The dynamics of the flavin, NADPH, and active site loops determine the mechanism of activation of class B flavin-dependent monooxygenases.

Flavin-dependent monooxygenases (FMOs) constitute a diverse enzyme family that catalyzes crucial hydroxylation, epoxidation, and Baeyer-Villiger reactions across various metabolic pathways in all domains of life. Due to the intricate nature of this enzyme family's mechanisms, some aspects of their functioning remain unknown. Here, we present the results of molecular dynamics computations, supplemented by a bioinformatics analysis, that clarify the early stages of their catalytic cycle. We have elucidated the intricate binding mechanism of NADPH and L-Orn to a class B monooxygenase, the ornithine hydroxylase from Aspergillus $$ Aspergillus $$ fumigatus $$ fumigatus $$ known as SidA. Our investigation involved a comprehensive characterization of the conformational changes associated with the FAD (Flavin Adenine Dinucleotide) cofactor, transitioning from the out to the in position. Furthermore, we explored the rotational dynamics of the nicotinamide ring of NADPH, shedding light on its role in facilitating FAD reduction, supported by experimental evidence. Finally, we also analyzed the extent of conservation of two Tyr-loops that play critical roles in the process.

Dynamics and Function of sRNA/mRNAs Under the Scrutiny of Computational Simulation Methods.

Molecular dynamics simulations have proved extremely useful in investigating the functioning of proteins with atomic-scale resolution. Many applications to the study of RNA also exist, and their number increases by the day. However, implementing MD simulations for RNA molecules in solution faces challenges that the MD practitioner must be aware of for the appropriate use of this tool. In this chapter, we present the fundamentals of MD simulations, in general, and the peculiarities of RNA simulations, in particular. We discuss the strengths and limitations of the technique and provide examples of its application to elucidate small RNA's performance.

On the Uses of PCA to Characterise Molecular Dynamics Simulations of Biological Macromolecules: Basics and Tips for an Effective Use.

Principal Component Analysis (PCA) is a procedure widely used to examine data collected from molecular dynamics simulations of biological macromolecules. It allows for greatly reducing the dimensionality of their configurational space, facilitating further qualitative and quantitative analysis. Its simplicity and relatively low computational cost explain its extended use. However, a judicious implementation of PCA requires the knowledge of its theoretical grounds as well as its weaknesses and capabilities. In this article, we review these issues and discuss several strategies developed over the last years to mitigate the main PCA flaws and enhance the reproducibility of its results.

Recognition and Binding of RsmE to an AGGAC Motif of RsmZ: Insights from Molecular Dynamics Simulations.

CsrA/RsmE is a post-transcriptional regulator protein widely distributed in bacteria. It impedes the expression of target mRNAs by attaching their 5' untranslated region. The translation is restored by small, noncoding RNAs that sequester CsrA/RsmE acting as sponges. In both cases, the protein recognizes and attaches to specific AGGAX and AXGGAX motifs, where X refers to any nucleotide. RsmZ of Pseudomonas protegens is one of these small RNAs. The structures of some of its complexes with RsmE were disclosed a few years ago. We have used umbrella sampling simulations to force the unbinding of RsmE from the AGGAC motif located in the single-stranded region sited between stem loops 2 and 3 of RsmZ. The calculations unveiled the identity of the main residues and nucleotides involved in the process. They also showed that the region adopts a hairpin-like conformation during the initial stages of the binding. The ability to acquire this conformation requires that the region has a length of at least nine nucleotides. Besides, we performed standard molecular dynamics simulations of the isolated fragments, analyzed their typical conformations, and characterized their movements. This analysis revealed that the free molecules oscillate along specific collective coordinates that facilitate the initial stages of the binding. The results strongly suggest that the flexibility of the single-stranded region of RsmZ crucially affects the ability of its binding motif to catch RsmE.

Ion Selectivity in P2X Receptors: A Comparison between hP2X3 and zfP2X4.

