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The Caenorhabditis elegans HMP-2/HMP-1 complex, akin to the mammalian β-catenin-α-catenin complex, serves as a critical mechanosensor at cell-cell adherens junctions, transducing tension between HMR-1 (also known as cadherin in mammals) and the actin cytoskeleton. Essential for embryonic development and tissue integrity in C. elegans, this complex experiences tension from both internal actomyosin contractility and external mechanical microenvironmental perturbations. While offering a valuable evolutionary comparison to its mammalian counterpart, the impact of tension on the mechanical stability of HMP-1 and HMP-2/HMP-1 interactions remains unexplored. In this study, we directly quantified the mechanical stability of full-length HMP-1 and its force-bearing modulation domains (M1-M3), as well as the HMP-2/HMP-1 interface. Notably, the M1 domain in HMP-1 exhibits significantly higher mechanical stability than its mammalian analog, attributable to interdomain interactions with M2-M3. Introducing salt bridge mutations in the M3 domain weakens the mechanical stability of the M1 domain. Moreover, the intermolecular HMP-2/HMP-1 interface surpasses its mammalian counterpart in mechanical stability, enabling it to support the mechanical activation of the autoinhibited M1 domain for mechanotransduction. Additionally, the phosphomimetic mutation Y69E in HMP-2 weakens the mechanical stability of the HMP-2/HMP-1 interface, compromising the force-transmission molecular linkage and its associated mechanosensing functions. Collectively, these findings provide mechanobiological insights into the C. elegans HMP-2/HMP-1 complex, highlighting the impact of salt bridges on mechanical stability in α-catenin and demonstrating the evolutionary conservation of the mechanical switch mechanism activating the HMP-1 modulation domain for protein binding at the single-molecule level. [ABSTRACT FROM AUTHOR]

Clostridium histolyticum is a Gram-positive anaerobic bacterium belonging to the Clostridium genus. It produces collagenase, an enzyme involved in breaking down collagen which is a key component of connective tissues. However, antimicrobial resistance (AMR) poses a great challenge in combating infections caused by this bacteria. The lengthy nature of traditional drug development techniques has resulted in a shift to computer-aided drug design and other modern drug discovery approaches. The above method offers a cost-effective means for gathering comprehensive information about how ligands interact with their target proteins. The objective of this study is to create novel, explicit drugs that specifically inhibit the C. histolyticum collagenase enzyme. Through structure-based virtual screening, a library containing 1830 compounds was screened to identify potential drug candidates against collagenase enzymes. Following that, molecular dynamic (MD) simulation was performed in an aqueous solution to evaluate the behavior of protein and ligand in a dynamic environment while density functional theory (DFT) analysis was executed to predict the molecular properties and structure of lead compounds, and the WaterSwap technique was utilized to obtain insights into the drug–protein interaction with water molecules. Furthermore, principal component analysis (PCA) was performed to reveal conformational changes, salt bridges to express electrostatic interaction and protein stability, and absorption, distribution, metabolism, excretion, and toxicity (ADMET) to assess the pharmacokinetics profile of top compounds and control molecules. Three potent drug candidates were identified MSID000001, MSID000002, MSID000003, and the control with a binding score of −10.7 kcal/mol, −9.8 kcal/mol, −9.5 kcal/mol, and −8 kcal/mol, respectively. Furthermore, Molecular Mechanics Poisson–Boltzmann Surface Area (MMPBSA) analysis of the simulation trajectories revealed energy scores of −79.54 kcal/mol, −73.99 kcal/mol, −62.26 kcal/mol, and −70.66 kcal/mol, correspondingly. The pharmacokinetics properties exhibited were under the acceptable range. The compounds hold the potential to be novel drugs; therefore, further investigation needs to be conducted to find out their anti-collagenase action against C. histolyticum infections and antibiotic resistance. [ABSTRACT FROM AUTHOR]

The extensive accumulation of polyethylene terephthalate (PET) has become a critical environmental issue. PET hydrolases can break down PET into its building blocks. Recently, we identified a glacial PET hydrolase GlacPETase sharing less than 31% amino acid identity with any known PET hydrolases. In this study, the crystal structure of GlacPETase was determined at 1.8 Å resolution, revealing unique structural features including a distinctive N-terminal disulfide bond and a specific salt bridge network. Site-directed mutagenesis demonstrated that the disruption of the N-terminal disulfide bond did not reduce GlacPETase’s thermostability or its catalytic activity on PET. However, mutations in the salt bridges resulted in changes in melting temperature ranging from −8°C to +2°C and the activity on PET ranging from 17.5% to 145.5% compared to the wild type. Molecular dynamics simulations revealed that these salt bridges stabilized the GlacPETase’s structure by maintaining their surrounding structure. Phylogenetic analysis indicated that GlacPETase represented a distinct branch within PET hydrolases-like proteins, with the salt bridges and disulfide bonds in this branch being relatively conserved. This research contributed to the improvement of our comprehension of the structural mechanisms that dictate the thermostability of PET hydrolases, highlighting the diverse characteristics and adaptability observed within PET hydrolases. IMPORTANCE The pervasive problem of polyethylene terephthalate (PET) pollution in various terrestrial and marine environments is widely acknowledged and continues to escalate. PET hydrolases, such as GlacPETase in this study, offered a solution for breaking down PET. Its unique origin and less than 31% identity with any known PET hydrolases have driven us to resolve its structure. Here, we report the correlation between its unique structure and biochemical properties, focusing on an N-terminal disulfide bond and specific salt bridges. Through site-directed mutagenesis experiments and molecular dynamics simulations, the roles of the N-terminal disulfide bond and salt bridges were elucidated in GlacPETase. This research enhanced our understanding of the role of salt bridges in the thermostability of PET hydrolases, providing a valuable reference for the future engineering of PET hydrolases. [ABSTRACT FROM AUTHOR]

Coiled coils are one of most common protein quaternary structures and represent the best understood relationship between amino acid sequence and protein conformation. Whereas the roles of residues at the canonical heptad positions the a, d, e, and g are understood in precise detail, conventional approaches often assume that the solvent‐exposed b‐, c‐, and f‐positions can be varied broadly for application‐specific purposes with minimal consequences. However, a growing body of evidence suggests that interactions among these b, c, and f residues can contribute substantially to coiled‐coil conformational stability. In the trimeric coiled coil described here, we find that b‐position Glu10 engages in a stabilizing long‐range synergistic interaction with c‐position Lys18 (ΔΔΔGf = −0.65 ± 0.02 kcal/mol). This favorable interaction depends strongly on the presence of two nearby f‐position residues: Lys 7 and Tyr14. Extensive mutational analysis of these residues in the presence of added salt versus denaturant suggests that this long‐range synergistic interaction is primarily electrostatic in origin, but also depends on the precise location and acidity of a side‐chain hydrogen‐bond donor within f‐position Tyr14. [ABSTRACT FROM AUTHOR]

Clostridium histolyticum is a Gram-positive anaerobic bacterium belonging to the Clostridium genus. It produces collagenase, an enzyme involved in breaking down collagen which is a key component of connective tissues. However, antimicrobial resistance (AMR) poses a great challenge in combating infections caused by this bacteria. The lengthy nature of traditional drug development techniques has resulted in a shift to computer-aided drug design and other modern drug discovery approaches. The above method offers a cost-effective means for gathering comprehensive information about how ligands interact with their target proteins. The objective of this study is to create novel, explicit drugs that specifically inhibit the C. histolyticum collagenase enzyme. Through structure-based virtual screening, a library containing 1830 compounds was screened to identify potential drug candidates against collagenase enzymes. Following that, molecular dynamic (MD) simulation was performed in an aqueous solution to evaluate the behavior of protein and ligand in a dynamic environment while density functional theory (DFT) analysis was executed to predict the molecular properties and structure of lead compounds, and the WaterSwap technique was utilized to obtain insights into the drug–protein interaction with water molecules. Furthermore, principal component analysis (PCA) was performed to reveal conformational changes, salt bridges to express electrostatic interaction and protein stability, and absorption, distribution, metabolism, excretion, and toxicity (ADMET) to assess the pharmacokinetics profile of top compounds and control molecules. Three potent drug candidates were identified MSID000001, MSID000002, MSID000003, and the control with a binding score of −10.7 kcal/mol, −9.8 kcal/mol, −9.5 kcal/mol, and −8 kcal/mol, respectively. Furthermore, Molecular Mechanics Poisson–Boltzmann Surface Area (MMPBSA) analysis of the simulation trajectories revealed energy scores of −79.54 kcal/mol, −73.99 kcal/mol, −62.26 kcal/mol, and −70.66 kcal/mol, correspondingly. The pharmacokinetics properties exhibited were under the acceptable range. The compounds hold the potential to be novel drugs; therefore, further investigation needs to be conducted to find out their anti-collagenase action against C. histolyticum infections and antibiotic resistance.

