α-Hydrazino Acid Insertion Governs Peptide Organization in Solution by Local Structure Ordering.

In this work, we have applied the concept of α-hydrazino acid insertion in a peptide sequence as a means of structurally organizing a potential protein-protein interactions (PPI) inhibitor. Hydrazino peptides characterized by the incorporation of an α-hydrazino acid at specific positions introduce an additional nitrogen atom into their backbone. This modification leads to a change in the electrostatic properties of the peptide and induces the restructuring of its hydrogen bonding network, resulting in conformational changes toward more stable structural motifs. Despite the successful use of synthetic hydrazino oligomers in binding to nucleic acids, the structural changes due to the incorporation of α-hydrazino acid into short natural peptides in solution are still poorly understood. Based on NMR data, we report structural models of p53-derived hydrazino peptides with elements of localized peptide structuring in the form of an α-, ß-, or γ-turn as a result of hydrazino modification in the peptide backbone. The modifications could potentially lead to the preorganization of a helical secondary peptide structure in a solution that is favorable for binding to a biological receptor. Spectroscopically, we observed that the ensemble averaged rapidly interconverting conformations, including isomerization of the E-Z hydrazide bond. This further increases the adaptability by expanding the conformational space of hydrazine peptides as potential protein-protein interaction antagonists.

Single Dispersion of Fe(H2O)2-Based Polyoxometalate on Polymeric Carbon Nitride for Biomimetic CH4 Photooxidation.

Direct methane conversion to value-added oxygenates under mild conditions with in-depth mechanism investigation has attracted wide interest. Inspired by methane monooxygenase, the K9Na2Fe(H2O)2{[γ-SiW9O34Fe(H2O)]}2·25H2O polyoxometalate (Fe-POM) with well-defined Fe(H2O)2 sites is synthesized to clarify the key role of Fe species and their microenvironment toward CH4 photooxidation. The Fe-POM can efficiently drive the conversion of CH4 to HCOOH with a yield of 1570.0 µmol gPOM -1 and 95.8% selectivity at ambient conditions, much superior to that of [Fe(H2O)SiW11O39]5- with Fe(H2O) active site, [Fe2SiW10O38(OH)]2 14- and [P8W48O184Fe16(OH)28(H2O)4]20- with multinuclear Fe-OH-Fe active sites. Single-dispersion of Fe-POM on polymeric carbon nitride (PCN) is facilely achieved to provide single-cluster functionalized PCN with well-defined Fe(H2O)2 site, the HCOOH yield can be improved to 5981.3 µmol gPOM -1. Systemic investigations demonstrate that the (WO)4-Fe(H2O)2 can supply Fe═O active center for C-H activation via forming (WO)4-Fea-Ot···CH4 intermediate, similar to that for CH4 oxidation in the monooxygenase. This work highlights a promising and facile strategy for single dispersion of ≈1-2 Å metal center with precise coordination microenvironment by uniformly anchoring nanoscale molecular clusters, which provides a well-defined model for in-depth mechanism research.

Insight into the Role of an α-Helix Cluster in Protoporphyrinogen IX Oxidase.

Protoporphyrinogen IX oxidase (PPO) is the last common enzyme in chlorophyll and heme biosynthesis pathways. In humans, point mutations on PPO are responsible for the dominantly inherited disorder disease variegate porphyria (VP). It is found that several VP-causing mutation sites are located on an α-helix cluster (consisting of α-5, α-6, and α-7 helix, named the G169 helix cluster) of human PPO, although these mutation sites are outside the active site of the human PPO. In this work, we investigated the role of the G169 helix cluster via site-directed mutagenesis, enzymatic kinetics, and computational studies. Kinetic studies showed that mutations on the G169 helix cluster affect the activity of PPO. The MD simulation showed that mutations on the G169 helix cluster reduced the activity of PPO by affecting the proper orientation of substrate protoporphyrinogen within the active site of PPO and possibly the dipole moment of the G169 helix cluster. Moreover, the mutation abolished the interaction between the mutated site and other residues, thus affecting the secondary structure and hydrogen bond interactions within the G169 helix cluster. These results indicated that the integrity of the G169 helix cluster is important for the stabilization of protoporphyrinogen within the active site of PPO to facilitate the interaction between protoporphyrinogen and cofactor FAD and provide a proper electrostatic environment for the activity of PPO. Our result provides new insight into understanding the relationship between the structure and function of PPO.