Charge discrimination in P2X receptors occurs in two stages. The first stage takes place in the extracellular vestibule. The second one happens as the ions travel across the pore. The search of the amino acids required to achieve these goals has focused on negatively charged residues conserved among the family members. This strategy, however, has afforded baffling results since residues that strongly influence ion selectivity in a given member are not present in others. This finding suggests that alternative family members could achieve the same goal using different molecular approaches. We have compared the mechanisms of charge discrimination in the extracellular vestibule of zebrafish P2X4 (zfP2X4) and human P2X3 (hP2X3), employing molecular dynamics simulations. In particular, we have analyzed how the mutation of residues D59 and D61 of zfP2X4 and residues E46, D53, and E57 of hP2X3 influence ion behavior. The results indicate that both D59 and D61 are required to confer the extracellular vestibule of zfP2X4 a preference for cations. In contrast, the presence of D53 suffices to provide that capacity to hP2X3. We also computed the potentials of mean force for the passage of Na+ and Cl- through the pore of hP2X3. These profiles were compared against those already available for zfP2X4. Altogether, the results provide a detailed description of the mechanisms employed by these receptors to discriminate between cations and anions.

Molecular Dynamics Simulations Unveil the Basis of the Sequential Binding of RsmE to the Noncoding RNA RsmZ.

CsrA/RsmE are dimeric proteins that bind to targeted mRNAs repressing translation. This mechanism modulates several metabolic pathways and allows bacteria to efficiently adjust their responses to environmental changes. In turn, small RNAs (sRNA) such as CsrB or RsmZ, restore translation by sequestering CsrA/RsmE dimers. Thus, these molecules act in tandem as a gene-expression regulatory system. Recently, a combined NMR-EPR approach solved the structure of part of RsmZ of Pseudomonas fluorescens, attached to three RsmE dimers. The study demonstrated that RsmE assembles onto RsmZ following a specific sequential order. The reasons underlying this peculiar behavior are still unclear. Here, we present a molecular dynamics analysis that explores the conformational diversity of RsmZ and RsmZ-RsmE complexes. The results reveal a clear pattern regarding the exposure of the alternative GGA binding motifs of RsmZ. This pattern is tuned by the attachment of RsmE dimers. Altogether, the observations provide a simple and convincing explanation for the order observed in the sequestration of RsmE dimers. Typical structures for RsmZ and RsmZ-RsmE complexes have been identified. Their characteristics concerning the exposure of the GGA sequences are presented and their most significant interactions are described.

Molecular diagnostic challenges for non-retinal developmental eye disorders in the United Kingdom.

Overall, approximately one-quarter of patients with genetic eye diseases will receive a molecular diagnosis. Patients with developmental eye disorders face a number of diagnostic challenges including phenotypic heterogeneity with significant asymmetry, coexisting ocular and systemic disease, limited understanding of human eye development and the associated genetic repertoire, and lack of access to next generation sequencing as regarded not to impact on patient outcomes/management with cost implications. Herein, we report our real world experience from a pediatric ocular genetics service over a 12 month period with 72 consecutive patients from 62 families, and that from a cohort of 322 patients undergoing whole genome sequencing (WGS) through the Genomics England 100,000 Genomes Project; encompassing microphthalmia, anophthalmia, ocular coloboma (MAC), anterior segment dysgenesis anomalies (ASDA), primary congenital glaucoma, congenital cataract, infantile nystagmus, and albinism. Overall molecular diagnostic rates reached 24.9% for those recruited to the 100,000 Genomes Project (73/293 families were solved), but up to 33.9% in the clinic setting (20/59 families). WGS was able to improve genetic diagnosis for MAC patients (15.7%), but not for ASDA (15.0%) and congenital cataracts (44.7%). Increased sample sizes and accurate human phenotype ontology (HPO) terms are required to improve diagnostic accuracy. The significant mixed complex ocular phenotypes distort these rates and lead to missed variants if the correct gene panel is not applied. Increased molecular diagnoses will help to explain the genotype-phenotype relationships of these developmental eye disorders. In turn, this will lead to improved integrated care pathways, understanding of disease, and future therapeutic development.

