Exploring Reactive Conformations of Coenzyme A during Binding and Unbinding to Pyruvate Formate-Lyase.

Pyruvate formate-lyase (PFL) is a glycyl radical enzyme that converts pyruvate and coenzyme A (CoA) into formate and acetyl-CoA in two half-reactions. Recently, we showed that the acetylation of the PFL active site in the first half-reaction induces subtle conformational changes, leading to the opening of a potential channel for CoA entry. Entry of CoA into the active site is crucial for the second half-reaction, involving the acetyl transfer to CoA, and the completion of the catalytic cycle.

Sharing Data from Molecular Simulations.

Given the need for modern researchers to produce open, reproducible scientific output, the lack of standards and best practices for sharing data and workflows used to produce and analyze molecular dynamics (MD) simulations has become an important issue in the field. There are now multiple well-established packages to perform molecular dynamics simulations, often highly tuned for exploiting specific classes of hardware, each with strong communities surrounding them, but with very limited interoperability/transferability options. Thus, the choice of the software package often dictates the workflow for both simulation production and analysis.

The Influence of Chemical Change on Protein Dynamics: A Case Study with Pyruvate Formate-Lyase.

Pyruvate formate-lyase (PFL) catalyzes the reversible conversion of pyruvate and coenzyme A (CoA) into formate and acetyl-CoA in two half-reactions. For the second half-reaction to take place, the S-H group of CoA must enter the active site of the enzyme to retrieve a protein-bound acetyl group. However, CoA is bound at the protein surface, whereas the active site is buried in the protein interior, some 20-30 Å away.

The reliability of molecular dynamics simulations of the multidrug transporter P-glycoprotein in a membrane environment.

Despite decades of research, the mechanism of action of the ABC multidrug transporter P-glycoprotein (P-gp) remains elusive. Due to experimental limitations, many researchers have turned to molecular dynamics simulation studies in order to investigate different aspects of P-gp function. However, such studies are challenging and caution is required when interpreting the results.

Structure of a lipid A phosphoethanolamine transferase suggests how conformational changes govern substrate binding.

Multidrug-resistant (MDR) gram-negative bacteria have increased the prevalence of fatal sepsis in modern times. Colistin is a cationic antimicrobial peptide (CAMP) antibiotic that permeabilizes the bacterial outer membrane (OM) and has been used to treat these infections. The OM outer leaflet is comprised of endotoxin containing lipid A, which can be modified to increase resistance to CAMPs and prevent clearance by the innate immune response.

Hydrophobic Shielding Drives Catalysis of Hydride Transfer in a Family of FH-Dependent Enzymes.

A family of flavin/deazaflavin-dependent oxidoreductases (FDORs) from mycobacteria has been recently characterized and found to play a variety of catalytic roles, including the activation of prodrugs such as the candidate anti-tuberculosis drug pretomanid (PA-824). However, our understanding of the catalytic mechanism used by these enzymes is relatively limited. To address this, we have used a combination of quantum mechanics and molecular dynamics calculations to study the catalytic mechanism of the activation of pretomanid by the deazaflavin-dependent nitroreductase (Ddn) from Mycobacterium tuberculosis.

Rv2074 is a novel F420 H2 -dependent biliverdin reductase in Mycobacterium tuberculosis.

Bilirubin is a potent antioxidant that is produced from the reduction of the heme degradation product biliverdin. In mammalian cells and Cyanobacteria, NADH/NADPH-dependent biliverdin reductases (BVRs) of the Rossmann-fold have been shown to catalyze this reaction. Here, we describe the characterization of Rv2074 from Mycobacterium tuberculosis, which belongs to a structurally and mechanistically distinct family of F420 H2 -dependent BVRs (F-BVRs) that are exclusively found in Actinobacteria.

Structural and dynamic perspectives on the promiscuous transport activity of P-glycoprotein.

The multidrug transporter P-glycoprotein (P-gp) is expressed in the blood-brain barrier endothelium where it effluxes a range of drug substrates, preventing their accumulation within the brain. P-gp has been studied extensively for 40 years because of its crucial role in the absorption, distribution, metabolism and elimination of a range of pharmaceutical compounds. Despite this, many aspects of the structure-function mechanism of P-gp are unresolved.

Identification of Possible Binding Sites for Morphine and Nicardipine on the Multidrug Transporter P-Glycoprotein Using Umbrella Sampling Techniques.

The multidrug transporter P-glycoprotein (P-gp) is central to the development of multidrug resistance in cancer. While residues essential for transport and binding have been identified, the location, composition, and specificity of potential drug binding sites are uncertain. Here molecular dynamics simulations are used to calculate the free energy profile for the binding of morphine and nicardipine to P-gp.

Calculation of the CD Spectrum of a Peptide from Its Conformational Phase Space: The Case of Met-enkephalin and Its Unnatural Analogue.

We have investigated the conformational phase spaces of both Met-enkephalin and Ada-enkephalin in 2,2,2-trifluoroethanol in order to connect them to their respective CD spectra. To this end, we have characterized the conformational preferences of the zwitterionic and neutral forms of Met-enkephalin and of both the R- and the S-epimers of Ada-enkephalin, as obtained by classical molecular dynamics. The CD spectrum for each peptide was subsequently obtained with a procedure of successive averaging, which accounts for the behavior of the solvent, the side chains, and the backbone variations of the peptides.

The Protonation States of the Active-Site Histidines in (6-4) Photolyase.

The active sites of the (6-4) photolyases contain two conserved histidine residues, which, in the Drosophila melanogaster enzyme, correspond to His365 and His369. While there are nine combinations in which the three possible protonation states of the two histidines (with protons on Nδ (HID), Nε (HIE), or both Nδ and Nε (HIP)) can be paired, there is presently no consensus as to which of these states is present, let alone mechanistically relevant. EPR hyperfine couplings for selected protons of the FADH(•) radical have previously been used to address this issue.

A compound QM/MM procedure: comparative performance on a pyruvate formate-lyase model system.

We present an ONIOM(G3:MM) method as an example of a technique capable of producing chemical accuracy in the quantum mechanical (QM) treatment with a molecular mechanical description context. By applying the method to small model systems, in which we are also able to calculate the pure QM G3-type results, it is possible to establish the reliability of the method as it applies to evaluating reaction mechanisms. By choosing small model systems that are relevant to the substrate mechanism of pyruvate formate-lyase, we are also able to discuss the inhibitory effect of oxamate and the relevance of an alternative H-abstraction mechanism in that context.

On the modeling of arginine-bound carboxylates: a case study with Pyruvate Formate-Lyase.

High level ab initio and density functional calculations have been employed to determine the most appropriate manner in which to truncate an arginine-bound carboxylate motif, using the substrate mechanism of Pyruvate Formate-Lyase as a case study. The results show that, both qualitatively and quantitatively, a neutral carboxylic acid provides a more realistic approximation to the salt bridge arrangement than does a bare anionic carboxylate substituent.