Insight into Tetramolecular DNA G-Quadruplexes Associated with ALS and FTLD: Cation Interactions and Formation of Higher-Ordered Structure.

The G4C2 hexanucleotide repeat expansion in the c9orf72 gene is a major genetic cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), with the formation of G-quadruplexes directly linked to the development of these diseases. Cations play a crucial role in the formation and structure of G-quadruplexes. In this study, we investigated the impact of biologically relevant potassium ions on G-quadruplex structures and utilized 15N-labeled ammonium cations as a substitute for K+ ions to gain further insights into cation binding and exchange dynamics. Through nuclear magnetic resonance spectroscopy and molecular dynamics simulations, we demonstrate that the single d(G4C2) repeat, in the presence of 15NH4+ ions, adopts a tetramolecular G-quadruplex with an all-syn quartet at the 5'-end. The movement of 15NH4+ ions through the central channel of the G-quadruplex, as well as to the bulk solution, is governed by the vacant cation binding site, in addition to the all-syn quartet at the 5'-end. Furthermore, the addition of K+ ions to G-quadruplexes folded in the presence of 15NH4+ ions induces stacking of G-quadruplexes via their 5'-end G-quartets, leading to the formation of stable higher-ordered species.

Unveiling the structural mechanism of a G-quadruplex pH-Driven switch.

The human telomere oligonucleotide, d[TAGGG(TTAGGG)2TTAGG] (TAGGG), can adopt two distinct 2-G-quartet G-quadruplex structures at pH 7.0 and 5.0, referred to as the TD and KDH+ forms, respectively. By using a combination of NMR and computational techniques, we determined high-resolution structures of both forms, which revealed unique loop architectures, base triples, and base pairs that play a crucial role in the pH-driven structural transformation of TAGGG. Our study demonstrated that TAGGG represents a reversible pH-driven switch system where the stability and pH-induced structural transformation of the G-quadruplexes are influenced by the terminal residues and base triples. Gaining insight into the factors that regulate the formation of G-quadruplexes and their pH-sensitive structural equilibrium holds great potential for the rational design of novel DNA based pH-driven switches. These advancements in understanding create exciting opportunities for applications in the field of nanotechnology, specifically in the development of bio-nano-motors.

An interaction network in Bacillus subtilis coproporphyrinogen oxidase is essential for the oxidation of protoporphyrinogen IX.

Coproporphyrinogen oxidase (CPO) plays important role in the biosynthesis of heme by catalyzing the coproporphyrinogen III to coproporphyrin III. However, in earlier research, it was regarded as the protoporphyrinogen oxidase (PPO) because it can also catalyze the oxidation of protoporphyrinogen IX to protoporphyrin IX. Identification of the commonalities in CPO and PPO would help us to get a further understanding of the enzyme function. In this work, we explored the role of a non-conserved residue, Asp65 in Bacillus subtilis CPO (bsCPO), whose corresponding residues in PPO from various species are neutral or positive residue (arginine in human PPO or asparagine in tobacco PPO, etc.). We found that Asp65 performs its function by forming a polar interaction network with its surrounding residues in bsCPO, which is important for enzymatic activity. This polar network maintains the substrate binding chamber and stabilizes the micro-environment of the isoalloxazine ring of FAD for the substrate-FAD interaction. Both the comparison of the crystal structures of bsCPO with PPO and our previous work showed that a similar polar interaction network is also present in PPOs. The results confirmed our conjecture that non-conserved residues can form a conserved element to maintain the function of CPO or PPO.

Discovery of (5-(Benzylthio)-4-(3-(trifluoromethyl)phenyl)-4H-1,2,4-triazol-3-yl) Methanols as Potent Phytoene Desaturase Inhibitors through Virtual Screening and Structure Optimization.

