Conformational Space of the Translocation Domain of Botulinum Toxin: Atomistic Modeling and Mesoscopic Description of the Coiled-Coil Helix Bundle.

The toxicity of botulinum multi-domain neurotoxins (BoNTs) arises from a sequence of molecular events, in which the translocation of the catalytic domain through the membrane of a neurotransmitter vesicle plays a key role. A recent structural study of the translocation domain of BoNTs suggests that the interaction with the membrane is driven by the transition of an α helical switch towards a ß hairpin. Atomistic simulations in conjunction with the mesoscopic Twister model are used to investigate the consequences of this proposition for the toxin-membrane interaction. The conformational mobilities of the domain, as well as the effect of the membrane, implicitly examined by comparing water and water-ethanol solvents, lead to the conclusion that the transition of the switch modifies the internal dynamics and the effect of membrane hydrophobicity on the whole protein. The central two α helices, helix 1 and helix 2, forming two coiled-coil motifs, are analyzed using the Twister model, in which the initial deformation of the membrane by the protein is caused by the presence of local torques arising from asymmetric positions of hydrophobic residues. Different torque distributions are observed depending on the switch conformations and permit an origin for the mechanism opening the membrane to be proposed.

New Crystal Form of Human Neuropilin-1 b1 Fragment with Six Electrostatic Mutations Complexed with KDKPPR Peptide Ligand.

Neuropilin 1 (NRP1), a cell-surface co-receptor of a number of growth factors and other signaling molecules, has long been the focus of attention due to its association with the development and the progression of several types of cancer. For example, the KDKPPR peptide has recently been combined with a photosensitizer and a contrast agent to bind NRP1 for the detection and treatment by photodynamic therapy of glioblastoma, an aggressive brain cancer. The main therapeutic target is a pocket of the fragment b1 of NRP1 (NRP1-b1), in which vascular endothelial growth factors (VEGFs) bind. In the crystal packing of native human NRP1-b1, the VEGF-binding site is obstructed by a crystallographic symmetry neighbor protein, which prevents the binding of ligands. Six charged amino acids located at the protein surface were mutated to allow the protein to form a new crystal packing. The structure of the mutated fragment b1 complexed with the KDKPPR peptide was determined by X-ray crystallography. The variant crystallized in a new crystal form with the VEGF-binding cleft exposed to the solvent and, as expected, filled by the C-terminal moiety of the peptide. The atomic interactions were analyzed using new approaches based on a multipolar electron density model. Among other things, these methods indicated the role played by Asp320 and Glu348 in the electrostatic steering of the ligand in its binding site. Molecular dynamics simulations were carried out to further analyze the peptide binding and motion of the wild-type and mutant proteins. The simulations revealed that specific loops interacting with the peptide exhibited mobility in both the unbound and bound forms.

Low-resolution description of the conformational space for intrinsically disordered proteins.

Intrinsically disordered proteins (IDP) are at the center of numerous biological processes, and attract consequently extreme interest in structural biology. Numerous approaches have been developed for generating sets of IDP conformations verifying a given set of experimental measurements. We propose here to perform a systematic enumeration of protein conformations, carried out using the TAiBP approach based on distance geometry. This enumeration was performed on two proteins, Sic1 and pSic1, corresponding to unphosphorylated and phosphorylated states of an IDP. The relative populations of the obtained conformations were then obtained by fitting SAXS curves as well as Ramachandran probability maps, the original finite mixture approach RamaMix being developed for this second task. The similarity between profiles of local gyration radii provides to a certain extent a converged view of the Sic1 and pSic1 conformational space. Profiles and populations are thus proposed for describing IDP conformations. Different variations of the resulting gyration radius between phosphorylated and unphosphorylated states are observed, depending on the set of enumerated conformations as well as on the methods used for obtaining the populations.

In Silico Conformational Features of Botulinum Toxins A1 and E1 According to Intraluminal Acidification.

Although botulinum neurotoxins (BoNTs) are among the most toxic compounds found in nature, their molecular mechanism of action is far from being elucidated. A key event is the conformational transition due to acidification of the interior of synaptic vesicles, leading to translocation of the BoNT catalytic domain into the neuronal cytosol. To investigate these conformational variations, homology modeling and atomistic simulations are combined to explore the internal dynamics of the sub-types BoNT/A1 (the most-used sub-type in medical applications) and BoNT/E1 (the most kinetically efficient sub-type). This first simulation study of di-chain BoNTs in closed and open states considers the effects of both neutral and acidic pH. The conformational mobility is driven by domain displacements of the ganglioside-binding site in the receptor binding domain, the translocation domain (HCNT) switch, and the belt α-helix, which present multiple conformations, depending on the primary sequence and the pH. Fluctuations of the belt α-helix are observed for closed conformations of the toxins and at acidic pH, while patches of more solvent-accessible residues appear under the same conditions in the core translocation domain HCNT. These findings suggest that, during translocation, the higher mobility of the belt could be transmitted to HCNT, leading to the favorable interaction of HCNT residues with the non-polar membrane environment.

