Elucidation of critical pH-dependent structural changes in Botulinum Neurotoxin E

Botulinum Neurotoxins (BoNT) are the most potent toxins currently known. However, they also have therapeutic applications for an increasing number of motor related conditions due to their specificity, and low diffusion into the system. Although the start- and end- points for the BoNT mechanism of action are well-studied, a critical step remains poorly understood. It is theorised that BoNTs undergo a pH-triggered conformational shift, activating the neurotoxin by priming it to form a transmembrane (TM) channel. To test this hypothesis, we combined molecular dynamic (MD) simulations and small-angle x-ray scattering (SAXS), revealing a new conformation of BoNT/E. This conformation was exclusively observed in simulations below pH 5.5, as determined by principal component analysis (PCA), and its theoretical SAXS profile matched an experimental SAXS profile obtained at pH 4. Additionally, a localised secondary structural change was observed in MD simulations below pH 5.5, in a region previously identified as instrumental for membrane insertion for BoNT/A. These changes were found at a critical pH value for BoNTs in vivo, and may be relevant for their therapeutic use

Structural Studies of pH Effects on Botulinum Toxins A & E

Botulinum neurotoxins (BoNTs) are responsible for botulism, a paralytic disease which can be lethal if not treated in time. They act by entering neurons and targeting the SNARE proteins (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor), which in turn blocks neurotransmission. However, these toxins can be repurposed for therapeutic use to treat a large number of conditions. The most studied serotypes are A and E (BoNT/A and BoNT/E, respectively), with notable differences in duration of action and domain spatial organisation. It has been shown that these toxins only exert their activity if the pH drops to 5 or lower, but it is unclear what effect the pH environment has on the toxin which drives this. Currently, the only available structural information on BoNTs is from X-ray crystallography which fixes the protein into a rigid crystal lattice. This gives limited information on its flexible regions, and no information about its dynamics and solution behaviour. To gain insight into this, molecular dynamic (MD) simulations were conducted under varying pH conditions. For BoNT/E, these simulations revealed a shift in conformational populations in solvated systems at pH ≤ 5 when compared to simulations at pH > 5, with the protein adopting a more extended conformation in the former. This was confirmed by analytical ultra-centrifugation (AUC), while small-angle X-ray scattering (SAXS) validated the two major conformations observed in the MD simulations. For BoNT/A, a major conformational change was not observed, but a rare event was identified by MD (in 0.014% of frames studied) which may explain the longer onset of action compared to BoNT/E. Another key difference between the two structures of BoNT/E and BoNT/A is the large number of contacts between a conserved region termed the “switch” and the binding domain (BD) in BoNT/A, which are absent in BoNT/E

Development of novel computational methods suitable for modelling intrinsically disordered proteins

PhD ThesisProteins without a stable tertiary structure are known as intrinsically disordered or metamorphic. These proteins denoted as IDPs – or protein domains denoted as IDRs – exert crucial roles in cellular signalling, growth and molecular recognition events. Due to their high plasticity, IDPs and IDRs are very challenging for experimental and computational structural studies. To enable these, all-atom molecular dynamics (MD) simulations are used, as they provide insight into structure and dynamics at the atomistic level of detail. However, the current generalist physical models (protein force fields and solvent models) used in MD simulations are unable to generate satisfactory ensembles for IDPs/IDRs when compared to existing experimental data. This work aimed to improve on the state-of-the-art accuracy for simulations of IDPs/IDRs without sacrificing accuracy for folded domains. Herein, the accuracy of several different force fields frequently used for simulations of proteins was compared, in simulations of both ordered and disordered systems. The results showed that each force field has strengths and limitations. Given the fact that interactions with the solvent are pivotal for accurate simulations of intrinsically disordered proteins, a novel solvation model was developed, denoted as Charge-Augmented 3 Point water model for Intrinsically disordered Proteins (CAIPi3P). CAIPi3P model was generated through systematic scanning of the dipole moment values calculated for the popular TIP3P three-point water model. By increasing the dipole magnitude, the agreement between experimental and calculated small-angle X-ray scattering (SAXS) curves was massively improved for a series of model IDRs. To further improve the simulations of proteins containing IDRs, a novel method to assemble force field parameters has been developed. Denoted as Hybrid_FF, it merges parameters from different established force-fields, performing well for structured and disordered regions (AMBER99SB-ILDN and AMBER03ws, respectively), parametrising each secondary structure differently. Testing these joint parameters for a series of IDR-containing proteins showed that such an approach improved the accuracy of the sampled configurations for long disordered regions. Finally, a software to estimate and analyse the transition dynamics of intrinsically disordered regions has been developed in this work. Named structural quantifier of entropy (SQuE), it uses a first-order approximation to the probability distribution to assess the structural entropy for protein transitions barriers. It is expected that tools developed in this study will generate more accurate IDP/IDR ensembles, broadening the range of biologically relevant systems amenable to atomistic molecular dynamics simulations

