Residues in the fructose-binding pocket are required for ketohexokinase-A activity.

Excessive fructose consumption is a primary contributor to the global surges in obesity, cancer, and metabolic syndrome. Fructolysis is not robustly regulated and is initiated by ketohexokinase (KHK). In this study, we determined the crystal structure of KHK-A, one of two human isozymes of KHK, in the apo-state at 1.85 Å resolution, and we investigated the roles of residues in the fructose-binding pocket by mutational analysis. Introducing alanine at D15, N42, or N45 inactivated KHK-A, whereas mutating R141 or K174 reduced activity and thermodynamic stability. Kinetic studies revealed that the R141A and K174A mutations reduced fructose affinity by 2- to 4-fold compared to WT KHK-A, without affecting ATP affinity. Molecular dynamics simulations provided mechanistic insights into the potential roles of the mutated residues in ligand coordination and the maintenance of an open state in one monomer and a closed state in the other. Protein-protein interactome analysis indicated distinct expression patterns and downregulation of partner proteins in different tumor tissues, warranting a reevaluation of KHK's role in cancer development and progression. The connections between different cancer genes and the KHK signaling pathway suggest that KHK is a potential target for preventing cancer metastasis. This study enhances our understanding of KHK-A's structure and function and offers valuable insights into potential targets for developing treatments for obesity, cancer, and metabolic syndrome.

Identification of novel allosteric sites of SARS-CoV-2 papain-like protease (PLpro) for the development of COVID-19 antivirals.

Coronaviruses such as SARS-CoV-2 encode a conserved papain-like protease (PLpro) that is crucial for viral replication and immune evasion, making it a prime target for antiviral drug development. In this study, three surface pockets on SARS-CoV-2 PLpro that may function as sites for allosteric inhibition were computationally identified. To evaluate the effects of these pockets on proteolytic activity, 52 residues were separately mutated to alanine. In Pocket 1, located between the Ubl and thumb domains, the introduction of alanine at T10, D12, T54, Y72, or Y83 reduced PLpro activity to <12% of that of WT. In Pocket 2, situated at the interface of the thumb, fingers, and palm domains, Q237A, S239A, H275A, and S278A inactivated PLpro. Finally, introducing alanine at five residues in Pocket 3, between the fingers and palm domains, inactivated PLpro: S212, Y213, Y251, K254, and Y305. Pocket 1 has a higher druggability score than Pockets 2 and 3. MD simulations showed that interactions within and between domains play critical roles in PLpro activity and thermal stability. The essential residues in Pockets 1 and 2 participate in a combination of intra- and inter-domain interactions. By contrast, the essential residues in Pocket three predominantly participate in inter-domain interactions. The most promising targets for therapeutic development are Pockets one and 3, which have the highest druggability score and the largest number of essential residues, respectively. Non-competitive inhibitors targeting these pockets may be antiviral agents against COVID-19 and related coronaviruses.

Enhancing predictions of protein stability changes induced by single mutations using MSA-based Language Models.

MOTIVATION: Protein Language Models offer a new perspective for addressing challenges in structural biology, while relying solely on sequence information. Recent studies have investigated their effectiveness in forecasting shifts in thermodynamic stability caused by single amino acid mutations, a task known for its complexity due to the sparse availability of data, constrained by experimental limitations. To tackle this problem, we introduce two key novelties: leveraging a Protein Language Model that incorporates Multiple Sequence Alignments to capture evolutionary information, and using a recently released mega-scale dataset with rigorous data pre-processing to mitigate overfitting. RESULTS: We ensure comprehensive comparisons by fine-tuning various pre-trained models, taking advantage of analyses such as ablation studies and baselines evaluation. Our methodology introduces a stringent policy to reduce the widespread issue of data leakage, rigorously removing sequences from the training set when they exhibit significant similarity with the test set. The MSA Transformer emerges as the most accurate among the models under investigation, given its capability to leverage co-evolution signals encoded in aligned homologous sequences. Moreover, the optimized MSA Transformer outperforms existing methods and exhibits enhanced generalization power, leading to a notable improvement in predicting changes in protein stability resulting from point mutations. AVAILABILITY AND IMPLEMENTATION: Code and data at https://github.com/RitAreaSciencePark/PLM4Muts. SUPPLEMENTARY INFORMATION: Supplementary Information is available at Bioinformatics online.