Using the marine natural product ptilocaulin as a starting point, the supramolecular behavior of N,N′‐bridged guanidinium nitrates in the crystalline state was investigated. For this purpose, various N,N′‐bridged guanidines were synthesized (typically by condensation of a 1,2‐ or 1,3‐diamine with cyanogen bromide) and subsequently converted into their nitrate salts with nitric acid. The crystal structures of 28 samples were determined by single crystal X‐ray crystallography and revealed the existence of a few privileged, recurring supramolecular motifs (hydrogen bond networks) formed by charge‐assisted self‐assembly. A topological analysis of possible pseudo‐2D‐patterns derived from the 1 : 1 ion pairs (generated by salt bridge formation between a N,N′‐bridged guanidinium cation and a nitrate anion) through additional H‐bonding uncovered only five conceivable regular structural motifs: (1) a linear chain, (2) an alternating (zig‐zag) chain, (3) a double chain, (4) a tape/ribbon‐type structure, and (5) a cyclotrimer. Experimentally, only the first four of these motives were observed while the cyclotrimer, as a non‐infinite and therefore particular super‐structure, did not occur. Other H‐bonding patterns were found (in three cases) when water was incorporated in the crystals. In two cases, polymorphism was observed. The identified supramolecular preferences prove the value of N,N′‐bridged guanidines as useful and easily tunable building blocks for supramolecular chemistry and crystal engineering. [ABSTRACT FROM AUTHOR]

The histidine-containing phosphocarrier (HPr) is a monomeric protein conserved in Gram-positive bacteria, which may be of mesophilic or thermophilic nature. In particular, the HPr protein from the thermophilic organism B. stearothermophilus is a good model system for thermostability studies, since experimental data, such as crystal structure and thermal stability curves, are available. However, its unfolding mechanism at higher temperatures is yet unclear at a molecular level. Therefore, in this work, we researched the thermal stability of this protein using molecular dynamics simulations, subjecting it to five different temperatures during a time span of 1 μs. The analyses of the structural parameters and molecular interactions were compared with those of the mesophilic homologue HPr protein from B. subtilis. Each simulation was run in triplicate using identical conditions for both proteins. The results showed that the two proteins lose stability as the temperature increases, but the mesophilic structure is more affected. We found that the salt bridge network formed by the triad of Glu3-Lys62-Glu36 residues and the salt bridge made up of Asp79-Lys83 ion pair are key factors to keep stable the thermophilic protein, maintaining the hydrophobic core protected and the structure packed. In addition, these molecular interactions neutralize the negative surface charge, acting as "natural molecular staples". [ABSTRACT FROM AUTHOR]

This study designed a practical approach of separating electrodes chambers to prove the influence of electrochemical oxidation on pyrene removal during electrokinetic (EK) remediation. The anode and cathode electrodes were arranged in two independent soil chambers, and the soil chambers were connected by salt bridges. Salt bridges electrokinetic (SBEK) remediation avoided acid–base neutralization and facilitated the intensity of the current. The results showed that 99.5% of pyrene was removed within 7 days in anodic chamber. The removal rate of pyrene was ∼99.6–99.9% at SBEK1 anode and 29.5–36.2% at SBEK1 cathode after 42 days. The changes in current, pH, and redox potential values indicated that an efficient "oxidizer" was formed in the anode chamber. Compared with conventional EK remediation, SBEK can take place under a suitable oxidizing condition because of increasing current intensity and decreasing soil pH in the anode chamber. Furthermore, SBEK with electrode polarity reversal test had better decontamination efficiency of pyrene than fixed electrodes. Removal rate of pyrene increased to 62.9% after 14 days with polarity reversal; correspondingly, the energy consumption of SBEK test was ∼13 times higher than that of the conventional EK test. The novel application of separated electrodes chambers appears to be a viable strategy for removing organic pollutants. [ABSTRACT FROM AUTHOR]

Microtubules are key players in several stages of the cell cycle and are also involved in the transportation of cellular organelles. Microtubules are polymerized by α/β tubulin dimers with a highly dynamic feature, especially at the plus ends of the microtubules. Therefore, understanding the interactions among tubulins is crucial for characterizing microtubule dynamics. Studying microtubule dynamics can help researchers make advances in the treatment of neurodegenerative diseases and cancer. In this study, we utilize a series of computational approaches to study the electrostatic interactions at the binding interfaces of tubulin monomers. Our study revealed that among all the four types of tubulin-tubulin binding modes, the electrostatic attractive interactions in the α/β tubulin binding are the strongest while the interactions of α/α tubulin binding in the longitudinal direction are the weakest. Our calculations explained that due to the electrostatic interactions, the tubulins always preferred to form α/β tubulin dimers. The interactions between two protofilaments are the weakest. Thus, the protofilaments are easily separated from each other. Furthermore, the important residues involved in the salt bridges at the binding interfaces of the tubulins are identified, which illustrates the details of the interactions in the microtubule. This study elucidates some mechanistic details of microtubule dynamics and also identifies important residues at the binding interfaces as potential drug targets for the inhibition of cancer cells. [ABSTRACT FROM AUTHOR]

Neutral (n) and zwitterionic (z) forms of cysteine monomers are combined in this work to extensively explore the potential energy surfaces for the formation of cysteine dimers in aqueous environments represented by a continuum. A simulated annealing search followed by optimization and characterization of the candidate structures afforded a total of 746 structurally different dimers held together via 80 different types of intermolecular contacts in 2894 individual non-covalent interactions as concluded from Natural Bond Orbitals (NBO), Quantum Theory of Atoms in Molecules (QTAIM) and Non-Covalent Interactions (NCI) analyses. This large pool of interaction possibilities includes the traditional primary hydrogen bonds and salt bridges which actually dictate the structures of the dimers, as well as the less common secondary hydrogen bonds, exotic X⋯Y (X = C, N, O, S) contacts, and H⋯H dihydrogen bonds. These interactions are not homogeneous but have rather complex distributions of strengths, interfragment distances and overall stabilities. Judging by their Gibbs bonding energies, most of the structures located here are suitable for experimental detection at room conditions. [ABSTRACT FROM AUTHOR]

Electrostatic interactions of enzymes and their effects on enzyme activity and stability are poorly understood in non-aqueous conditions. Here, we investigate the contribution of the electrostatic interactions on the stability and activity of enzymes in the non-aqueous environment using molecular dynamics simulations. Lipase was selected as active and lysozyme as inactive model enzymes in non-aqueous media. Hexane was used as a common non-aqueous solvent model. In agreement with the previous experiments, simulations show that lysozyme has more structural instabilities than lipase in hexane. The number of hydrogen bonds and salt bridges of both enzymes is dramatically increased in hexane. In contrast to the other opinions, we show that the increase of the electrostatic interactions in non-aqueous media is not so favorable for enzymatic function and stability. In this condition, the newly formed hydrogen bonds and salt bridges can partially denature the local structure of the enzymes. For lysozyme, the changes in electrostatic interactions occur in all domains including the active site cleft, which leads to enzyme inactivation and destabilization. Interestingly, most of the changes in electrostatic interactions of lipase occur far from the active site regions. Therefore, the active site entrance regions remain functional in hexane. The results of this study reveal how the changes in electrostatic interactions can affect enzyme stability and activity in non-aqueous conditions. Moreover, we show for the first time how some enzymes, such as lipase, remain active in a non-aqueous environment.Communicated by Ramaswamy H. Sarma.