Phytoene desaturase (PDS) is not only an important enzyme in the biosynthesis of carotenoids but also a promising target for herbicide discovery. However, in recent years, no expected PDS inhibitors with new scaffolds have been reported. Hence, a solution for developing PDS inhibitors is to search for new compounds with novel chemotypes based on the PDS protein structure. In this work, we integrated structure-based virtual screening, structure-guided optimization, and biological evaluation to discover some PDS inhibitors with novel chemotypes. It is noteworthy that the highly potent compound 1b, 1-(4-chlorophenyl)-2-((5-(hydroxymethyl)-4-(3-(trifluoromethyl)phenyl)-4H-1,2,4-triazol-3-yl)thio)ethan-1-one, exhibited a broader spectrum of post-emergence herbicidal activity at 375-750 g/ha against six kinds of weeds than the commercial PDS inhibitor diflufenican. Surface plasmon resonance (SPR) assay showed that the affinity of our compound 1b (KD = 65.9 µM) to PDS is slightly weaker but at the same level as diflufenican (KD = 38.3 µM). Meanwhile, determination of the phytoene content and PDS mRNA quantification suggested that 1b could induce PDS mRNA reduction and phytoene accumulation. Moreover, 1b also caused the increase of reactive oxygen species (ROS) and the change of ROS-associated enzyme activity in albino leaves. Hence, all these results indicated the feasibility of PDS protein structure-based virtual screen and structure optimization to search for highly potent PDS inhibitors with novel chemotypes for weed control.

Prediction and molecular field view of drug resistance in HIV-1 protease mutants.

Conquering the mutational drug resistance is a great challenge in anti-HIV drug development and therapy. Quantitatively predicting the mutational drug resistance in molecular level and elucidating the three dimensional structure-resistance relationships for anti-HIV drug targets will help to improve the understanding of the drug resistance mechanism and aid the design of resistance evading inhibitors. Here the MB-QSAR (Mutation-dependent Biomacromolecular Quantitative Structure Activity Relationship) method was employed to predict the molecular drug resistance of HIV-1 protease mutants towards six drugs, and to depict the structure resistance relationships in HIV-1 protease mutants. MB-QSAR models were constructed based on a published data set of Ki values for HIV-1 protease mutants against drugs. Reliable MB-QSAR models were achieved and these models display both well internal and external prediction abilities. Interpreting the MB-QSAR models supplied structural information related to the drug resistance as well as the guidance for the design of resistance evading drugs. This work showed that MB-QSAR method can be employed to predict the resistance of HIV-1 protease caused by polymorphic mutations, which offer a fast and accurate method for the prediction of other drug target within the context of 3D structures.

Molecular dynamics analysis of a flexible loop at the binding interface of the SARS-CoV-2 spike protein receptor-binding domain.

Since the identification of the SARS-CoV-2 virus as the causative agent of the current COVID-19 pandemic, considerable effort has been spent characterizing the interaction between the Spike protein receptor-binding domain (RBD) and the human angiotensin converting enzyme 2 (ACE2) receptor. This has provided a detailed picture of the end point structure of the RBD-ACE2 binding event, but what remains to be elucidated is the conformation and dynamics of the RBD prior to its interaction with ACE2. In this work, we utilize molecular dynamics simulations to probe the flexibility and conformational ensemble of the unbound state of the receptor-binding domain from SARS-CoV-2 and SARS-CoV. We have found that the unbound RBD has a localized region of dynamic flexibility in Loop 3 and that mutations identified during the COVID-19 pandemic in Loop 3 do not affect this flexibility. We use a loop-modeling protocol to generate and simulate novel conformations of the CoV2-RBD Loop 3 region that sample conformational space beyond the ACE2 bound crystal structure. This has allowed for the identification of interesting substates of the unbound RBD that are lower energy than the ACE2-bound conformation, and that block key residues along the ACE2 binding interface. These novel unbound substates may represent new targets for therapeutic design.

The hydrogen bonding network involved Arg59 in human protoporphyrinogen IX oxidase is essential for enzyme activity.