pH profiles of 3-chymotrypsin-like protease (3CLpro) from SARS-CoV-2 elucidate its catalytic mechanism and a histidine residue critical for activity.

3-Chymotrypsin-like protease (3CLpro) is a promising drug target for coronavirus disease 2019 and related coronavirus diseases because of the essential role of this protease in processing viral polyproteins after infection. Understanding the detailed catalytic mechanism of 3CLpro is essential for designing effective inhibitors of infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Molecular dynamics studies have suggested pH-dependent conformational changes of 3CLpro, but experimental pH profiles of SARS-CoV-2 3CLpro and analyses of the conserved active-site histidine residues have not been reported. In this work, pH-dependence studies of the kinetic parameters of SARS-CoV-2 3CLpro revealed a bell-shaped pH profile with 2 pKa values (6.9 ± 0.1 and 9.4 ± 0.1) attributable to ionization of the catalytic dyad His41 and Cys145, respectively. Our investigation of the roles of conserved active-site histidines showed that different amino acid substitutions of His163 produced inactive enzymes, indicating a key role of His163 in maintaining catalytically active SARS-CoV-2 3CLpro. By contrast, the H164A and H172A mutants retained 75% and 26% of the activity of WT, respectively. The alternative amino acid substitutions H172K and H172R did not recover the enzymatic activity, whereas H172Y restored activity to a level similar to that of the WT enzyme. The pH profiles of H164A, H172A, and H172Y were similar to those of the WT enzyme, with comparable pKa values for the catalytic dyad. Taken together, the experimental data support a general base mechanism of SARS-CoV-2 3CLpro and indicate that the neutral states of the catalytic dyad and active-site histidine residues are required for maximum enzyme activity.

Impact of Confinement within a Hydrogel Mesh on Protein Thermodynamic Stability and Aggregation Kinetics.

Though protein stability and aggregation have been well characterized in dilute solutions, the influence of a confining environment that exists (e.g., in intercellular and tissue spaces and therapeutic formulations) on the protein structure is largely unknown. Herein, the effects of confinement on stability and aggregation were explored for proteins of different sizes, stability, and hydrophobicity when encapsulated in hydrophilic poly(ethylene glycol) hydrogels. Denaturation curves show linear correlations between confinement size (mesh size) and thermodynamic stability, i.e., unfolding free energy and surface area accessible for solvation (represented by m-value). Two clusters of protein types are identifiable from these correlations; the clusters are defined by differences in protein stability, surface area, and aggregation propensity. Proteins with higher stability, larger surface area, and lower aggregation propensity (e.g., lysozyme and myoglobin) are less affected by the confinement imposed by mesh size than proteins with lower stability, smaller surface area, and higher aggregation propensity (e.g., growth hormone and aldehyde dehydrogenase). According to aggregation kinetics measured by thioflavin T fluorescence, confinement in smaller mesh sizes resulted in slower aggregation rates than that in larger mesh sizes. Compared to that in buffer solution, the confinement of a hydrophobic protein (e.g., human insulin) in the hydrogels accelerates protein aggregation. Conversely, the confinement of a hydrophilic protein (e.g., human amylin) in the hydrogels decelerates or prevents aggregation, with the rates of aggregation inversely proportional to mesh size. These findings provide new insights into protein conformational stability in confined microenvironments relevant to various cellular, tissue, and therapeutics scenarios.

Detailed investigation of the binding abilities of the heterodomain of a multiHis cyclopeptide toward Cu(II) ions.