Zwitterionic polymer brushes are not a practical choice since their ionic response mechanisms are unclear, despite their great potential for surface antifouling modification. Therefore, atomic force microscopy and molecular dynamics simulations investigated the ionic response of the surface electrical properties, hydration properties, and protein adhesion of three types of zwitterionic brushes. The surface of PMPC (poly(2-methacryloyloxyethyl phosphorylcholine)) and PSBMA (poly(sulfobetaine methacrylate)) zwitterionic polymer brushes in salt solution exhibits a significant accumulation of cations, which results in a positive shift in the surface potential. In contrast, the surface of PSBMA polymer brushes demonstrates no notable change in potential. Furthermore, divalent Ca 2+ enhances protein adhesion to polymer brushes by Ca 2+ bridges. Conversely, monovalent Na + diminishes the number of salt bridges between PSBMA and PCBMA (poly(carboxybetaine methacrylate)) zwitterionic polymer brushes and proteins via a competitive adsorption mechanism, thereby reducing protein adhesion. A summary of polymer brush material selection and design concepts in a salt solution environment is provided based on the salt response law of protein adhesion resistance of various zwitterionic materials. This work closes a research gap on the response mechanism of zwitterionic polymer brushes' antifouling performance in a salt solution environment, significantly advancing the practical use of these brushes., (© 2024 Wiley‐VCH GmbH.)

Thermophilic proteins have various practical applications in theoretical research and in industry. In recent years, the demand for thermophilic proteins on an industrial scale has been increasing; therefore, the engineering of thermophilic proteins has become a hot direction in the field of protein engineering. However, the exact mechanism of thermostability of proteins is not yet known, for engineering thermophilic proteins knowing the basis of thermostability is necessary. In order to understand the basis of the thermostability in proteins, we have made a statistical analysis of the sequences, secondary structures, hydrogen bonds, salt bridges, DHA (Donor–Hydrogen–Accepter) angles, and bond lengths of ten pairs of thermophilic proteins and their non-thermophilic orthologous. Our findings suggest that polar amino acids contribute to thermostability in proteins by forming hydrogen bonds and salt bridges which provide resistance against protein denaturation. Short bond length and a wider DHA angle provide greater bond stability in thermophilic proteins. Moreover, the increased frequency of aromatic amino acids in thermophilic proteins contributes to thermal stability by forming more aromatic interactions. Additionally, the coil, helix, and loop in the secondary structure also contribute to thermostability. [ABSTRACT FROM AUTHOR]

Utilizing the "ideal" ionic liquid salt bridge to measure Gibbs energies of transfer of silver ions between the solvents water, acetonitrile, propylene carbonate and dimethylformamide results in a consistent data set with a precision of 0.6 kJ mol−1 over 87 measurements in 10 half‐cells. This forms the basis for a coherent experimental thermodynamic framework of ion solvation chemistry. In addition, we define the solvent independent peabsH2O ‐ and the EabsH2O values that account for the electronating potential of any redox system similar to the pHabsH2O value of a medium that accounts for its protonating potential. This EabsH2O scale is thermodynamically well‐defined enabling a straightforward comparison of the redox potentials (reducities) of all media with respect to the aqueous redox potential scale, hence unifying all conventional solvents′ redox potential scales. Thus, using the Gibbs energy of transfer of the silver ion published herein, one can convert and unify all hitherto published redox potentials measured, for example, against ferrocene, to the EabsH2O scale. [ABSTRACT FROM AUTHOR]

Native electrospray ionization-mass spectrometry, with gas-phase activation and solution compositions that partially release subcomplexes, can elucidate topologies of macromolecular assemblies. That so much complexity can be preserved in gas-phase assemblies is remarkable, although a long-standing conundrum has been the differences between their gas- and solution-phase decompositions. Collision-induced dissociation of multimeric noncovalent complexes typically distributes products asymmetrically (i.e., by ejecting a single subunit bearing a large percentage of the excess charge). That unexpected behavior has been rationalized as one subunit "unfolding" to depart with more charge. We present an alternative explanation based on heterolytic ion-pair scission and rearrangement, a mechanism that inherently partitions charge asymmetrically. Excessive barriers to dissociation are circumvented in this manner, when local charge rearrangements access a lower-barrier surface. An implication of this ion pair consideration is that stability differences between high- and low-charge state ions usually attributed to Coulomb repulsion may, alternatively, be conveyed by attractive forces from ion pairs (salt bridges) stabilizing low-charge state ions. Should the number of ion pairs be roughly inversely related to charge, symmetric dissociations would be favored from highly charged complexes, as observed. Correlations between a gas-phase protein's size and charge reflect the quantity of restraining ion pairs. Collisionally-facilitated salt bridge rearrangement (SaBRe) may explain unusual size "contractions" seen for some activated, low charge state complexes. That some low-charged multimers preferentially cleave covalent bonds or shed small ions to disrupting noncovalent associations is also explained by greater ion pairing in low charge state complexes. Graphical Abstract ᅟ.

Fungal aryl-alcohol oxidases (AAOs) are attractive biocatalysts because they selectively oxidize a broad range of aromatic and aliphatic allylic primary alcohols while yielding hydrogen peroxide as the only by-product. However, their use is hampered by challenging and often unsuccessful heterologous expression. Production of PeAAO1 from Pleurotus eryngii ATCC 90787 in Pichia pastoris failed, while PeAAO2 from P. eryngii P34 with an amino acid identity of 99% was expressed at high yields. By successively introducing mutations in PeAAO1 to mimic the sequence of PeAAO2, the double mutant PeAAO1 ER with mutations K583E and Q584R was constructed, that was successfully expressed in P. pastoris. Functional expression was enhanced up to 155 U/l via further replacements D361N (variant NER) or V367A (variant AER). Fed-batch cultivation of recombinant P. pastoris yielded up to 116 mg/l of active variants. Glycosylated PeAAO1 variants demonstrated high stability and catalytic efficiencies similar to PeAAO2. Interestingly, P. pastoris expressing PeAAO1 variant ER contained roughly 13 gene copies but showed similar volumetric activity as NER and AER with one to two gene copies and four times lower mRNA levels. Additional H-bonds and salt bridges introduced by mutations K583E and Q584R might facilitate heterologous expression by enhanced protein folding. Key points • PeAAO1 not expressed in P. pastoris and PeAAO2 well-expressed in Pichia differ at 7 positions. • Expression of PeAAO1 in P. pastoris achieved through mutagenesis based on PeAAO2 sequence. • Combination of K583E and Q584R is essential for expression of PeAAO1 in P. pastoris. [ABSTRACT FROM AUTHOR]