Protoporphyrinogen IX oxidase (PPO) is the last common enzyme in chlorophyll and heme biosynthesis pathways. In human, point mutations on PPO are responsible for the dominantly inherited disorder disease, Variegate Porphyria (VP). Of the VP-causing mutation site, the Arg59 is by far the most prevalent VP mutation residue identified. Multiple sequences alignment of PPOs shows that the Arg59 of human PPO (hPPO) is not conserved, and experiments have shown that the equivalent residues in PPO from various species are essential for enzymatic activity. In this work, it was proposed that the Arg59 performs its function by forming a hydrogen-bonding (HB) network around it in hPPO, and we investigated the role of the HB network via site-directed mutagenesis, enzymatic kinetics and computational studies. We found the integrity of the HB network around Arg59 is important for enzyme activity. The HB network maintains the substrate binding chamber by holding the side chain of Arg59, while it stabilizes the micro-environment of the isoalloxazine ring of FAD, which is favorable for the substrate-FAD interaction. Our result provides a new insight to understanding the relationship between the structure and function for hPPO that non-conserved residues can form a conserved element to maintain the function of protein.

NMR unveils an N-terminal interaction interface on acetylated-α-synuclein monomers for recruitment to fibrils.

Amyloid fibril formation of α-synuclein (αS) is associated with multiple neurodegenerative diseases, including Parkinson's disease (PD). Growing evidence suggests that progression of PD is linked to cell-to-cell propagation of αS fibrils, which leads to seeding of endogenous intrinsically disordered monomer via templated elongation and secondary nucleation. A molecular understanding of the seeding mechanism and driving interactions is crucial to inhibit progression of amyloid formation. Here, using relaxation-based solution NMR experiments designed to probe large complexes, we probe weak interactions of intrinsically disordered acetylated-αS (Ac-αS) monomers with seeding-competent Ac-αS fibrils and seeding-incompetent off-pathway oligomers to identify Ac-αS monomer residues at the binding interface. Under conditions that favor fibril elongation, we determine that the first 11 N-terminal residues on the monomer form a common binding site for both fibrils and off-pathway oligomers. Additionally, the presence of off-pathway oligomers within a fibril seeding environment suppresses seeded amyloid formation, as observed through thioflavin-T fluorescence experiments. This highlights that off-pathway αS oligomers can act as an auto-inhibitor against αS fibril elongation. Based on these data taken together with previous results, we propose a model in which Ac-αS monomer recruitment to the fibril is driven by interactions between the intrinsically disordered monomer N terminus and the intrinsically disordered flanking regions (IDR) on the fibril surface. We suggest that this monomer recruitment may play a role in the elongation of amyloid fibrils and highlight the potential of the IDRs of the fibril as important therapeutic targets against seeded amyloid formation.

Pre-folded structures govern folding pathways of human telomeric G-quadruplexes.

Understanding the mechanism by which biological macromolecules fold into their functional native conformations represents a problem of fundamental interest. DNA oligonucleotides derived from human telomeric repeat d[TAGGG(TTAGGG)3] and d[TAGGG(TTAGGG)3TT] fold into G-quadruplexes through diverse steps. Varying the pH and temperature by the use of nuclear magnetic resonance and other methods enabled detection of pre-folded structures that exist in solution before completely formed G-quadruplexes upon addition of cations. Pre-folded structures are in general hard to detect, however their knowledge is crucial to set up folding pathways into final structure since they are believed to be a starting point. Unexpectedly well-defined pre-folded structures composed of base triples for both oligonucleotides were detected at certain pH and temperature. These kinds of structures were up to now only hypothesized as intermediates in the folding process. All revealed pre-folded structures irrespective of the pH and temperature exhibited one common structural feature that could govern folding process.

GC ends control topology of DNA G-quadruplexes and their cation-dependent assembly.

GCn and GCnCG, where n = (G2AG4AG2), fold into well-defined, dimeric G-quadruplexes with unprecedented folding topologies in the presence of Na+ ions as revealed by nuclear magnetic resonance spectroscopy. Both G-quadruplexes exhibit unique combination of structural elements among which are two G-quartets, A(GGGG)A hexad and GCGC-quartet. Detailed structural characterization uncovered the crucial role of 5'-GC ends in formation of GCn and GCnCG G-quadruplexes. Folding in the presence of 15NH4+ and K+ ions leads to 3'-3' stacking of terminal G-quartets of GCn G-quadruplexes, while 3'-GC overhangs in GCnCG prevent dimerization. Results of the present study expand repertoire of possible G-quadruplex structures. This knowledge will be useful in DNA sequence design for nanotechnological applications that may require specific folding topology and multimerization properties.

