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.

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.

Modification in hydrophobic packing of HAMP domain induces a destabilization of the auto-phosphorylation site in the histidine kinase CpxA.

The histidine kinases belong to the family of two-component systems, which serves in bacteria to couple environmental stimuli to adaptive responses. Most of the histidine kinases are homodimers, in which the HAMP and DHp domains assemble into an elongated helical region flanked by two CA domains. Recently, X-ray crystallographic structures of the cytoplasmic region of the Escherichia coli histidine kinase CpxA were determined and a phosphotransferase-defective mutant, M228V, located in HAMP, was identified. In the present study, we recorded 1 µs molecular dynamics trajectories to compare the behavior of the WT and M228V protein dimers. The M228V modification locally induces the appearance of larger voids within HAMP as well as a perturbation of the number of voids within DHp, thus destabilizing the HAMP and DHp hydrophobic packing. In addition, a disruption of the stacking interaction between F403 located in the lid of the CA domain involved in the auto-phosphorylation and R296 located in the interacting DHp region, is more often observed in the presence of the M228V modification. Experimental modifications R296A and R296D of CpxA have been observed to reduce also the CpxA activity. These observations agree with the destabilization of the R296/F403 stacking, and could be the sign of the transmission of a conformational event taking place in HAMP to the auto-phosphorylation site of histidine kinase. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 670-682, 2016.

Functional motions modulating VanA ligand binding unraveled by self-organizing maps.

The VanA D-Ala:D-Lac ligase is a key enzyme in the emergence of high level resistance to vancomycin in Enterococcus species and methicillin-resistant Staphylococcus aureus. It catalyzes the formation of D-Ala-D-Lac instead of the vancomycin target, D-Ala-D-Ala, leading to the production of modified, low vancomycin binding affinity peptidoglycan precursors. Therefore, VanA appears as an attractive target for the design of new antibacterials to overcome resistance. The catalytic site of VanA is delimited by three domains and closed by an ω-loop upon enzymatic reaction. The aim of the present work was (i) to investigate the conformational transition of VanA associated with the opening of its ω-loop and of a part of its central domain and (ii) to relate this transition with the substrate or product binding propensities. Molecular dynamics trajectories of the VanA ligase of Enterococcus faecium with or without a disulfide bridge distant from the catalytic site revealed differences in the catalytic site conformations with a slight opening. Conformations were clustered with an original machine learning method, based on self-organizing maps (SOM), which revealed four distinct conformational basins. Several ligands related to substrates, intermediates, or products were docked to SOM representative conformations with the DOCK 6.5 program. Classification of ligand docking poses, also performed with SOM, clearly distinguished ligand functional classes: substrates, reaction intermediates, and product. This result illustrates the acuity of the SOM classification and supports the quality of the DOCK program poses. The protein-ligand interaction features for the different classes of poses will guide the search and design of novel inhibitors.

The redundancy of NMR restraints can be used to accelerate the unfolding behavior of an SH3 domain during molecular dynamics simulations.

BACKGROUND: The simulation of protein unfolding usually requires recording long molecular dynamics trajectories. The present work aims to figure out whether NMR restraints data can be used to probe protein conformations in order to accelerate the unfolding simulation. The SH3 domain of nephrocystine (nph SH3) was shown by NMR to be destabilized by point mutations, and was thus chosen to illustrate the proposed method. RESULTS: The NMR restraints observed on the WT nph SH3 domain were sorted from the least redundant to the most redundant ones. Protein NMR conformations were then calculated with: (i) the set full including all NMR restraints measured on nph SH3, (ii) the set reduced where the least redundant restraints with respect to the set full were removed, (iii) the sets random where randomly picked-up restraints were removed. From each set of conformations, we recorded series of 5-ns MD trajectories. The ß barrel architecture of nph SH3 in the trajectories starting from sets (i) and (iii) appears to be stable. On the contrary, on trajectories based on the set (ii), a displacement of the hydrophobic core residues and a variation of the ß barrel inner cavity profile were observed. The overall nph SH3 destabilization agrees with previous experimental and simulation observations made on other SH3 domains. The destabilizing effect of mutations was also found to be enhanced by the removal of the least redundant restraints. CONCLUSIONS: We conclude that the NMR restraint redundancy is connected to the instability of the SH3 nph domain. This restraint redundancy generalizes the contact order parameter, which is calculated from the contact map of a folded protein and was shown in the literature to be correlated to the protein folding rate. The relationship between the NMR restraint redundancy and the protein folding is also reminiscent of the previous use of the Gaussian Network Model to predict protein folding parameters.