Type IV collagen, a principal constituent of basement membranes, consists of six distinct α chains that assemble into both ABC and AAB-type heterotrimers. While collagen-like peptides have been investigated for heterotrimer formation, the construction of ABC-type heterotrimeric collagen mimetic peptides remains a formidable challenge, primarily due to the intricate composition and arrangement of the chains. We have herein for the first time reported the development of a versatile triblock peptide system to mimic ABC-type heterotrimeric collagen stabilized by salt bridges. The triblock peptides A, B, and C incorporate functional natural type IV collagen sequences in the center, along with charged amino acids at their N and C-terminals. By leveraging electrostatic repulsion at these charged termini, the formation of homotrimers is effectively inhibited, while stable ABC-type heterotrimers are generated through the establishment of salt bridges between oppositely charged terminals. Circular dichroism (CD) spectroscopy demonstrated that peptides A, B, and C existed as individual monomers, while they effectively formed stable ABC-type heterotrimers upon being mixed at a molar ratio of 1:1:1. Additionally, fluorescence quenching results indicated that fluorescence-labeled peptides A′, B′, and C′ formed ABC-type heterotrimer, exhibiting comparable thermal stability as determined by CD spectroscopy. Molecular dynamics simulations elucidated the role of salt bridges between arginine and aspartic acid residues at N- and C-terminals in maintaining a unique chain register in the ABC-type heterotrimers. These triblock peptides offer a robust approach for replicating the structural and functional characteristics of type IV collagen, with promising applications in elucidating the biological roles and pathologies associated with heterotrimeric collagen. [ABSTRACT FROM AUTHOR]

Microbial Fuel Cell (MFC) is one tool that uses microbes to produce electrical energy. The main components of MFC support are anodes, cathodes, and salt bridges. In this paper, the application of tropical fruit waste to MFC is presented. Tropical fruit waste used here are pineapples, oranges, bananas, watermelons, mangos, and papayas. The fruit waste is extracted and fermented to produce microbial. The microbial produces ions attached to the anode. Measurement of system current and voltage is carried out using a digital multimeter. In pineapple substrate MFC, Voc has a maximum of 485 mV, maximum current density is 163 mA/m2 and maximum power density of 11mW/m2. The orange substrate obtained Voc maximum of 805 mV, maximum current density of 661 mA/m2 and maximum power density of 62 mW/m2. Voc banana substrate has a maximum of 312 mV, maximum current density of 118 mA/m2 and maximum power density of 5.9 mW/m2. The Voc watermelon substrate has a maximum of 451 mV, maximum current density of 306 mA/m2 and maximum power density of 18.6 mW/m2. Voc mango substrate has a maximum of 586 mV, maximum current density of 229 mA/m2 and maximum power density of 4.3 mW/m2. Voc papaya substrate is a maximum of 338 mV, maximum current density of 58 mA/m2 and maximum power density of 2.9 mW/m2. These results show the potential for renewable electricity sources.

Abstract Background The lignin peroxidase isozyme H8 from the white-rot fungus Phanerochaete chrysosporium (LiPH8) demonstrates a high redox potential and can efficiently catalyze the oxidation of veratryl alcohol, as well as the degradation of recalcitrant lignin. However, native LiPH8 is unstable under acidic pH conditions. This characteristic is a barrier to lignin depolymerization, as repolymerization of phenolic products occurs simultaneously at neutral pH. Because repolymerization of phenolics is repressed at acidic pH, a highly acid-stable LiPH8 could accelerate the selective depolymerization of recalcitrant lignin. Results The engineered LiPH8 was in silico designed through the structural superimposition of surface-active site-harboring LiPH8 from Phanerochaete chrysosporium and acid-stable manganese peroxidase isozyme 6 (MnP6) from Ceriporiopsis subvermispora. Effective salt bridges were probed by molecular dynamics simulation and changes to Gibbs free energy following mutagenesis were predicted, suggesting promising variants with higher stability under extremely acidic conditions. The rationally designed variant, A55R/N156E-H239E, demonstrated a 12.5-fold increased half-life under extremely acidic conditions, 9.9-fold increased catalytic efficiency toward veratryl alcohol, and a 7.8-fold enhanced lignin model dimer conversion efficiency compared to those of native LiPH8. Furthermore, the two constructed salt bridges in the variant A55R/N156E-H239E were experimentally confirmed to be identical to the intentionally designed LiPH8 variant using X-ray crystallography (PDB ID: 6A6Q). Conclusion Introduction of strong ionic salt bridges based on computational design resulted in a LiPH8 variant with markedly improved stability, as well as higher activity under acidic pH conditions. Thus, LiPH8, showing high acid stability, will be a crucial player in biomass valorization using selective depolymerization of lignin.

Buried salt bridges widely exist in protein structures but are rarely used in synthetic systems for molecular recognition in water. By mimicking the binding pocket of bioreceptors, we designed and synthesized a pair of endo‐functionalized macrocyclic hosts with secondary ammonium groups in a hydrophobic cavity. We found that these macrocycles are able to selectively recognize carboxylic acids in water through salt bridges and the hydrophobic effect. Moreover, it was demonstrated that these macrocyclic receptors can be used in circular‐dichroism‐based optical chirality sensing of chiral carboxylic acids and fluorescent sensing of phenylpyruvic acid—a biomarker for phenylketonuria. This research showcases that buried salt bridges can be effectively used by endo‐functionalized macrocyclic hosts for molecular recognition in water, where solvent screening on polar noncovalent interactions is high. [ABSTRACT FROM AUTHOR]

Experimental evidence suggests that the folding and aggregation of the amyloid beta-protein (Abeta) into oligomers is a key pathogenetic event in Alzheimer's disease. Inhibiting the pathologic folding and oligomerization of Abeta could be effective in the prevention and treatment of Alzheimer's disease. Here, using all-atom molecular dynamics simulations in explicit solvent, we probe the initial stages of folding of a decapeptide segment of Abeta, Abeta(21-30), shown experimentally to nucleate the folding process. In addition, we examine the folding of a homologous decapeptide containing an amino acid substitution linked to hereditary cerebral hemorrhage with amyloidosis-Dutch type, [Gln-22]Abeta(21-30). We find that: (i) when the decapeptide is in water, hydrophobic interactions and transient salt bridges between Lys-28 and either Glu-22 or Asp-23 are important in the formation of a loop in the Val-24-Lys-28 region of the wild-type decapeptide; (ii) in the presence of salt ions, salt bridges play a more prominent role in the stabilization of the loop; (iii) in water with a reduced density, the decapeptide forms a helix, indicating the sensitivity of folding to different aqueous environments; and (iv) the "Dutch" peptide in water, in contrast to the wild-type peptide, fails to form a long-lived Val-24-Lys-28 loop, suggesting that loop stability is a critical factor in determining whether Abeta folds into pathologic structures.

Experimental evidence suggests that the folding and aggregation of the amyloid beta-protein (Abeta) into oligomers is a key pathogenetic event in Alzheimer's disease. Inhibiting the pathologic folding and oligomerization of Abeta could be effective in the prevention and treatment of Alzheimer's disease. Here, using all-atom molecular dynamics simulations in explicit solvent, we probe the initial stages of folding of a decapeptide segment of Abeta, Abeta(21-30), shown experimentally to nucleate the folding process. In addition, we examine the folding of a homologous decapeptide containing an amino acid substitution linked to hereditary cerebral hemorrhage with amyloidosis-Dutch type, [Gln-22]Abeta(21-30). We find that: (i) when the decapeptide is in water, hydrophobic interactions and transient salt bridges between Lys-28 and either Glu-22 or Asp-23 are important in the formation of a loop in the Val-24-Lys-28 region of the wild-type decapeptide; (ii) in the presence of salt ions, salt bridges play a more prominent role in the stabilization of the loop; (iii) in water with a reduced density, the decapeptide forms a helix, indicating the sensitivity of folding to different aqueous environments; and (iv) the "Dutch" peptide in water, in contrast to the wild-type peptide, fails to form a long-lived Val-24-Lys-28 loop, suggesting that loop stability is a critical factor in determining whether Abeta folds into pathologic structures.