Cyclopeptides hold significant relevance in various fields of science and medicine, due to their unique structural properties and diverse biological activities. Cyclic peptides, characterized by intrinsically higher conformational order, exhibit remarkable stability and resistance to proteolytic degradation, making them attractive candidates for developing targeted drug delivery systems. The aim of this work is to elucidate the unique coordination properties of the multi-His cyclic peptide with c(HDHKHPHHKHHP) sequence (HDCP - heterodomain cyclopeptide). This peptide, indeed, is able to form homo- and hetero-dinuclear complexes in a wide pH range, being thus a good chelator for Cu(II) ions. Herein, we present the results of a combined study, involving potentiometric, spectroscopic (UV-Vis, CD, and EPR), and computational investigations, on its coordination properties. To better understand the interaction pattern with Cu(II) metal ions, two other peptides, each one bearing only one of the two binding domains of HDCP are also considered in this study: c(HDHKHPGGKGGP) = CP1, c(GKGGKPHHKHHP) = CP2, which share sequence fragments of HDCP and allow separate investigations of its coordination domains.

TMH Stab-pred: Predicting the stability of α-helical membrane proteins using sequence and structural features.

The folding and stability of transmembrane proteins (TMPs) are governed by the insertion of secondary structural elements into the cell membrane followed by their assembly. Understanding the important features that dictate the stability of TMPs is important for elucidating their functions. In this work, we related sequence and structure-based parameters with free energy (ΔG0) of α-helical membrane proteins. Our results showed that the free energy transfer of hydrophobic peptides, relative contact order, total interaction energy, number of hydrogen bonds and lipid accessibility of transmembrane regions are important for stability. Further, we have developed multiple-regression models to predict the stability of α-helical membrane proteins using these features and our method can predict the stability with a correlation and mean absolute error (MAE) of 0.89 and 1.21 kcal/mol, respectively, on jack-knife test. The method was validated with a blind test set of three recently reported experimental ΔG0, which could predict the stability within an average MAE of 0.51 kcal/mol. Further, we developed a webserver for predicting the stability and it is freely available at (https://web.iitm.ac.in/bioinfo2/TMHS/). The importance of selected parameters and limitations are discussed.

Key Allosteric and Active Site Residues of SARS-CoV-2 3CLpro Are Promising Drug Targets.

The main protease of SARS-CoV-2, 3-chymotrypsin-like protease (3CLpro), is a prominent target for antiviral development due to its essential role in the viral life cycle. Research has largely focused on competitive inhibitors of 3CLpro that target the active site. However, allosteric sites distal to the peptide substrate-binding region are also potential targets for the design of reversible noncompetitive inhibitors. Computational analyses have examined the importance of key contacts at allosteric sites of 3CLpro, but these contacts have not been validated experimentally. In this work, four druggable pockets spanning the surface of SARS-CoV-2 3CLpro were predicted: pocket 1 is the active site, whereas pockets 2, 3, and 4 are located away from the active site at the interface of domains II and III. Site-directed alanine mutagenesis of selected residues with important structural interactions revealed that 7 of 13 active site residues (N28, R40, Y54, S147, Y161, D187 and Q192) and 7 of 12 allosteric site residues (T111, R131, N133, D197, N203, D289 and D295) are essential for maintaining catalytically active and thermodynamically stable 3CLpro. Alanine substitution at these key amino acid residues inactivated or reduced the activity of 3CLpro. In addition, the thermodynamic stability of 3CLpro decreased in the presence of some of these mutations. This work provides experimental validation of essential contacts in the active and allosteric sites of 3CLpro that could be targeted with competitive and noncompetitive inhibitors as new therapeutics against COVID-19.

Stability and Strength of Monolayer Polymeric C60.