Positively Charged Residues in the Head Domain of P2X4 Receptors Assist the Binding of ATP.

P2X receptors are a family of trimeric cationic channels located in the membrane of mammalian cells. They open in response to the binding of ATP. The differences between the closed and open structures have been described in detail for some members of the family. However, the order in which the conformational changes take place as ATP enters the binding cleft, and the residues involved in the intermediate stages, are still unknown. Here, we present the results of umbrella sampling simulations aimed to elucidate the sequence of conformational changes that occur during the reversible binding of ATP to the P2X4 receptor. The simulations also provided information about the interactions that develop in the course of the process. In particular, they revealed the existence of a metastable state which assists the binding. This state is stabilized by positively charged residues located in the head domain of the receptor. Based on these findings, we propose a novel mechanism for the capture of ATP by P2X4 receptors.

A Quasi-Classical Evaluation of the J-Shifting Approximation for the Reactive Cross Sections of F + CHD3 and F + CH4.

We evaluated the accuracy of the J-shifting approximation to estimate reactant state-selected cross sections for the F+CH4 → HF+CH3 and F+CHD3 → HF+CD3/DF+CHD2 reactions. In particular, we analyzed how the rotational state of methane influences the quality of the approximation. The systems were considered in full dimensionality. Since full-quantum scattering calculations are still unfeasible for these reactions, we employed quasi-classical trajectories (QCT) to calculate the cross sections. The characteristics of the Born-Oppenheimer potential energy surface of these reactions pose a great challenge to the assumptions of the J-shifting approach. In spite of this, we found that it performs well for both reactions if the methane molecule is in the rotational ground state. However, when methane is rotationally excited, the approach affords good results for the F+CH4 system but clearly fails for F+CHD3. The reasons for this failure will be discussed, and a simple procedure to recover good estimators for the cross sections from J = 0 calculations will be introduced.

Molecular Dynamics Simulations of Substrate Release from Trypanosoma cruzi UDP-Galactopyranose Mutase.

The enzyme UDP-galactopyranose mutase (UGM) represents a promising drug target for the treatment of infections with Trypanosoma cruzi. We have computed the Potential of Mean Force for the release of UDP-galactopyranose from UGM, using Umbrella Sampling simulations. The simulations revealed the conformational changes that both substrate and enzyme undergo during the process. It was determined that the galactopyranose portion of the substrate is highly mobile and that the opening/closing of the active site occurs in stages. Previously uncharacterized interactions with highly conserved residues were also identified. These findings provide new pieces of information that contribute to the rational design of drugs against T. cruzi.

Charge Discrimination in P2X4 Receptors Occurs in Two Consecutive Stages.

P2X receptors are a group of trimeric cationic channels that are activated by adenosine 5'-triphosphate. They perform critical roles in the membranes of mammalian cells, and their improper functioning is associated with numerous diseases. Despite the vast amount of research devoted to them, several aspects of their operation are currently unclear, including the causes of their charge selectivity. We present the results of molecular dynamics simulation, which shed light on this issue for the case of P2X4 channels. We examined in detail the behavior of Na+ and Cl- ions inside the receptor. The examination reveals that charge discrimination occurs in two stages. First, cations bear precedence over anions to enter the extracellular vestibule. Then, cations at the extracellular vestibule are more likely to cross the pore than anions in an equivalent position. In this manner, a thorough but straightforward analysis of computational simulations suggests a stepwise mechanism, without a unique determinant factor.

Non-adiabatic effects in F + CHD3 reactive scattering.