We report microsecond timescale ligand field molecular dynamics simulations of the copper complexes of three known mutants of the amyloid-β peptide, E22G, E22Q and E22K, alongside the naturally occurring sequence. We find that all three mutants lead to formation of less compact structures than the wild-type: E22Q is the most similar to the native peptide, while E22G and especially E22K are markedly different in size, shape and stability. Turn and coil structures dominate all structures studied but subtle differences in helical and β-sheet distribution are noted, especially in the C-terminal region. The origin of these changes is traced to disruption of key salt bridges: in particular, the Asp23-Lys28 bridge that is prevalent in the wild-type is absent in E22G and E22K, while Lys22 in the latter mutant forms a strong association with Asp23. We surmise that the drastically different pattern of salt bridges in the mutants lead to adoption of a different structural ensemble of the peptide backbone, and speculate that this might affect the ability of the mutant peptides to aggregate in the same manner as known for the wild-type.

Guanylate binding proteins (GBPs) belong to the dynamin-related superfamily and exhibit various functions in the fight against infections. The functions of the human guanylate binding protein 1 (hGBP1) are tightly coupled to GTP hydrolysis and dimerization. Despite known crystal structures of the hGBP1 monomer and GTPase domain dimer, little is known about the dynamics of hGBP1. To gain a mechanistic understanding of hGBP1, we performed sub-millisecond multi-resolution molecular dynamics simulations of both the hGBP1 monomer and dimer. We found that hGBP1 is a highly flexible protein that undergoes a hinge motion similar to the movements observed for other dynamin-like proteins. Another large-scale motion was observed for the C-terminal helix α13, providing a molecular view for the α13–α13 distances previously reported for the hGBP1 dimer. Most of the loops of the GTPase domain were found to be flexible, revealing why GTP binding is needed for hGBP1 dimerization to occur. [ABSTRACT FROM AUTHOR]

Epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor important in diverse biological processes including cell proliferation and survival. Upregulation of EGFR activity due to over-expression or mutation is widely implicated in cancer. Activating somatic mutations of the EGFR kinase are postulated to affect the conformation and/or stability of the protein, shifting the EGFR inactive-active state equilibrium towards the activated state. Here, we examined a common EGFR deletion mutation, Δ746ELREA750, which is frequently observed in non-small cell lung cancer patients. By using molecular dynamics simulation, we investigated the structural effects of the mutation that lead to the experimentally reported increases in kinase activity. Simulations of the active form wild-type and ΔELREA EGFRs revealed the deletion stabilizes the αC helix of the kinase domain, which is located adjacent to the deletion site, by rigidifying the flexible β3-αC loop that accommodates the ELREA sequence. Consequently, the αC helix is stabilized in the “αC-in” active conformation that would prolong the time of the activated state. Moreover, in the mutant kinase, a salt bridge between E762 and K745, which is key for EGFR activity, was also stabilized during the simulation. Additionally, the interaction between EGFR and ATP was favored by ΔELREA EGFR over wild-type EGFR, as reflected by the number of hydrogen bonds formed and the free energy of binding. Simulation of inactive EGFR suggested the deletion would promote a shift from the inactive conformation towards active EGFR, which is supported by the inward movement of the αC helix. The MDS results also align with the effects of tyrosine kinase inhibitors on ΔELREA and wild-type EGFR lung cancer cell lines, where more pronounced inhibition was observed against ΔELREA than for wild-type EGFR by inhibitors recognizing the active kinase conformation. [ABSTRACT FROM AUTHOR]

Mycolic acids are long chain alpha-alkyl beta-hydroxy fatty acids that are major constituents of the cell wall of Mycobacterium tuberculosis. M. tuberculosis produces three main types of mycolic acids, alpha mycolic acids and keto and methoxy mycolic acids. Cycloproponated mycolic acids make the cell wall less permeable, contribute to antibiotic resistance and host immunomodulation and protect from injury. Cyclopropanation is catalyzed by enzymes of the Cyclopropane Mycolic Acid Synthase (CMAS) family. In the current study, we addressed two CMAS enzymes, proximal alpha cyclopropane mycolic acid synthase (PcaA/CmaA3) and keto cyclopropane Mycolic acid synthase (CmaA2). All-atom Molecular Dynamics (MD) simulations were performed for these enzymes for a timeframe of 100ns each (in triplicate), using GROMACS. Based on the PDB structures of apo and holo states of related CMAS enzymes, we generated a framework which helped us correlate active or inactive states of the enzymes to different conformations sampled by the enzymes during MD simulations. Dynamics suggested that the free or unbound enzymes have intrinsic memory and sample different states of catalysis even in the absence of the substrate/cofactor. Additionally, we find that F200, P201 and W204 may have functional significance. MD simulation of CmaA2 was performed with the objective of gaining insights into the putative role of a loop insert. Analysis showed that acidic residues of this loop possibly play an important role during the active state by forming salt bridges. The insights gained in this study can potentially be utilized for design of effective inhibitors against CMAS enzymes. Image 1 • Framework generated for active state conformation of CMAS enzymes of M. tuberculosis. • Free enzyme MD simulation of CmaA3 samples active state conformation around 61ns. • Residues of potential significance in CmaA3 – F200, P201, W204 discussed. • Salt bridge profile of D152 and D156 of the loop insert region of CmaA2 investigated. • Insights gained potentially useful for design of inhibitors against CMAS enzymes. [ABSTRACT FROM AUTHOR]

Hitherto, how salt bridges contribute towards the structure-function of endo -mannanases has not been demonstrated. In the present study, we revealed that ManB-1601 (GH26 endo -mannanase from Bacillus sp.) has eight salt bridges which are highly conserved among GH26 endo-mannanases from Bacillus spp. Disruption of salt bridges do not alter overall structure, optimum pH and temperature of ManB-1601. Among the salt bridges, elimination of K95/E156 and E171/R221 pair decreased the substrate affinity and catalytic efficiency of ManB-1601. Differential scanning calorimetry and isothermal equilibrium denaturation studies suggested that salt bridges do not markedly contribute towards thermal (∆T m 0 to −5 °C) and conformational stability (∆∆G −0.37 to −1.25 Kcal mol−1) of ManB-1601. Interestingly, salt bridges were found to prominently contribute towards kinetic stability of ManB-1601 as salt bridge mutants exhibited drastic reduction in half-life of enzyme inactivation (T 1/2) at 66 °C (1.1 to 6-fold) and 70 °C (4.09 to 22.5-fold). Molecular dynamic simulations studies showed that salt bridges contribute towards maintaining the biological activity against thermal denaturation by rigidifying the active site. Our study on ManB-1601 suggest that even in the case salt bridges do not confer thermal and conformational stabilities they may serve as crucial structural element s for enzyme functioning by contributing towards kinetic stability. • Salt bridges are conserved among GH26 endo-mannanase from Bacillus spp. • In ManB-1601, salt bridges do not contribute towards thermodynamic stability. • Prominent contribution of salt bridges towards the kinetic stability of ManB-1601. • Salt bridges rigidify the loops around the active site of ManB-1601. [ABSTRACT FROM AUTHOR]

The light-promoted recovery of epidermal barrier of skin was evaluated by the associated recovery of transepidermal potential (TEP), the potential difference between the surface and dermis of skin, by using porcine skin samples. An accelerated recovery of TEP was observed by irradiation of red light with the irradiance of 40 mW/cm2 and a duration of > 10 min. The influence of the light stimulation to the surroundings (~ 20 mm) was also observed. The irradiations of blue and purple lights were ineffective in accelerating the barrier recovery. These characteristics of the light stimulation would be useful for the design of effective and safe phototherapy devices for skin. The present study proves that the TEP can serve as a spatiotemporal indicator of the epidermal barrier function. [ABSTRACT FROM AUTHOR]