Two-dimensional fullerene networks have been synthesized in several forms, and it is unknown which monolayer form is stable under ambient conditions. Using first-principles calculations, I show that the believed stability of the quasi-tetragonal phases is challenged by mechanical, dynamic, or thermodynamic stability. For all temperatures, the quasi-hexagonal phase is thermodynamically the least stable. However, the relatively high dynamic and mechanical stabilities suggest that the quasi-hexagonal phase is intrinsically stronger than the other phases under various strains. The origin of the high stability and strength of the quasi-hexagonal phase can be attributed to the strong covalent C-C bonds that strongly hold the linked C60 clusters together, enabling the closely packed hexagonal network. These results rationalize the experimental observations that so far only the quasi-hexagonal phase has been exfoliated experimentally as monolayers.

NMR Dynamic View of the Stabilization of the WW4 Domain by Neutral NaCl and Kosmotropic Na2SO4 and NaH2PO4.

The Hofmeister series categorizes ions based on their effects on protein stability, yet the microscopic mechanism remains a mystery. In this series, NaCl is neutral, Na2SO4 and Na2HPO4 are kosmotropic, while GdmCl and NaSCN are chaotropic. This study employs CD and NMR to investigate the effects of NaCl, Na2SO4, and Na2HPO4 on the conformation, stability, binding, and backbone dynamics (ps-ns and µs-ms time scales) of the WW4 domain with a high stability and accessible side chains at concentrations ≤ 200 mM. The results indicated that none of the three salts altered the conformation of WW4 or showed significant binding to the four aliphatic hydrophobic side chains. NaCl had no effect on its thermal stability, while Na2SO4 and Na2HPO4 enhanced the stability by ~5 °C. Interestingly, NaCl only weakly interacted with the Arg27 amide proton, whereas Na2SO4 bound to Arg27 and Phe31 amide protons with Kd of 32.7 and 41.6 mM, respectively. Na2HPO4, however, bound in a non-saturable manner to Trp9, His24, and Asn36 amide protons. While the three salts had negligible effects on ps-ns backbone dynamics, NaCl and Na2SO4 displayed no effect while Na2HPO4 significantly increased the µs-ms backbone dynamics. These findings, combined with our recent results with GdmCl and NaSCN, suggest a microscopic mechanism for the Hofmeister series. Additionally, the data revealed a lack of simple correlation between thermodynamic stability and backbone dynamics, most likely due to enthalpy-entropy compensation. Our study rationalizes the selection of chloride and phosphate as the primary anions in extracellular and intracellular spaces, as well as polyphosphate as a primitive chaperone in certain single-cell organisms.

Thermodynamic Stability Theories of Irreversible Processes and the Fourth Law of Thermodynamics.

Three approaches for determining the thermodynamic stability of irreversible processes are described in generalized formulations. The simplest is the Gibbs-Duhem theory, specialized to irreversible trajectories, which uses the concept of virtual displacement in the reverse direction. Its only drawback is that even a trajectory leading to an explosion is identified as a thermodynamically stable motion. In the second approach, we use a thermodynamic Lyapunov function and its time rate from the Lyapunov thermodynamic stability theory (LTS, previously known as CTTSIP). In doing so, we demonstrate that the second differential of entropy, a frequently used Lyapunov function, is useful only for investigating the stability of equilibrium states. Nonequilibrium steady states do not qualify. Without using explicit perturbation coordinates, we further identify asymptotic thermodynamic stability and thermodynamic stability under constantly acting disturbances of unperturbed trajectories as well as of nonequilibrium steady states. The third approach is also based on the Lyapunov function from LTS, but here we additionally use the rates of perturbation coordinates, based on the Gibbs relations and without using their explicit expressions, to identify not only asymptotic thermodynamic stability but also thermodynamic stability under constantly acting disturbances. Only those trajectories leading to an infinite rate of entropy production (unstable states) are excluded from this conclusion. Finally, we use these findings to formulate the Fourth Law of thermodynamics based on the thermodynamic stability. It is a comprehensive statement covering all nonequilibrium trajectories, close to as well as far from equilibrium. Unlike previous suggested "fourth laws", this one meets the same level of generality that is associated with the original zeroth to third laws. The above is illustrated using the Schlögl reaction with its multiple steady states in certain regions of operation.