Structural and Functional Studies on BK(Ca) Channels

The long term goal of this research is to study the structure and function of the BKCa channels, by focusing on the effect of a single residue mutation, the epilepsy mutation. BKCa channels are potassium channels, activated by voltage, Ca2+ and Mg2+ ions. These factors control the opening and closing of the channel pore and thus regulate the large K+ current passing through them. Recently, a mutation D434G in humans, was found to make the channel hyperactive and more sensitive to the Ca2+ ions. The single residue mutation, resulting from a substitution of an Asp to Gly, was found to be linked with epilepsy and paroxysmal dyskinesia. The central focus of this thesis is to identify the molecular mechanism behind the structural and functional changes caused by this mutation. Using comparative modeling and molecular dynamics simulations, it is revealed that the epilepsy mutation reduces the flexibility of the channel protein and drives it to a rigid conformation. The loss in dynamics is seen around the Ca2+ binding site which reflects its direct impact on the Ca2+ activation of the channel. Comparison with experimental results show that the change in dynamics is targeted to regions which possibly connects the Ca2+ –binding site to the pore and thus transfer this effect to the pore. The thesis also presents a new method of representation of cations in computational techniques, the multisite cation model. The model presents improvement in the reproduction of accurate structural and thermodynamical properties of ion–mediated mechanisms. The successful implementation of the model in protein and water systems show that the model will prove very useful in increasing the accuracy and precision of metal mediated simulations and energy calculations

Protein Flexibility in Structure-Based Drug Design: Method Development and Novel Mechanisms for Inhibiting HIV-1 Protease.

Structure-based drug design (SBDD) has emerged as an important tool in drug discovery research. Traditionally, SBDD is based on a static crystal structure of the target protein. However, a protein in solution exists as an ensemble of energetically accessible conformations and is best described when all states are represented. Upon ligand binding, further conformational changes in the receptor can be induced. While ligand flexibility can be accurately reproduced, replicating the innumerable degrees of freedom of the protein is impractical due to limitations in computational power. Previously, Carlson et al. developed a robust method to generate receptor-based pharmacophore models based on an ensemble of protein conformations. The use of multiple protein structures (MPS) allows a range of conformational space that can be assumed by the protein to be sampled and hence, simulates the inherent flexibility of a binding site in a computationally feasible manner. Small molecule probes are used to map energetically favorable regions of each protein active site, and the MPS are then overlaid to identify the most important, chemically relevant features conserved across the conformations. Here, we have refined the MPS method by developing techniques to optimize different steps in the procedure. First, we outline tools to properly overlay flexible proteins based on the rigid regions of the structure by incorporating a Gaussian weight into a standard RMSD alignment. Atoms that barely move between the two conformations will have a greater weighting than those that have a large displacement. Using HIV-1 protease (HIV-1p) as a test case, we next examine the use of various sources of MPS: snapshots of an apo structure across a molecular dynamics simulation, a bound NMR ensemble, and a collection of bound crystal structures. Finally, we implement a simple ranking metric into the MPS method to quantify ligand overlap with a contour-based representation of the pharmacophore model. Overlapping in a region of the active site dense with pharmacophore spheres results in a higher ranking of a ligand pose. The refined MPS method and other computational techniques are then applied to study HIV-1p and investigate a novel inhibition mechanism by modulating its conformational behavior.Ph.D.Medicinal ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/57666/2/kdamm_1.pd

A Commemorative Issue in Honor of Professor Nick Hadjiliadis: Metal Complex Interactions with Nucleic Acids and/or DNA

This Special Issue of the International Journal of Molecular Science comprises a comprehensive study on “Metal Complex Interactions with Nucleic Acids and/or DNA”. This Special Issue has been inspired by the important contribution of Prof. Nick Hadjiliadis to the field of palladium or/and platinum/nucleic acid interactions. It covers a selection of recent research and review articles in the field of metal complex interactions with nucleic acids and/or DNA. Moreover, this Special Issue on "Metal Complexes Interactions with Nucleic Acids and/or DNA" provides an overview of this increasingly diverse field, presenting recent developments and the latest research with particular emphasis on metal-based drugs and metal ion toxicity