The effect of non-adiabatic transitions on the F(2P) + CHD3(ν1) → DF + CHD2 and F(2P) + CHD3(ν1) → HF + CD3 reactions is investigated. The dynamics of the nuclei was simulated using trajectory surface hopping and a vibronically and spin-orbit coupled diabatic potential energy matrix. To facilitate the calculations, the fewest switching algorithm of Tully was adapted to the use of a complex diabatic potential energy matrix. For reactions of CHD3 with ground state fluorine atoms, F(2P3/2), the ratio between the previously computed adiabatic cross sections and the non-adiabatic ones was found to range from 1.4 to 2.1. The actual ratio depends on the translational energy and the initial vibrational state of CHD3. The total reactivity of CHD3(ν1 = 1) was found to be always larger than that of CHD3(ν1=0) mainly because of the increase in the cross sections for the HF + CD3 channel. Thus, the inclusion of non-adiabatic transitions in the theoretical treatment cannot resolve the existing disagreement between theory and experiment. Cross sections for the reaction of CHD3 with spin-orbit excited fluorine atoms, F(2P1/2), were found to be significantly smaller than the ones for reaction with F(2P3/2).

Consistent Principal Component Modes from Molecular Dynamics Simulations of Proteins.

Principal component analysis is a technique widely used for studying the movements of proteins using data collected from molecular dynamics simulations. In spite of its extensive use, the technique has a serious drawback: equivalent simulations do not afford the same PC-modes. In this article, we show that concatenating equivalent trajectories and calculating the PC-modes from the concatenated one significantly enhances the reproducibility of the results. Moreover, the consistency of the modes can be systematically improved by adding more individual trajectories to the concatenated one.

The Dynamic Behavior of the P2X4 Ion Channel in the Closed Conformation.

We present the results of a detailed molecular dynamics study of the closed form of the P2X4 receptor. The fluctuations observed in the simulations were compared with the changes that occur in the transition from the closed to the open structure. To get further insight on the opening mechanism, the actual displacements were decomposed into interchain motions and intrachain deformations. This analysis revealed that the iris-like expansion of the transmembrane helices mainly results from interchain motions that already take place in the closed conformation. However, these movements cannot reach the amplitude required for the opening of the channel because they are impeded by interactions occurring around the ATP binding pocket. This suggests that the union of ATP produces distortions in the chains that eliminate the restrictions on the interchain displacements, leading to the opening of the pore.

Communication: Mode specific quantum dynamics of the F + CHD3 → HF + CD3 reaction.

The mode specific reactivity of the F + CHD3 → HF + CD3 reaction is investigated using an eight-dimensional quantum dynamical model on a recently developed ab initio based full-dimensional potential energy surface. Our results indicate prominent resonance structures at low collision energies and absence of an energy threshold in reaction probabilities. It was also found that excitation of the C-D stretching or CD3 umbrella mode has a relatively small impact on reactivity. On the other hand, the excitation of the C-H vibration (v1) in CHD3 is shown to significantly increase the reactivity, which, like several recent quasi-classical trajectory studies, is at odds with the available experimental data. Possible sources of the disagreement are discussed.

A Quasiclassical Study of the F((2)P) + CHD3 (ν1 = 0,1) Reactive System on an Accurate Potential Energy Surface.

Quasiclassical trajectories (QCT) have been employed to elucidate the effect of exciting the C-H bond in F + CHD3 collisions. The calculations were performed on a new potential energy surface that accurately describes the van der Waals complexes in the entrance channel of the reaction. It was found that exciting the C-H bond significantly enhances the yield of HF + CD3, whereas it has a minor effect on the production of DF + CHD2. Therefore, the net effect is that the total reactivity increases upon excitation. This result strongly contradicts recent experimental findings. Significant differences in regard to the yield of each product channel were also found between QCT results calculated with the new surface and those obtained with the surface previously developed by Czakó et al. This shows that relatively small variations in the topography of the entrance channel can result in huge discrepancies in the predicted DF/HF branching ratio. However, in regard to other attributes of the reaction, the agreement between QCT results computed with different surfaces, and between them and experimental results, is good. For the F + CHD3 → HF + CD3 reaction, at a collisional energy of 9.0 kcal/mol, experiments and QCT calculations agree, indicating that the extra energy deposited in the C-H bond is channelled into the HF product. In addition, the angular distribution of CD3 is backward oriented and is not sensitive to the excitation of the C-H bond.