Key dimer interface residues impact the catalytic activity of 3CLpro, the main protease of SARS-CoV-2.

3C-like protease (3CLpro) processes and liberates functional viral proteins essential for the maturation and infectivity of severe acute respiratory syndrome coronavirus 2, the virus responsible for COVID-19. It has been suggested that 3CLpro is catalytically active as a dimer, making the dimerization interface a target for antiviral development. Guided by structural analysis, here we introduced single amino acid substitutions at nine residues at three key sites of the dimer interface to assess their impact on dimerization and activity. We show that at site 1, alanine substitution of S1 or E166 increased by twofold or reduced relative activity, respectively. At site 2, alanine substitution of S10 or E14 eliminated activity, whereas K12A exhibited ∼60% relative activity. At site 3, alanine substitution of R4, E290, or Q299 eliminated activity, whereas S139A exhibited 46% relative activity. We further found that the oligomerization states of the dimer interface mutants varied; the inactive mutants R4A, R4Q, S10A/C, E14A/D/Q/S, E290A, and Q299A/E were present as dimers, demonstrating that dimerization is not an indication of catalytically active 3CLpro. In addition, present mostly as monomers, K12A displayed residual activity, which could be attributed to the conspicuous amount of dimer present. Finally, differential scanning calorimetry did not reveal a direct relationship between the thermodynamic stability of mutants with oligomerization or catalytic activity. These results provide insights on two allosteric sites, R4/E290 and S10/E14, that may promote the design of antiviral compounds that target the dimer interface rather than the active site of severe acute respiratory syndrome coronavirus 2 3CLpro.

GC heterogeneity reveals sequence-structures evolution of angiosperm ITS2.

BACKGROUND: Despite GC variation constitutes a fundamental element of genome and species diversity, the precise mechanisms driving it remain unclear. The abundant sequence data available for the ITS2, a commonly employed phylogenetic marker in plants, offers an exceptional resource for exploring the GC variation across angiosperms. RESULTS: A comprehensive selection of 8666 species, comprising 165 genera, 63 families, and 30 orders were used for the analyses. The alignment of ITS2 sequence-structures and partitioning of secondary structures into paired and unpaired regions were performed using 4SALE. Substitution rates and frequencies among GC base-pairs in the paired regions of ITS2 were calculated using RNA-specific models in the PHASE package. The results showed that the distribution of ITS2 GC contents on the angiosperm phylogeny was heterogeneous, but their increase was generally associated with ITS2 sequence homogenization, thereby supporting the occurrence of GC-biased gene conversion (gBGC) during the concerted evolution of ITS2. Additionally, the GC content in the paired regions of the ITS2 secondary structure was significantly higher than that of the unpaired regions, indicating the selection of GC for thermodynamic stability. Furthermore, the RNA substitution models demonstrated that base-pair transformations favored both the elevation and fixation of GC in the paired regions, providing further support for gBGC. CONCLUSIONS: Our findings highlight the significance of secondary structure in GC investigation, which demonstrate that both gBGC and structure-based selection are influential factors driving angiosperm ITS2 GC content.

Predicting the stability of mutant proteins by computational approaches: an overview.

A very large number of computational methods to predict the change in thermodynamic stability of proteins due to mutations have been developed during the last 30 years, and many different web servers are currently available. Nevertheless, most of them suffer from severe drawbacks that decrease their general reliability and, consequently, their applicability to different goals such as protein engineering or the predictions of the effects of mutations in genetic diseases. In this review, we have summarized all the main approaches used to develop these tools, with a survey of the web servers currently available. Moreover, we have also reviewed the different assessments made during the years, in order to allow the reader to check directly the different performances of these tools, to select the one that best fits his/her needs, and to help naïve users in finding the best option for their needs.