Computational and biochemical analysis of type IV pilus dynamics and stability.

Type IV pili (T4P) are distinctive dynamic filaments at the surface of many bacteria that can rapidly extend and retract and withstand strong forces. T4P are important virulence factors in many human pathogens, including Enterohemorrhagic Escherichia coli (EHEC). The structure of the EHEC T4P has been determined by integrating nuclear magnetic resonance (NMR) and cryo-electron microscopy data. To better understand pilus assembly, stability, and function, we performed a total of 108 ms all-atom molecular dynamics simulations of wild-type and mutant T4P. Extensive characterization of the conformational landscape of T4P in different conditions of temperature, pH, and ionic strength is complemented with targeted mutagenesis and biochemical analyses. Our simulations and NMR experiments reveal a conserved set of residues defining a calcium-binding site at the interface between three pilin subunits. Calcium binding enhances T4P stability ex vivo and in vitro, supporting the role of this binding site as a potential pocket for drug design.

Conformational Insights into the Control of CNF1 Toxin Activity by Peptidyl-Prolyl Isomerization: A Molecular Dynamics Perspective.

The cytotoxic necrotizing factor 1 (CNF1) toxin from uropathogenic Escherichia coli constitutively activates Rho GTPases by catalyzing the deamidation of a critical glutamine residue located in the switch II (SWII). In crystallographic structures of the CNF1 catalytic domain (CNF1CD), surface-exposed P768 and P968 peptidyl-prolyl imide bonds (X-Pro) adopt an unusual cis conformation. Here, we show that mutation of each proline residue into glycine abrogates CNF1CD in vitro deamidase activity, while mutant forms of CNF1 remain functional on RhoA in cells. Using molecular dynamics simulations coupled to protein-peptide docking, we highlight the long-distance impact of peptidyl-prolyl cis-trans isomerization on the network of interactions between the loops bordering the entrance of the catalytic cleft. The energetically favorable isomerization of P768 compared with P968, induces an enlargement of loop L1 that fosters the invasion of CNF1CD catalytic cleft by a peptide encompassing SWII of RhoA. The connection of the P968 cis isomer to the catalytic cysteine C866 via a ladder of stacking interactions is alleviated along the cis-trans isomerization. Finally, the cis-trans conversion of P768 favors a switch of the thiol side chain of C866 from a resting to an active orientation. The long-distance impact of peptidyl-prolyl cis-trans isomerizations is expected to have implications for target modification.

Tandem domain structure determination based on a systematic enumeration of conformations.

Protein structure determination is undergoing a change of perspective due to the larger importance taken in biology by the disordered regions of biomolecules. In such cases, the convergence criterion is more difficult to set up and the size of the conformational space is a obstacle to exhaustive exploration. A pipeline is proposed here to exhaustively sample protein conformations using backbone angle limits obtained by nuclear magnetic resonance (NMR), and then to determine the populations of conformations. The pipeline is applied to a tandem domain of the protein whirlin. An original approach, derived from a reformulation of the Distance Geometry Problem is used to enumerate the conformations of the linker connecting the two domains. Specifically designed procedure then permit to assemble the domains to the linker conformations and to optimize the tandem domain conformations with respect to two sets of NMR measurements: residual dipolar couplings and paramagnetic resonance enhancements. The relative populations of optimized conformations are finally determined by fitting small angle X-ray scattering (SAXS) data. The most populated conformation of the tandem domain is a semi-closed one, fully closed and more extended conformations being in minority, in agreement with previous observations. The SAXS and NMR data show different influences on the determination of populations.

Secondary structure assignment of proteins in the absence of sequence information.