Understanding and Kinetic Modeling of Complex Degradation Pathways in the Solid Dosage Form: The Case of Saxagliptin.

Drug substance degradation kinetics in solid dosage forms is rarely mechanistically modeled due to several potential micro-environmental and manufacturing related effects that need to be integrated into rate laws. The aim of our work was to construct a model capable of predicting individual degradation product concentrations, taking into account also formulation composition parameters. A comprehensive study was done on active film-coated tablets, manufactured by layering of the drug substance, a primary amine compound saxagliptin, onto inert tablet cores. Formulation variables like polyethylene glycol (PEG) 6000 amount and film-coat polymer composition are incorporated into the model, and are connected to saxagliptin degradation, via formation of reactive impurities. Derived reaction equations are based on mechanisms supported by ab initio calculations of individual reaction activation energies. Alongside temperature, relative humidity, and reactant concentration, the drug substance impurity profile is dependent on micro-environmental pH, altered by formation of acidic PEG degradation products. A consequence of pH lowering, due to formation of formic acid, is lower formation of main saxagliptin degradation product epi-cyclic amidine, a better resistance of formulation to high relative humidity conditions, and satisfactory tablet appearance. Discovered insights enhance the understanding of degradational behavior of similarly composed solid dosage forms on overall drug product quality and may be adopted by pharmaceutical scientists for the design of a stable formulation.

Molecular underpinnings of integrin binding to collagen-mimetic peptides containing vascular Ehlers-Danlos syndrome-associated substitutions.

Collagens carry out critical extracellular matrix (ECM) functions by interacting with numerous cell receptors and ECM components. Single glycine substitutions in collagen III, which predominates in vascular walls, result in vascular Ehlers-Danlos syndrome (vEDS), leading to arterial, uterine, and intestinal rupture and an average life expectancy of <50 years. Collagen interactions with integrin α2ß1 are vital for platelet adhesion and activation; however, how these interactions are impacted by vEDS-associated mutations and by specific amino acid substitutions is unclear. Here, we designed collagen-mimetic peptides (CMPs) with previously reported Gly → Xaa (Xaa = Ala, Arg, or Val) vEDS substitutions within a high-affinity integrin α2ß1-binding motif, GROGER. We used these peptides to investigate, at atomic-level resolution, how these amino acid substitutions affect the collagen III-integrin α2ß1 interaction. Using a multitiered approach combining biological adhesion assays, CD, NMR, and molecular dynamics (MD) simulations, we found that these substitutions differentially impede human mesenchymal stem cell spreading and integrin α2-inserted (α2I) domain binding to the CMPs and were associated with triple-helix destabilization. Although an Ala substitution locally destabilized hydrogen bonding and enhanced mobility, it did not significantly reduce the CMP-integrin interactions. MD simulations suggested that bulkier Gly → Xaa substitutions differentially disrupt the CMP-α2I interaction. The Gly → Arg substitution destabilized CMP-α2I side-chain interactions, and the Gly → Val change broke the essential Mg2+ coordination. The relationship between the loss of functional binding and the type of vEDS substitution provides a foundation for developing potential therapies for managing collagen disorders.

trans-Diastereoselective Ru(II)-Catalyzed Asymmetric Transfer Hydrogenation of α-Acetamido Benzocyclic Ketones via Dynamic Kinetic Resolution.

A highly efficient enantio- and diastereoselective catalyzed asymmetric transfer hydrogenation via dynamic kinetic resolution (DKR-ATH) of α,ß-dehydro-α-acetamido and α-acetamido benzocyclic ketones to ent- trans-ß-amido alcohols is disclosed employing a new ansa-Ru(II) complex of an enantiomerically pure syn- N, N-ligand, i.e. ent- syn-ULTAM-(CH2)3Ph. DFT calculations of the transition state structures revealed an atypical two-pronged substrate attractive stabilization engaging the commonly encountered CH/π electrostatic interaction and a new additional O═S═O···HNAc H-bond hence favoring the trans-configured products.

Effect of Ribose Conformation on RNA Cleavage via Internal Transesterification.

