader peptide showed that it is necessary for complete modification by XncB.

Unraveling the complexity: Advanced methods in analyzing DNA, RNA, and protein interactions.

Exploring the intricate interplay within and between nucleic acids, as well as their interactions with proteins, holds pivotal significance in unraveling the molecular complexities steering cancer initiation and progression. To investigate these interactions, a diverse array of highly specific and sensitive molecular techniques has been developed. The selection of a particular technique depends on the specific nature of the interactions. Typically, researchers employ an amalgamation of these different techniques to obtain a comprehensive and holistic understanding of inter- and intramolecular interactions involving DNA-DNA, RNA-RNA, DNA-RNA, or protein-DNA/RNA. Examining nucleic acid conformation reveals alternative secondary structures beyond conventional ones that have implications for cancer pathways. Mutational hotspots in cancer often lie within sequences prone to adopting these alternative structures, highlighting the importance of investigating intra-genomic and intra-transcriptomic interactions, especially in the context of mutations, to deepen our understanding of oncology. Beyond these intramolecular interactions, the interplay between DNA and RNA leads to formations like DNA:RNA hybrids (known as R-loops) or even DNA:DNA:RNA triplex structures, both influencing biological processes that ultimately impact cancer. Protein-nucleic acid interactions are intrinsic cellular phenomena crucial in both normal and pathological conditions. In particular, genetic mutations or single amino acid variations can alter a protein's structure, function, and binding affinity, thus influencing cancer progression. It is thus, imperative to understand the differences between wild-type (WT) and mutated (MT) genes, transcripts, and proteins. The review aims to summarize the frequently employed methods and techniques for investigating interactions involving nucleic acids and proteins, highlighting recent advancements and diverse adaptations of each technique.

Genotype-phenotype correlations of neurofibromatosis type 1: a cross-sectional study from a large Chinese cohort.

Neurofibromatosis type 1 (NF1) is a highly heterogeneous autosomal genetic disorder characterized by a broad spectrum of clinical and molecular manifestations. The correlations between genotype and phenotype in NF1 remain elusive. This study aimed to elucidate genotype-phenotype associations in a large Chinese cohort of NF1 patients. We included NF1 patients from our center who underwent genetic testing for NF1 variants and systemic examination. Genotype-phenotype correlation analyses were performed, focusing on variation types and involved neurofibromin domains. A total of 195 patients were enrolled, comprising 105 males and 90 females, with a median age of 18years. Truncating variants, single amino acid variations, and splicing variants accounted for 139/195 (71.3%), 23/195 (11.8%), and 33/195 (16.9%), respectively. Patients with splicing variants exhibited a significantly higher prevalence of spinal plexiform neurofibromas (spinal PNF) than those with truncating variants (76.4% vs. 51.8%; p = 0.022). Variations affecting the PKC domain were associated with higher rates of cutaneous neurofibromas (CNF) (100% vs. 64.9%, p < 0.001), Lisch nodules (100% vs. 61.2%, p < 0.001), plexiform neurofibromas (PNF) (100% vs. 95.7%, p = 0.009), and psychiatric disorders (11.8% vs. 1.6%, p = 0.042). Patients with mutations in the CSRD had an elevated risk of secondary primary malignancies (11.6% vs. 2.8%, p = 0.015). GRD involvement might enhance the risk of Lisch nodules (76.9% vs. 53.7%, p = 0.044). Variations in the Sec14-PH domain were correlated with a higher rate of CNF (76.8% vs. 58.6%, p = 0.014). Additionally, we found that the p.R1748* variants carry a high risk of malignancy. Our study suggested some novel genotype-phenotype correlations within a Chinese cohort, providing innovative insights into this complex field that may contribute to genetic counseling, risk stratification, and clinical management for the NF1 population.