Motivation: The structure of proteins is organized in a hierarchy among which the secondary structure elements, α-helix, ß-strand and loop, are the basic bricks. The determination of secondary structure elements usually requires the knowledge of the whole structure. Nevertheless, in numerous experimental circumstances, the protein structure is partially known. The detection of secondary structures from these partial structures is hampered by the lack of information about connecting residues along the primary sequence. Results: We introduce a new methodology to estimate the secondary structure elements from the values of local distances and angles between the protein atoms. Our method uses a message passing neural network, named Sequoia, which allows the automatic prediction of secondary structure elements from the values of local distances and angles between the protein atoms. This neural network takes as input the topology of the given protein graph, where the vertices are protein residues, and the edges are weighted by values of distances and pseudo-dihedral angles generalizing the backbone angles ϕ and ψ. Any pair of residues, independently of its covalent bonds along the primary sequence of the protein, is tagged with this distance and angle information. Sequoia permits the automatic detection of the secondary structure elements, with an F1-score larger than 80% for most of the cases, when α helices and ß strands are predicted. In contrast to the approaches classically used in structural biology, such as DSSP, Sequoia is able to capture the variations of geometry at the interface of adjacent secondary structure element. Due to its general modeling frame, Sequoia is able to handle graphs containing only C α atoms, which is particularly useful on low resolution structural input and in the frame of electron microscopy development. Availability and implementation: Sequoia source code can be found at https://github.com/Khalife/Sequoia with additional documentation. Supplementary information: Supplementary data are available at Bioinformatics Advances online.

Analyzing In Silico the Relationship Between the Activation of the Edema Factor and Its Interaction With Calmodulin.

Molecular dynamics (MD) simulations have been recorded on the complex between the edema factor (EF) of Bacilllus anthracis and calmodulin (CaM), starting from a structure with the orthosteric inhibitor adefovir bound in the EF catalytic site. The starting structure has been destabilized by alternately suppressing different co-factors, such as adefovir ligand or ions, revealing several long-distance correlations between the conformation of CaM, the geometry of the CaM/EF interface, the enzymatic site and the overall organization of the complex. An allosteric communication between CaM/EF interface and the EF catalytic site, highlighted by these correlations, was confirmed by several bioinformatics approaches from the literature. A network of hydrogen bonds and stacking interactions extending from the helix V of of CaM, and the residues of the switches A, B and C, and connecting to catalytic site residues, is a plausible candidate for the mediation of allosteric communication. The greatest variability in volume between the different MD conditions was also found for cavities present at the EF/CaM interface and in the EF catalytic site. The similarity between the predictions from literature and the volume variability might introduce the volume variability as new descriptor of allostery.

Systematic Exploration of Protein Conformational Space Using a Distance Geometry Approach.

The optimization approaches classically used during the determination of protein structure encounter various difficulties, especially when the size of the conformational space is large. Indeed, in such a case, algorithmic convergence criteria are more difficult to set up. Moreover, the size of the search space makes it difficult to achieve a complete exploration. The interval branch-and-prune (iBP) approach, based on the reformulation of the distance geometry problem (DGP) provides a theoretical frame for the generation of protein conformations, by systematically sampling the conformational space. When an appropriate subset of interatomic distances is known exactly, this worst-case exponential-time algorithm is provably complete and fixed-parameter tractable. These guarantees, however, immediately disappear as distance measurement errors are introduced. Here we propose an improvement of this approach: threading-augmented interval branch-and-prune (TAiBP), where the combinatorial explosion of the original iBP approach arising from its exponential complexity is alleviated by partitioning the input instances into consecutive peptide fragments and by using self-organizing maps (SOMs) to obtain clusters of similar solutions. A validation of the TAiBP approach is presented here on a set of proteins of various sizes and structures. The calculation inputs are a uniform covalent geometry extracted from force field covalent terms, the backbone dihedral angles with error intervals, and a few long-range distances. For most of the proteins smaller than 50 residues and interval widths of 20°, the TAiBP approach yielded solutions with RMSD values smaller than 3 Å with respect to the initial protein conformation. The efficiency of the TAiBP approach for proteins larger than 50 residues will require the use of nonuniform covalent geometry and may have benefits from the recent development of residue-specific force-fields.

Structural Biology and Molecular Modeling to Analyze the Entry of Bacterial Toxins and Virulence Factors into Host Cells.

Understanding the functions and mechanisms of biological systems is an outstanding challenge. One way to overcome it is to combine together several approaches such as molecular modeling and experimental structural biology techniques. Indeed, the interplay between structural and dynamical properties of the system is crucial to unravel the function of molecular machinery's. In this review, we focus on how molecular simulations along with structural information can aid in interpreting biological data. Here, we examine two different cases: (i) the endosomal translocation toxins (diphtheria, tetanus, botulinum toxins) and (ii) the activation of adenylyl cyclase inside the cytoplasm (edema factor, CyA, ExoY).