The gastrointestinal pathogen, Clostridioides difficile, initiates infection when its metabolically dormant spore form germinates in the mammalian gut. While most spore-forming bacteria use transmembrane germinant receptors to sense nutrient germinants, C. difficile is thought to use the soluble pseudoprotease, CspC, to detect bile acid germinants. To gain insight into CspC’s unique mechanism of action, we solved its crystal structure. Guided by this structure, we identified CspC mutations that confer either hypo- or hyper-sensitivity to bile acid germinant. Surprisingly, hyper-sensitive CspC variants exhibited bile acid-independent germination as well as increased sensitivity to amino acid and/or calcium co-germinants. Since mutations in specific residues altered CspC’s responsiveness to these different signals, CspC plays a critical role in regulating C. difficile spore germination in response to multiple environmental signals. Taken together, these studies implicate CspC as being intimately involved in the detection of distinct classes of co-germinants in addition to bile acids and thus raises the possibility that CspC functions as a signaling node rather than a ligand-binding receptor. [ABSTRACT FROM AUTHOR]

Multiple microsecond-length molecular dynamics simulations of complexes of Al(III) with amyloid-β (Aβ) peptides of varying length are reported, employing a non-bonded model of Al-coordination to the peptide, which is modelled using the AMBER ff14SB forcefield. Individual simulations reach equilibrium within 100 to 400 ns, as determined by root mean square deviations, leading to between 2.1 and 2.7 μs of equilibrated data. These reveal a compact set of configurations, with radius of gyration similar to that of the metal free peptide but larger than complexes with Cu, Fe and Zn. Strong coordination through acidic residues Glu3, Asp7 and Glu11 is maintained throughout all trajectories, leading to average coordination numbers of approximately 4 to 5. Helical conformations predominate, particularly in the longer Al-Aβ40 and Al-Aβ42 peptides, while β-strand forms are rare. Binding of the small, highly charged Al(III) ion to acidic residues in the N-terminus strongly disrupts their ability to engage in salt bridges, whereas residues outside the metal binding region engage in salt bridges to similar extent to the metal-free peptide, including the Asp23-Lys28 bridge known to be important for formation of fibrils. High helical content and disruption of salt bridges leads to characteristic tertiary structure, as shown by heat maps of contact between residues as well as representative clusters of trajectories. [ABSTRACT FROM AUTHOR]

Cadherins are homophilic cell-cell adhesion molecules whose aberrant expression has often been shown to correlate with different stages of tumor progression. In this work, we investigate the interaction of two peptidomimetic ligands with the extracellular portion of human E-cadherin using a combination of NMR and computational techniques. Both ligands have been previously developed as mimics of the tetrapeptide sequence Asp1-Trp2-Val3-Ile4 of the cadherin adhesion arm, and have been shown to inhibit E-cadherin-mediated adhesion in epithelial ovarian cancer cells with millimolar potency. To sample a set of possible interactions of these ligands with the E-cadherin extracellular portion, STD-NMR experiments in the presence of two slightly different constructs, the wild type E-cadherin-EC1-EC2 fragment and the truncated E-cadherin-(Val3)-EC1-EC2 fragment, were carried out at three temperatures. Depending on the protein construct, a different binding epitope of the ligand and also a different temperature effect on STD signals were observed, both suggesting an involvement of the Asp1-Trp2 protein sequence among all the possible binding events. To interpret the experimental results at the atomic level and to probe the role of the cadherin adhesion arm in the dynamic interaction with the peptidomimetic ligand, a computational protocol based on docking calculations and molecular dynamics simulations was applied. In agreement with NMR data, the simulations at different temperatures unveil high variability/dynamism in ligand-cadherin binding, thus explaining the differences in ligand binding epitopes. In particular, the modulation of the signals seems to be dependent on the protein flexibility, especially at the level of the adhesive arm, which appears to participate in the interaction with the ligand. Overall, these results will help the design of novel cadherin inhibitors that might prevent the swap dimer formation by targeting both the Trp2 binding pocket and the adhesive arm residues. [ABSTRACT FROM AUTHOR]

The highly extreme conditions of the lower convective layer in the Atlantis II (ATII) Deep brine pool of the Red Sea make it an ideal environment for the search for novel enzymes that can function under extreme conditions. In the current study, we isolated a novel sequence of a thioredoxin reductase (TrxR) enzyme from the metagenomic dataset established from the microbial community that resides in the lower convective layer of Atlantis II. The gene was cloned, expressed and characterized for redox activity, halophilicity, and thermal stability. The isolated thioredoxin reductase (ATII-TrxR) was found to belong to the high-molecular-weight class of thioredoxin reductases. A search for conserved domains revealed the presence of an extra domain (Crp) in the enzyme sequence. Characterization studies of ATII-TrxR revealed that the enzyme was halophilic (maintained activity at 4 M NaCl), thermophilic (optimum temperature was 65°C) and thermostable (60% of its activity was retained at 70°C). Additionally, the enzyme utilized NADH in addition to NADPH as an electron donor. In conclusion, a novel thermostable and halophilic thioredoxin reductase has been isolated with a unique sequence that adapts to the harsh conditions of the brine pools making this protein a good candidate for biological research and industrial applications. [ABSTRACT FROM AUTHOR]

Hexameric arginine repressor, ArgR, is the feedback regulator of bacterial L-arginine regulons, and sensor of L-arg that controls transcription of genes for its synthesis and catabolism. Although ArgR function, as well as its secondary, tertiary, and quaternary structures, is essentially the same in E. coli and B. subtilis, the two proteins differ significantly in sequence, including residues implicated in the response to L-arg. Molecular dynamics simulations are used here to evaluate the behavior of intact B. subtilis ArgR with and without L-arg, and are compared with prior MD results for a domain fragment of E. coli ArgR. Relative to its crystal structure, B. subtilis ArgR in absence of L-arg undergoes a large-scale rotational shift of its trimeric subassemblies that is very similar to that observed in the E. coli protein, but the residues driving rotation have distinct secondary and tertiary structural locations, and a key residue that drives rotation in E. coli is missing in B. subtilis. The similarity of trimer rotation despite different driving residues suggests that a rotational shift between trimers is integral to ArgR function. This conclusion is supported by phylogenetic analysis of distant ArgR homologs reported here that indicates at least three major groups characterized by distinct sequence motifs but predicted to undergo a common rotational transition. The dynamic consequences of L-arg binding for transcriptional activation of intact ArgR are evaluated here for the first time in two-microsecond simulations of B. subtilis ArgR. L-arg binding to intact B. subtilis ArgR causes a significant further shift in the angle of rotation between trimers that causes the N-terminal DNA-binding domains lose their interactions with the C-terminal domains, and is likely the first step toward adopting DNA-binding-competent conformations. The results aid interpretation of crystal structures of ArgR and ArgR-DNA complexes.

Ion pairs (also known as salt bridges) of electrostatically interacting cationic and anionic moieties are important for proteins and nucleic acids to perform their function. Although numerous three-dimensional structures show ion pairs at functionally important sites of biological macromolecules and their complexes, the physicochemical properties of the ion pairs are not well understood. Crystal structures typically show a single state for each ion pair. However, recent studies have revealed the dynamic nature of the ion pairs of the biological macromolecules. Biomolecular ion pairs undergo dynamic transitions between distinct states in which the charged moieties are either in direct contact or separated by water. This dynamic behavior is reasonable in light of the fundamental concepts that were established for small ions over the last century. In this review, we introduce the physicochemical concepts relevant to the ion pairs and provide an overview of the recent advancement in biophysical research on the ion pairs of biological macromolecules.