Equiatomic binary phases of Copper-Rare Earth Elements. An overview of monocuprides from first-principles calculations.

Equiatomic binary phases of copper with rare earth (RE) elements exhibit either primitive cubic ( P m 3 ‾ m ${Pm\bar 3m}$ ) or orthorhombic (Pnma) structures and in some cases both. By using density functional theory (DFT), we calculated the enthalpies of formation along the series of RE elements combined equimolarly with copper. For RE from Sc to Lu, the calculated enthalpies of formation fall in the range -49.8 kJ/mol for LuCu to -9.1 kJ/mol for the least thermodynamically stable CeCu. Except NdCu, all the other cubic or orthorhombic compounds exhibit lattice stability. Either forms of NdCu indicated lattice instability. Along the Sc-group, the hypothetical primitive cubic and orthorhombic forms of LuCu are found thermodynamically and mechanically stable. The overall trend of the formation enthalpies as a function of the Meyer Periodic Number is consistent with the energy trend of the 4 f-orbital filling as moving from Sc to Lu monocuprides. In addition, the calculated Gibbs free energies indicate that the thermodynamic stability is largely due to the entropic contributions. All standard DFT calculations were also repeated with DFT+U to better describe the correlation between the 5d-4f and 3d shells of RECu compounds. It has been found that DFT+U slightly affects the enthalpies of formation of RECu binaries. Moreover, DFT+U shifts up the f-band energies of RECu with light RE elements (such as La, Ce and Pr) and in contrast lowers them in the case of RECu with heavy RE elements from Nd to Lu.

Sequential loss-on-ignition as a simple method for evaluating the stability of soil organic matter under actual environmental conditions.

Soil organic matter (SOM) is one of the largest carbon (C) reservoirs on Earth, and therefore its stability attracts a great deal of interest from the perspective of the global C cycle. This study examined the applicability of loss-on-ignition with a stepwise increase in temperature (SIT-LOI) of soil to evaluate the stability of SOM using soil samples having different organic matter (OM) and mineral contents and different mean residence times (MRTs) for SOM. The responses of SOM to the SIT-LOI varied depending on the samples but were all successfully approximated by a liner regression model as a function of the temperature of LOI. The slope value in the liner model that determines the residual potential of carbon during the SIT-LOI highly correlated with MRT of SOM, suggesting that this value reflects the overall stability of SOM over a range of soil properties. This hypothesis was consistent with the observation that Δ14C values of SOM decreased with increasing LOI temperature and thus, older, slower-cycling SOM was preferentially left in the soil samples by SIT-LOI. Additionally, the hypothesis was also supported by the significant correlations (p < 0.01) between the slope value and OM and mineral contents in the samples because these components are considered to regulate SOM stability. In addition to the regression analysis of the SIT-LOI data, changes in carbon to nitrogen (C/N) and carbon to hydrogen (C/H) ratios and stable carbon isotope signatures (δ13C) of the samples were investigated. The results suggest that the mineral association of SOM is an important factor characterizing the response of SOM to LOI. Hence, it was concluded that SIT-LOI is a simple and useful method for evaluating the stability of SOM under actual environmental conditions.

Greigite (Fe3S4) is thermodynamically stable: Implications for its terrestrial and planetary occurrence.

Iron sulfide minerals are widespread on Earth and likely in planetary bodies in and beyond our solar system. Using measured enthalpies of formation for three magnetic iron sulfide phases: bulk and nanophase Fe3S4 spinel (greigite), and its high-pressure monoclinic phase, we show that greigite is a stable phase in the Fe-S phase diagram at ambient temperature. The thermodynamic stability and low surface energy of greigite supports the common occurrence of fine-grained Fe3S4 in many anoxic terrestrial settings. The high-pressure monoclinic phase, thermodynamically metastable below about 3 GPa, shows a calculated negative P-T slope for its formation from the spinel. The stability of these three phases suggests their potential existence on Mercury and their magnetism may contribute to its present magnetic field.