Publisher Correction: Genomic insights into the 2016-2017 cholera epidemic in Yemen.

In the HTML version of this Letter, the affiliations for authors Andrew S. Azman, Dhirendra Kumar and Thandavarayan Ramamurthy were inverted (the PDF and print versions of the Letter were correct); the affiliations have been corrected online.

Minimal NMR distance information for rigidity of protein graphs.

Nuclear Magnetic Resonance (NMR) experiments provide distances between nearby atoms of a protein molecule. The corresponding structure determination problem is to determine the 3D protein structure by exploiting such distances. We present a new order on the atoms of the protein, based on information from the chemistry of proteins and NMR experiments, which allows us to formulate the problem as a combinatorial search. Additionally, this order tells us what kind of NMR distance information is crucial to understand the cardinality of the solution set of the problem and its computational complexity.

Genomic insights into the 2016-2017 cholera epidemic in Yemen.

Yemen is currently experiencing, to our knowledge, the largest cholera epidemic in recent history. The first cases were declared in September 2016, and over 1.1 million cases and 2,300 deaths have since been reported1. Here we investigate the phylogenetic relationships, pathogenesis and determinants of antimicrobial resistance by sequencing the genomes of Vibrio cholerae isolates from the epidemic in Yemen and recent isolates from neighbouring regions. These 116 genomic sequences were placed within the phylogenetic context of a global collection of 1,087 isolates of the seventh pandemic V. cholerae serogroups O1 and O139 biotype El Tor2-4. We show that the isolates from Yemen that were collected during the two epidemiological waves of the epidemic1-the first between 28 September 2016 and 23 April 2017 (25,839 suspected cases) and the second beginning on 24 April 2017 (more than 1 million suspected cases)-are V. cholerae serotype Ogawa isolates from a single sublineage of the seventh pandemic V. cholerae O1 El Tor (7PET) lineage. Using genomic approaches, we link the epidemic in Yemen to global radiations of pandemic V. cholerae and show that this sublineage originated from South Asia and that it caused outbreaks in East Africa before appearing in Yemen. Furthermore, we show that the isolates from Yemen are susceptible to several antibiotics that are commonly used to treat cholera and to polymyxin B, resistance to which is used as a marker of the El Tor biotype.

Conformational sampling of CpxA: Connecting HAMP motions to the histidine kinase function.

In the histidine kinase family, the HAMP and DHp domains are considered to play an important role into the transmission of signal arising from environmental conditions to the auto-phosphorylation site and to the binding site of response regulator. Several conformational motions inside HAMP have been proposed to transmit this signal: (i) the gearbox model, (ii) α helices rotations, pistons and scissoring, (iii) transition between ordered and disordered states. In the present work, we explore by temperature-accelerated molecular dynamics (TAMD), an enhanced sampling technique, the conformational space of the cytoplasmic region of histidine kinase CpxA. Several HAMP motions, corresponding to α helices rotations, pistoning and scissoring have been detected and correlated to the segmental motions of HAMP and DHp domains of CpxA.

Closed-Locked and Apo-Resting State Structures of the Human α7 Nicotinic Receptor: A Computational Study.

Nicotinic acetylcholine receptors, belonging to the Cys-loop superfamily of ligand-gated ion channels (LGICs), are membrane proteins present in neurons and at neuromuscular junctions. They are responsible for signal transmission, and their function is regulated by neurotransmitters, agonists, and antagonists drugs. A detailed knowledge of their conformational transition in response to ligand binding is critical to understanding the basis of ligand-receptor interaction, in view of new pharmacological approaches to control receptor activity. However, the scarcity of experimentally derived structures of human channels makes this perspective extremely challenging. To contribute overcoming this issue, we have recently reported structural models for the open and the desensitized states of the human α7 nicotinic receptor. Here, we provide all-atom structural models of the same receptor in two different nonconductive states. The first structure, built via homology modeling and relaxed with extensive Molecular Dynamics simulations, represents the receptor bound to the natural antagonist α-conotoxin ImI. After comparison with available experimental data and computational models of other eukaryotic LGICs, we deem it consistent with the "closed-locked" state. The second model, obtained with simulations from the spontaneous relaxation of the open, agonist-bound α7 structure after ligand removal, recapitulates the characteristics of the apo-resting state of the receptor. These results add to our previous work on the active and desensitized state conformations, contributing to the structural characterization of the conformational landscape of the human α7 receptor and suggesting benchmarks to discriminate among conformations found in experiments or in simulations of LGICs. In particular key interactions at the interface between the extracellular domain and the transmembrane domain are identified, that could be critical to the α7 receptor function.