CRISPR-Cas systems have become widely used across all fields of biology as a genome engineering tool. With its recent demonstration in the Gram positive industrial workhorse Bacillus subtilis, this tool has become an attractive option for rapid, markerless strain engineering of industrial production hosts. Previously described strategies for CRISPR-Cas9 genome editing in B. subtilis have involved chromosomal integrations of Cas9 and single guide RNA expression cassettes, or construction of large plasmids for simultaneous transformation of both single guide RNA and donor DNA. Here we use a flexible, co-transformation approach where the single guide RNA is inserted in a plasmid for Cas9 co-expression, and the donor DNA is supplied as a linear PCR product observing an editing efficiency of 76%. This allowed multiple, rapid rounds of in situ editing of the subtilisin E gene to incorporate a salt bridge triad present in the Bacillus clausii thermotolerant homolog, M-protease. A novel subtilisin E variant was obtained with increased thermotolerance and activity. [ABSTRACT FROM AUTHOR]

Malaria is a life-threatening disease spread by mosquitoes. Plasmodium falciparum M1 alanyl aminopeptidase (PfM1-AAP) is a promising target for the treatment of malaria. The recently solved crystal structures of PfM1-AAP revealed that the buried active site can be accessed through two channel openings: a short N-terminal channel with the length of 8 Å and a long C-terminal channel with the length of 30 Å. It is unclear, however, how substrates and inhibitors migrate to the active site and a product of cleavage leaves. Here, we study the molecular mechanism of substrate and inhibitor migration to the active site and the product release using steered molecular dynamics simulations. We identified a stepwise passage of substrates and inhibitors in the C-terminal channel of PfM1-AAP, involving (I) ligand recognition at the opening of the channel, (II) ionic translation to the ‘water reservoir’, (III) ligand reorientation in the ‘water reservoir’ and (IV) passage in a suitable conformation into the active site. Endorsed by enzymatic analysis of functional recombinant PfM1-AAP and mutagenesis studies, our novel ligand-residue binding network analysis has identified the functional residues controlling ligand migration within the C-terminal channel of PfM1-AAP. Furthermore, from unbinding simulations of the Arg product we propose a charge repulsion as the driving force to expel the product out from the N-terminal channel of PfM1-AAP. Our work paves the way towards the design of a novel class of PfM1-AAP inhibitors based on preventing substrate entry to the active site. [ABSTRACT FROM AUTHOR]

The MHC class II region in humans is highly polymorphic. Each MHC molecule is formed by an α and a β chain, produced by different genes, creating an antigen-binding groove. In the groove there are several pockets into which antigens anchor and fit. The affinity of this fitting determines the recognition specificity of a given peptide. Here, based on our previous results about the association of MHC class II with the WNV disease, we examined the role of the binding pockets of HLA-DPA1, -DQA1 and–DRB1 in the severe form of the disease. In HLA-DQA1, variants in all pockets 1, 6 and 9 were found to be associated with either protection and/or susceptibility to neuroinvasion caused by WNV. Similarly, pockets 7, 9 and 10 in HLA-DRB1 were associated with severe disease. Protein modeling of these molecules revealed structural and functional differences among alleles with opposite roles concerning the development of the disease. Different amino acids in positions α52 and α66 (HLA-DQA1) significantly influenced the peptide binding while DYWLR/EFA combination (HLA-DRB1) was associated with neuronal damage. Further studies could help us understand the selectivity of pocket variants in order to create suitable peptides for an effective response. [ABSTRACT FROM AUTHOR]

Substrate permissiveness has long been regarded as the raw materials for the evolution of new enzymatic functions. In land plants, hydroxycinnamoyltransferase (HCT) is an essential enzyme of the phenylpropanoid metabolism. Although essential enzymes are normally associated with high substrate specificity, HCT can utilize a variety of non-native substrates. To examine the structural and dynamic basis of substrate permissiveness in this enzyme, we report the crystal structure of HCT from Selaginella moellendorffii and molecular dynamics (MD) simulations performed on five orthologous HCTs from several major lineages of land plants. Through altogether 17-μs MD simulations, we demonstrate the prevalent swing motion of an arginine handle on a submicrosecond timescale across all five HCTs, which plays a key role in native substrate recognition by these intrinsically promiscuous enzymes. Our simulations further reveal how a non-native substrate of HCT engages a binding site different from that of the native substrate and diffuses to reach the catalytic center and its co-substrate. By numerically solving the Smoluchowski equation, we show that the presence of such an alternative binding site, even when it is distant from the catalytic center, always increases the reaction rate of a given substrate. However, this increase is only significant for enzyme-substrate reactions heavily influenced by diffusion. In these cases, binding non-native substrates ‘off-center’ provides an effective rationale to develop substrate permissiveness while maintaining the native functions of promiscuous enzymes. [ABSTRACT FROM AUTHOR]

RIP2, one of the RIP kinases, interacts with p75 neurotrophin receptor, regulating the neuron survival, and with NOD1 and NOD2 proteins, causing the innate immune response against gram-negative and gram-positive bacteria via its caspase recruitment domain (CARD). This makes RIP2 a prospective target for novel therapies, aimed to modulate the inflammatory diseases and neurogenesis/neurodegeneration. Several studies report the problems with the stability of human RIP2 CARD and its production in bacterial hosts, which is a prerequisite for the structural investigation with solution NMR spectroscopy. In the present work, we report the high yield production and refolding protocols and resolve the structure of rat RIP2 CARD. The structure reveals the important differences to the previously published conformation of the homologous human protein. Using solution NMR, we characterized the intramolecular mobility and pH-dependent behavior of RIP2 CARD, and found the propensity of the protein to form high-order oligomers at physiological pH while being monomeric under acidic conditions. The oligomerization of protein may be explained, based on the electrostatic properties of its surface. Analysis of the structure and sequences of homologous proteins reveals the residues which are significant for the unusual fold of RIP2 CARD domains from different species. The high-throughput protein production/refolding protocols and proposed explanation for the protein oligomerization, provide an opportunity to design the stabilized variants of RIP2 CARD, which could be used to study the structural details of RIP2/NOD1/NOD2 interaction and perform the rational drug design. [ABSTRACT FROM AUTHOR]

Several recent studies indicate that small Open Reading Frames (sORFs) embedded within multiple eukaryotic non-coding RNAs can be translated into bioactive peptides of up to 100 amino acids in size. However, the functional roles of the 607 Stress Induced Peptides (SIPs) previously identified from 189 Transcriptionally Active Regions (TARs) in Arabidopsis thaliana remain unclear. To provide a starting point for functional annotation of these plant-derived peptides, we performed a large-scale prediction of peptide binding sites on protein surfaces using coarse-grained peptide docking. The docked models were subjected to further atomistic refinement and binding energy calculations. A total of 530 peptide-protein pairs were successfully docked. In cases where a peptide encoded by a TAR is predicted to bind at a known ligand or cofactor-binding site within the protein, it can be assumed that the peptide modulates the ligand or cofactor-binding. Moreover, we predict that several peptides bind at protein-protein interfaces, which could therefore regulate the formation of the respective complexes. Protein-peptide binding analysis further revealed that peptides employ both their backbone and side chain atoms when binding to the protein, forming predominantly hydrophobic interactions and hydrogen bonds. In this study, we have generated novel predictions on the potential protein-peptide interactions in A. thaliana, which will help in further experimental validation. [ABSTRACT FROM AUTHOR]

Calcium ions (Ca2+) play an important role in the toxicity of the cysteine-rich and cationic antifungal protein PAF from Penicillium chrysogenum: high extracellular Ca2+ levels reduce the toxicity of PAF in the sensitive model fungus Neurospora crassa in a concentration dependent way. However, little is known about the mechanistic details of the Ca2+ ion impact and the Ca2+ binding capabilities of PAF outside the fungal cell, which might be the reason for the activity loss. Using nuclear magnetic resonance (NMR), isothermal titration calorimetry and molecular dynamics (MD) simulations we demonstrated that PAF weakly, but specifically binds Ca2+ ions. MD simulations of PAF predicted one major Ca2+ binding site at the C-terminus involving Asp53 and Asp55, while Asp19 was considered as putative Ca2+ binding site. The exchange of Asp19 to serine had little impact on the Ca2+ binding, however caused the loss of antifungal activity, as was shown in our recent study. Now we replaced the C-terminal aspartates and expressed the serine variant PAFD53S/D55S. The specific Ca2+ binding affinity of PAFD53S/D55S decreased significantly if compared to PAF, whereas the antifungal activity was retained. To understand more details of Ca2+ interactions, we investigated the NMR and MD structure/dynamics of the free and Ca2+-bound PAF and PAFD53S/D55S. Though we found some differences between these protein variants and the Ca2+ complexes, these effects cannot explain the observed Ca2+ influence. In conclusion, PAF binds Ca2+ ions selectively at the C-terminus; however, this Ca2+ binding does not seem to play a direct role in the previously documented modulation of the antifungal activity of PAF. [ABSTRACT FROM AUTHOR]