Insights into the enzymatic mechanism of 6-phosphogluconolactonase from Trypanosoma brucei using structural data and molecular dynamics simulation.

Trypanosoma brucei is the causative agent of African sleeping sickness. Current work for the development of new drugs against this pathology includes evaluation of enzymes of the pentose phosphate pathway (PPP), which first requires a clear understanding of their function and mechanism of action. In this context, we focused on T. brucei 6-phosphogluconolactonase (Tb6PGL), which converts delta-6-phosphogluconolactone into 6-phosphogluconic acid in the second step of the PPP. We have determined the crystal structure of Tb6PGL in complex with two ligands, 6-phosphogluconic acid and citrate, at 2.2 A and 2.0 A resolution, respectively. We have performed molecular dynamics (MD) simulations on Tb6PGL in its empty form and in complex with delta-6-phosphogluconolactone, its natural ligand. Analysis of the structural data and MD simulations allowed us to propose a detailed enzymatic mechanism for 6PGL enzymes.

Three dimensional structure and implications for the catalytic mechanism of 6-phosphogluconolactonase from Trypanosoma brucei.

Enzymes from the pentose phosphate pathway (PPP) are potential drug targets for the development of new drugs against Trypanosoma brucei, the causative agent of African sleeping disease: for instance, the 6-phosphogluconate dehydrogenase is currently studied actively for such purposes. Structural and functional studies are necessary to better characterize the associated enzymes and compare them to their human homologues, in order to undertake structure-based drug design studies on such targets. In this context, the crystal structure of 6-phosphogluconolactonase (6PGL) from T. brucei, the second enzyme from PPP, was determined at 2.1 Angstroms resolution. Comparison of its sequence and structure to other related proteins in the 6PGL family with a known structure (Thermotoga maritima Tm6GPL 1PBT and Vibrio cholerae Vc6PGL (1Y89), which have not been discussed in print), or in the glucosamine-6-phosphate-deaminase family (hexameric Escherichia coli 1DEA and monomeric Bacillus subtilis 2BKV), allowed the identification of the 6PGL active site. In addition to the analysis of the crystal structure, 3D NMR interaction studies and docking experiments are reported here. Key residues involved in substrate binding or in catalysis were identified.

Determination of dihedral Psi angles in large proteins by combining NH(N)/C(alpha)H(alpha) dipole/dipole cross-correlation and chemical shifts.

We propose a strategy based on the combination of experimental NH(N)/C(alpha)H(alpha) dipole/dipole cross-correlated relaxation rates and chemical shift analysis for the determination of Psi torsion angles in proteins. The method allows the determination of a dihedral angle that is not easily accessible by nuclear magnetic resonance (NMR). The measurement of dihedral angle restraints can be used for structure calculation, which is known to improve the quality of NMR structures. The method is of particular interest in the case of large proteins, for which spectral assignment of the nuclear Overhauser effect spectra, and therefore straightforward structural determination, is out of reach. One advantage of the method is that it is reasonably simple to implement, and could be used in association with other methods aiming at obtaining structural information on complex systems, such as residual dipolar coupling measurements. An illustrative example is analyzed in the case of the 30-kDa protein 6-phosphogluconolactonase.