RNA cleavage via internal transesterification is a fundamental reaction involved in RNA processing and metabolism, and the regulation thereof. Herein, the influence of ribose conformation on this reaction was investigated with conformationally constrained ribonucleotides. RNA cleavage rates were found to decrease in the order South-constrained ribonucleotide > native ribonucleotide ≫ North-constrained counterpart, indicating that the ribose conformation plays an important role in modulating RNA cleavage via internal transesterification.

A combined variable temperature 600 MHz NMR/MD study of the calcium release agent cyclic adenosine diphosphate ribose (cADPR): Structure, conformational analysis, and thermodynamics of the conformational equilibria.

A combined variable temperature 600 MHz NMR/molecular dynamics study of the Ca2+-release agent cyclic adenosine 5'-diphosphate ribose (cADPR) was conducted. In addition to elucidating the major and minor orientations of the conformationally flexible furanose rings, γ- (C4'-C5'), and ß- (C5'-O5') bonds, the thermodynamics (ΔHo, ΔSo) associated with each of these conformational equilibria were determined. Both furanose rings were biased towards a south conformation (64-74%) and both ß-bonds heavily favored trans conformations. The R-ring γ-bond was found to exist almost exclusively as the γ+ conformer, whereas the A-ring γ-bond was a mixture of the γ+ and γt conformers, with the trans conformer being slightly favored. Enthalpic factors accounted for most of the observed conformational preferences, although the R-ring furanose exists as its major conformation based solely on entropic factors. There was excellent agreement between the NMR and MD results, particularly with regard to the conformer identities, but the MD showed a bias towards γ+ conformers. The MD results showed that both N-glycosidic χ-bonds are exclusively syn. Collectively the data allowed for the construction of a model for cADPR in which many of the conformationally flexible units in fact effectively adopt single orientations and where most of the conformational diversity resides in its A-ring furanose and γ-bond.

Computational and NMR spectroscopy insights into the conformation of cyclic di-nucleotides.

Cyclic di-nucleotides (CDNs) are second messengers in bacteria and metazoan that are as such controlling important biological processes. Here the conformational space of CDNs was explored systematically by a combination of extensive conformational search and DFT calculations as well as NMR methods. We found that CDNs adopt pre-organized conformations in solution in which the ribose conformations are North type and glycosidic bond conformations are anti type. The overall flexibility of CDNs as well as the backbone torsion angles depend on the cyclization of the phosphodiester bond. Compared to di-nucleotides, CDNs display high rigidity in the macrocyclic moieties. Structural comparison studies demonstrate that the pre-organized conformations of CDNs highly resemble the biologically active conformations. These findings provide information for the design of small molecules to modulate CDNs signalling pathways in bacteria or as vaccine adjuvants. The rigidity of the backbone of CDNs enables the design of high order structures such as molecular cages based on CDNs analogues.

Molecular Simulations Bring New Insights into Protoporphyrinogen IX Oxidase/Protoporphyrinogen IX Interaction Modes.

Protoporphyrinogen IX oxidase (PPO, EC 1.3.3.4) catalyzes the oxidation of protoporphyrinogen IX (protogen IX) to protoporphyrin IX (proto IX) in the haem/chlorophyll biosynthetic pathway. Although extensive studies of PPO have already afforded many insights into its biological function and its significance to agriculture and medicine, details of the enzymatic mechanism as well as the nature of the specific amino acids involved in substrate binding still remain largely unknown due to the lack of structural information about protogen IX binding to PPO. In this study, we carried out a detailed and systematic investigation on the binding mode of protogen IX in the Nicotiana tabacum PPO (mtPPO) by performing a computational docking followed by molecular simulations, quantum mechanics calculations, and an integrated analysis. The proposed binding mode was consistent with experimental studies, and several potential key residues that have not been investigated in previous studies, such as Thr70, Arg233, Ser235, Ser474 and Lys477, were identified. In addition, we compared the binding modes of protogen IX in mtPPO and Homo sapiens PPO, and found their differences. Considering the low sequence identity and the differences of biochemical properties among the PPOs from various species, the investigation could provide a valuable basis for the design of PPO inhibitors with high potency and species-selectivity.