Salt-Specific Suppression of the Cold Denaturation of Thermophilic Multidomain Initiation Factor 2.

Thermophilic proteins and enzymes are attractive for use in industrial applications due to their resistance against heat and denaturants. Here, we report on a thermophilic protein that is stable at high temperatures (Ttrs, hot 67 °C) but undergoes significant unfolding at room temperature due to cold denaturation. Little is known about the cold denaturation of thermophilic proteins, although it can significantly limit their applications. We investigated the cold denaturation of thermophilic multidomain protein translation initiation factor 2 (IF2) from Thermus thermophilus. IF2 is a GTPase that binds to ribosomal subunits and initiator fMet-tRNAfMet during the initiation of protein biosynthesis. In the presence of 9 M urea, measurements in the far-UV region by circular dichroism were used to capture details about the secondary structure of full-length IF2 protein and its domains during cold and hot denaturation. Cold denaturation can be suppressed by salt, depending on the type, due to the decreased heat capacity. Thermodynamic analysis and mathematical modeling of the denaturation process showed that salts reduce the cooperativity of denaturation of the IF2 domains, which might be associated with the high frustration between domains. This characteristic of high interdomain frustration may be the key to satisfying numerous diverse contacts with ribosomal subunits, translation factors, and tRNA.

Symplectic Geometry Aspects of the Parametrically-Dependent Kardar-Parisi-Zhang Equation of Spin Glasses Theory, Its Integrability and Related Thermodynamic Stability.

A thermodynamically unstable spin glass growth model described by means of the parametrically-dependent Kardar-Parisi-Zhang equation is analyzed within the symplectic geometry-based gradient-holonomic and optimal control motivated algorithms. The finitely-parametric functional extensions of the model are studied, and the existence of conservation laws and the related Hamiltonian structure is stated. A relationship of the Kardar-Parisi-Zhang equation to a so called dark type class of integrable dynamical systems, on functional manifolds with hidden symmetries, is stated.

Formation mechanism of typical aromatic sulfuric anhydrides and their potential role in atmospheric nucleation process.

Sulfuric anhydrides, generated from the cycloaddition reaction of SO3 with carboxylic acids, have been revealed to be potential participants in the nucleation process of new particle formation (NPF). Hence the reaction mechanisms of typical aromatic acids (benzoic acid (BA), phenylacetic acid (PAA), phthalic acid (PA), isophthalic acid (mPA), and terephthalic acid (PTA)) with SO3 to generate the corresponding aromatic sulfuric anhydrides were investigated by density functional theory calculations at the level of M06-2X/6-311++G(3df,3pd). As a result, these reactions were found to be feasible in the gas phase with barriers of 0.34, 0.30, 0.18, 0.08 and 0.12 kcal/mol to generate corresponding aromatic sulfuric anhydrides, respectively. The thermodynamic stabilities of clusters containing aromatic sulfuric anhydrides and atmospheric nucleation precursors (sulfuric acid, ammonia and dimethylamine) were further analyzed to identify the potential role of aromatic sulfuric anhydrides in NPF. As the thermodynamic stability of a cluster depends on both the number and strength of hydrogen bonds, the greater stability of the interactions between atmospheric nucleation precursors and aromatic sulfuric anhydrides than with aromatic acids make aromatic sulfuric anhydrides potential participators in the nucleation process of NPF. Moreover, compared with BA, the addition of a -CH2- functional group in PAA has little influence on the reaction barrier with SO3 but an inhibitive effect on the thermodynamic stability of clusters. The position of the two -COOH functional groups in PA, mPA and PTA does not have a consistent impact on the reaction barrier with SO3 or the thermodynamic stability.