A mutated KRAS protein is frequently observed in human cancers. Traditionally, the oncogenic properties of KRAS missense mutants at position 12 (G12X) have been considered as equal. Here, by assessing the probabilities of occurrence of all KRAS G12X mutations and KRAS dynamics we show that this assumption does not hold true. Instead, our findings revealed an outstanding mutational bias. We conducted a thorough mutational analysis of KRAS G12X mutations and assessed to what extent the observed mutation frequencies follow a random distribution. Unique tissue-specific frequencies are displayed with specific mutations, especially with G12R, which cannot be explained by random probabilities. To clarify the underlying causes for the nonrandom probabilities, we conducted extensive atomistic molecular dynamics simulations (170 μs) to study the differences of G12X mutations on a molecular level. The simulations revealed an allosteric hydrophobic signaling network in KRAS, and that protein dynamics is altered among the G12X mutants and as such differs from the wild-type and is mutation-specific. The shift in long-timescale conformational dynamics was confirmed with Markov state modeling. A G12X mutation was found to modify KRAS dynamics in an allosteric way, which is especially manifested in the switch regions that are responsible for the effector protein binding. The findings provide a basis to understand better the oncogenic properties of KRAS G12X mutants and the consequences of the observed nonrandom frequencies of specific G12X mutations. [ABSTRACT FROM AUTHOR]

Actinoporins sticholysin I and sticholysin II (St I, St II) are proposed to lyse model and biomembranes via toroidal pore formation by their N-terminal domain. Based on the hypothesis that peptide fragments can reproduce the structure and function of this domain, the behavior of peptides containing St I residues 12–31 (StI12-31), St II residues 11–30 (StII11-30), and its TOAC-labeled analogue (N-TOAC-StII11-30) was examined. Molecular modeling showed a good match with experimental structures, indicating amphipathic α-helices in the same regions as in the toxins. CD spectra revealed that the peptides were essentially unstructured in aqueous solution, acquiring α-helical conformation upon interaction with micelles and large unilamellar vesicles (LUV) of variable lipid composition. Fluorescence quenching studies with NBD-containing lipids indicated that N-TOAC-StII11-30’s nitroxide moiety is located in the membranes polar head group region. Pyrene-labeled phospholipid inter-leaflet redistribution suggested that the peptides form toroidal pores, according to the mechanism of action proposed for the toxins. Binding occurred only to negatively charged LUV, indicating the importance of electrostatic interactions; in contrast the peptides bound to both negatively charged and zwitterionic micelles, pointing to a lesser influence of these interactions. In addition, differences between bilayers and micelles in head group packing and in curvature led to differences in peptide-membrane interaction. We propose that the peptides topography in micelles resembles that of the toxins in the toroidal pore. The peptides mimicked the toxins permeabilizing activity, St II peptides being more effective than StI12-31. To our knowledge, this is the first demonstration that differences in the toxins N-terminal amphipathic α-helix play a role in the difference between St I and St II activities. [ABSTRACT FROM AUTHOR]

The adherens junctions between epithelial cells involve a protein complex formed by E-cadherin, β-catenin, α-catenin and F-actin. The stability of this complex was a puzzle for many years, since in vitro studies could reconstitute various stable subsets of the individual proteins, but never the entirety. The missing ingredient turned out to be mechanical tension: a recent experiment that applied physiological forces to the complex with an optical tweezer dramatically increased its lifetime, a phenomenon known as catch bonding. However, in the absence of a crystal structure for the full complex, the microscopic details of the catch bond mechanism remain mysterious. Building on structural clues that point to α-catenin as the force transducer, we present a quantitative theoretical model for how the catch bond arises, fully accounting for the experimental lifetime distributions. The underlying hypothesis is that force induces a rotational transition between two conformations of α-catenin, overcoming a significant energy barrier due to a network of salt bridges. This transition allosterically regulates the energies at the interface between α-catenin and F-actin. The model allows us to predict these energetic changes, as well as highlighting the importance of the salt bridge rotational barrier. By stabilizing one of the α-catenin states, this barrier could play a role in how the complex responds to additional in vivo binding partners like vinculin. Since significant conformational energy barriers are a common feature of other adhesion systems that exhibit catch bonds, our model can be adapted into a general theoretical framework for integrating structure and function in a variety of force-regulated protein complexes. [ABSTRACT FROM AUTHOR]

Opioids represent a highly-abused and highly potent class of drugs that have become a significant threat to public safety. Often there are little to no pharmacological and toxicological data available for new, illicitly used and abused opioids, and this has resulted in a growing number of serious adverse events, including death. The large influx of new synthetic opioids permeating the street-drug market, including fentanyl and fentanyl analogs, has generated the need for a fast and effective method to evaluate the risk a substance poses to public safety. In response, the US FDA’s Center for Drug Evaluation and Research (CDER) has developed a rapidly-deployable, multi-pronged computational approach to assess a drug’s risk to public health. A key component of this approach is a molecular docking model to predict the binding affinity of biologically uncharacterized fentanyl analogs to the mu opioid receptor. The model was validated by correlating the docking scores of structurally diverse opioids with experimentally determined binding affinities. Fentanyl derivatives with sub-nanomolar binding affinity at the mu receptor (e.g. carfentanil and lofentanil) have significantly lower binding scores, while less potent fentanyl derivatives have increased binding scores. The strong correlation between the binding scores and the experimental binding affinities suggests that this approach can be used to accurately predict the binding strength of newly identified fentanyl analogs at the mu receptor in the absence of in vitro data and may assist in the temporary scheduling of those substances that pose a risk to public safety. [ABSTRACT FROM AUTHOR]

Organic hydroperoxide resistance (Ohr) enzymes are highly efficient Cys-based peroxidases that play central roles in bacterial response to fatty acid hydroperoxides and peroxynitrite, two oxidants that are generated during host-pathogen interactions. In the active site of Ohr proteins, the conserved Arg (Arg19 in Ohr from Xylella fastidiosa) and Glu (Glu51 in Ohr from Xylella fastidiosa) residues, among other factors, are involved in the extremely high reactivity of the peroxidatic Cys (Cp) toward hydroperoxides. In the closed state, the thiolate of Cp is in close proximity to the guanidinium group of Arg19. Ohr enzymes can also assume an open state, where the loop containing the catalytic Arg is far away from Cp and Glu51. Here, we aimed to gain insights into the putative structural switches of the Ohr catalytic cycle. First, we describe the crystal structure of Ohr from Xylella fastidiosa (XfOhr) in the open state that, together with the previously described XfOhr structure in the closed state, may represent two snapshots along the coordinate of the enzyme-catalyzed reaction. These two structures were used for the experimental validation of molecular dynamics (MD) simulations. MD simulations employing distinct protonation states and in silico mutagenesis indicated that the polar interactions of Arg19 with Glu51 and Cp contributed to the stabilization of XfOhr in the closed state. Indeed, Cp oxidation to the disulfide state facilitated the switching of the Arg19 loop from the closed to the open state. In addition to the Arg19 loop, other portions of XfOhr displayed high mobility, such as a loop rich in Gly residues. In summary, we obtained a high correlation between crystallographic data, MD simulations and biochemical/enzymatic assays. The dynamics of the Ohr enzymes are unique among the Cys-based peroxidases, in which the active site Arg undergoes structural switches throughout the catalytic cycle, while Cp remains relatively static. [ABSTRACT FROM AUTHOR]