Rendering protein mutation movies with MutAmore

BackgroundThe success of AlphaFold2 in reliable protein three-dimensional (3D) structure prediction, assists the move of structural biology toward studies of protein dynamics and mutational impact on structure and function. This transition needs tools that qualitatively assess alternative 3D conformations.ResultsWe introduce MutAmore, a bioinformatics tool that renders individual images of protein 3D structures for, e.g., sequence mutations into a visually intuitive movie format. MutAmore streamlines a pipeline casting single amino-acid variations (SAVs) into a dynamic 3D mutation movie providing a qualitative perspective on the mutational landscape of a protein. By default, the tool first generates all possible variants of the sequence reachable through SAVs (L*19 for proteins with L residues). Next, it predicts the structural conformation for all L*19 variants using state-of-the-art models. Finally, it visualizes the mutation matrix and produces a color-coded 3D animation. Alternatively, users can input other types of variants, e.g., from experimental structures.ConclusionMutAmore samples alternative protein configurations to study the dynamical space accessible from SAVs in the post-AlphaFold2 era of structural biology. As the field shifts towards the exploration of alternative conformations of proteins, MutAmore aids in the understanding of the structural impact of mutations by providing a flexible pipeline for the generation of protein mutation movies using current and future structure prediction models.

A quantitative model for virus uncoating predicts influenza A infectivity

For virus infection of new host cells, the disassembly of the protective outer protein shell (capsid) is a critical step, but the mechanisms and host-virus interactions underlying the dynamic, active, and regulated uncoating process are largely unknown. Here, we develop an experimentally supported, multiscale kinetics model that elucidates mechanisms of influenza A virus (IAV) uncoating in cells. Biophysical modeling demonstrates that interactions between capsid M1 proteins, host histone deacetylase 6 (HDAC6), and molecular motors can physically break the capsid in a tug-of-war mechanism. Biochemical analysis and biochemical-biophysical modeling identify unanchored ubiquitin chains as essential and allow robust prediction of uncoating efficiency in cells. Remarkably, the different infectivity of two clinical strains can be ascribed to a single amino acid variation in M1 that affects binding to HDAC6. By identifying crucial modules of viral infection kinetics, the mechanisms and models presented here could help formulate novel strategies for broad-range antiviral treatment.

Semirational engineering of Cytophaga hutchinsonii polyphosphate kinase for developing a cost-effective, robust, and efficient adenosine 5'-triphosphate regeneration system.

The adenosine 5'-triphosphate (ATP) regeneration system can significantly reduce the cost of many biocatalytic processes. Numerous studies have endeavored to utilize the ATP regeneration system based on Cytophaga hutchinsonii PPK (ChPPK). However, the wild-type ChPPK enzyme possesses limitations such as low enzymatic activity, poor stability, and limited substrate tolerance, impeding its application in catalytic reactions. To enhance the performance of ChPPK, we employed a semi-rational design approach to obtain the variant ChPPK/A79G/S106C/I108F/L285P. The enzymatic kinetic parameters and the catalytic performance in the synthesis of nicotinamide mononucleotide demonstrated that the variant ChPPK/A79G/S106C/I108F/L285P exhibited superior enzymatic properties than the wild-type enzyme. All data indicated that our engineered ATP regeneration system holds inherent potential for implementation in biocatalytic processes.

Enhancement of recombinant adeno-associated virus activity by improved stoichiometry and homogeneity of capsid protein assembly

Studies of recombinant adeno-associated virus (rAAV) revealed the mixture of full particles with different densities in rAAV. There are no conclusive results because of the lack of quantitative stoichiometric viral proteins, encapsidated DNA, and particle level analyses. We report the first comprehensive characterization of low- and high-density rAAV serotype 2 particles. Capillary gel electrophoresis showed high-density particles possessing a designed DNA encapsidated in the capsid composed of (VP1+ VP2)/VP3= 0.27, whereas low-density particles have the same DNA but with a different capsid composition of (VP1+ VP2)/VP3= 0.31, supported by sedimentation velocity-analytical ultracentrifugation and charge detection-mass spectrometry. Invitro analysis demonstrated that the low-density particles had 8.9% higher transduction efficacy than that of the particles before fractionation. Further, based on our recent findings of VP3 clip, we created rAAV2 single amino acid variants of the transcription start methionine of VP3 (M203V) and VP3 clip (M211V). The rAAV2-M203V variant had homogeneous particles with higher (VP1+VP2)/VP3 values (0.35) and demonstrated 24.7% higher transduction efficacy compared with the wild type. This study successfully provided highly functional rAAV by the extensive fractionation from the mixture of rAAV2 full particles or by the single amino acid replacement.