Ordering Protein Contact Matrices.

Numerous biophysical approaches provide information about residues spatial proximity in proteins. However, correct assignment of the protein fold from this proximity information is not straightforward if the spatially close protein residues are not assigned to residues in the primary sequence. Here, we propose an algorithm to assign such residue numbers by ordering the columns and lines of the raw protein contact matrix directly obtained from proximity information between unassigned amino acids. The ordering problem is formatted as the search of a trail within a graph connecting protein residues through the nonzero contact values. The algorithm performs in two steps: (i) finding the longest trail of the graph using an original dynamic programming algorithm, (ii) clustering the individual ordered matrices using a self-organizing map (SOM) approach. The combination of the dynamic programming and self-organizing map approaches constitutes a quite innovative point of the present work. The algorithm was validated on a set of about 900 proteins, representative of the sizes and proportions of secondary structures observed in the Protein Data Bank. The algorithm was revealed to be efficient for noise levels up to 40%, obtaining average gaps of about 20% at maximum between ordered and initial matrices. The proposed approach paves the ways toward a method of fold prediction from noisy proximity information, as TM scores larger than 0.5 have been obtained for ten randomly chosen proteins, in the case of a noise level of 10%. The methods has been also validated on two experimental cases, on which it performed satisfactorily.

re-TAMD: exploring interactions between H3 peptide and YEATS domain using enhanced sampling.

BACKGROUND: Analysis of preferred binding regions of a ligand on a protein is important for detecting cryptic binding pockets and improving the ligand selectivity. RESULT: The enhanced sampling approach TAMD has been adapted to allow a ligand to unbind from its native binding site and explore the protein surface. This so-called re-TAMD procedure was then used to explore the interaction between the N terminal peptide of histone H3 and the YEATS domain. Depending on the length of the peptide, several regions of the protein surface were explored. The peptide conformations sampled during the re-TAMD correspond to peptide free diffusion around the protein surface. CONCLUSIONS: The re-TAMD approach permitted to get information on the relative influence of different regions of the N terminal peptide of H3 on the interaction between H3 and YEATS.

Molecular Modeling of the Catalytic Domain of CyaA Deepened the Knowledge of Its Functional Dynamics.

Although CyaA has been studied for over three decades and revealed itself to be a very good prototype for developing various biotechnological applications, only a little is known about its functional dynamics and about the conformational landscape of this protein. Molecular dynamics simulations helped to clarify the view on these points in the following way. First, the model of interaction between AC and calmodulin (CaM) has evolved from an interaction centered on the surface between C-CaM hydrophobic patch and the α helix H of AC, to a more balanced view, in which the C-terminal tail of AC along with the C-CaM Calcium loops play an important role. This role has been confirmed by the reduction of the affinity of AC for calmodulin in the presence of R338, D360 and N347 mutations. In addition, enhanced sampling studies have permitted to propose a representation of the conformational space for the isolated AC. It remains to refine this representation using structural low resolution information measured on the inactive state of AC. Finally, due to a virtual screening study on another adenyl cyclase from Bacillus anthracis, weak inhibitors of AC have been discovered.

Building Graphs To Describe Dynamics, Kinetics, and Energetics in the d-ALa:d-Lac Ligase VanA.

The d-Ala:d-Lac ligase, VanA, plays a critical role in the resistance of vancomycin. Indeed, it is involved in the synthesis of a peptidoglycan precursor, to which vancomycin cannot bind. The reaction catalyzed by VanA requires the opening of the so-called "ω-loop", so that the substrates can enter the active site. Here, the conformational landscape of VanA is explored by an enhanced sampling approach: the temperature-accelerated molecular dynamics (TAMD). Analysis of the molecular dynamics (MD) and TAMD trajectories recorded on VanA permits a graphical description of the structural and kinetics aspects of the conformational space of VanA, where the internal mobility and various opening modes of the ω-loop play a major role. The other important feature is the correlation of the ω-loop motion with the movements of the opposite domain, defined as containing the residues A149-Q208. Conformational and kinetic clusters have been determined and a path describing the ω-loop opening was extracted from these clusters. The determination of this opening path, as well as the relative importance of hydrogen bonds along the path, permit one to propose some key residue interactions for the kinetics of the ω-loop opening.