New insights into the meaning and usefulness of principal component analysis of concatenated trajectories.

A comparison between different conformations of a given protein, relating both structure and dynamics, can be performed in terms of combined principal component analysis (combined-PCA). To that end, a trajectory is obtained by concatenating molecular dynamics trajectories of the individual conformations under comparison. Then, the principal components are calculated by diagonalizing the correlation matrix of the concatenated trajectory. Since the introduction of this approach in 1995 it has had a large number of applications. However, the interpretation of the eigenvectors and eigenvalues so obtained is based on intuitive foundations, because analytical expressions relating the concatenated correlation matrix with those of the individual trajectories under consideration have not been provided yet. In this article, we present such expressions for the cases of two, three, and an arbitrary number of concatenated trajectories. The formulas are simple and show what is to be expected and what is not to be expected from a combined-PCA. Their correctness and usefulness is demonstrated by discussing some representative examples. The results can be summarized in a simple sentence: the correlation matrix of a concatenated trajectory is given by the average of the individual correlation matrices plus the correlation matrix of the individual averages. From this it follows that the combined-PCA of trajectories belonging to different free energy basins provides information that could also be obtained by alternative and more straightforward means.

QM/MM molecular dynamics study of the galactopyranose → galactofuranose reaction catalysed by Trypanosoma cruzi UDP-galactopyranose mutase.

The enzyme UDP-Galactopyranose Mutase (UGM) catalyses the conversion of galactopyranose into galactofuranose. It is known to be critical for the survival and proliferation of several pathogenic agents, both prokaryotic and eukaryotic. Among them is Trypanosoma cruzi, the parasite responsible for Chagas' disease. Since the enzyme is not present in mammals, it appears as a promising target for the design of drugs to treat this illness. A precise knowledge of the mechanism of the catalysed reaction would be crucial to assist in such design. In this article we present a detailed study of all the putative steps of the mechanism. The study is based on QM/MM free energy calculations along properly selected reaction coordinates, and on the analysis of the main structural changes and interactions taking place at every step. The results are discussed in connection with the experimental evidence and previous theoretical studies.

Resonances in the entrance channel of the elementary chemical reaction of fluorine and methane.

Extending the fully quantum-state-resolved description of elementary chemical reactions beyond three or four atom systems is a crucial issue in fundamental chemical research. Reactions of methane with F, Cl, H or O are key examples that have been studied prominently. In particular, reactive resonances and nonintuitive mode-selective chemistry have been reported in experimental studies for the F+CH4 →HF+CH3 reaction. By investigating this reaction using transition-state spectroscopy, this joint theoretical and experimental study provides a clear picture of resonances in the F+CH4 system. This picture is deduced from high-resolution slow electron velocity-map imaging (SEVI) spectra and accurate full-dimensional (12D) quantum dynamics simulations in the picosecond regime.

Free-energy computations identify the mutations required to confer trans-sialidase activity into Trypanosoma rangeli sialidase.

Trypanosoma rangeli's sialidase (TrSA) and Trypanosoma cruzi's trans-sialidase (TcTS) are members of the glycoside hydrolase family 33 (GH-33). They share 70% of sequence identity and their crystallographic Cα RMSD is 0.59 Å. Despite these similarities they catalyze different reactions. TcTS transfers sialic acid between glycoconjugates while TrSA can only cleave sialic acid from sialyl-glyconjugates. Significant effort has been invested into unraveling the differences between TrSA and TcTS, and into conferring TrSA with trans-sialidase activity through appropriate point mutations. Recently, we calculated the free-energy change for the formation of the covalent intermediate (CI) in TcTS and performed an energy decomposition analysis of that process. In this article we present a similar study for the formation of the CI in TrSA, as well as in a quintuple mutant (TrSA5mut), which has faint trans-sialidase activity. The comparison of these new results with those previously obtained for TcTS allowed identifying five extra mutations to be introduced in TrSA5mut that should create a mutant (TrSA10mut ) with high trans-sialidase activity.