Hydrophobin Film Structure for HFBI and HFBII and Mechanism for Accelerated Film Formation

Peer reviewe

Conformation and dynamics of human urotensin II and urotensin related peptide in aqueous solution

Conformation and dynamics of the vasoconstrictive peptides human urotensin II (UII) and urotensin related peptide (URP) have been investigated by both unrestrained and enhanced-sampling molecular-dynamics (MD) simulations and NMR spectroscopy. These peptides are natural ligands of the G-protein coupled urotensin II receptor (UTR) and have been linked to mammalian pathophysiology. UII and URP cannot be characterized by a single structure but exist as an equilibrium of two main classes of ring conformations, <i>open</i> and <i>folded</i>, with rapidly interchanging subtypes. The <i>open</i> states are characterized by turns of various types centered at K⁸Y⁹ or F⁶W⁷ predominantly with no or only sparsely populated transannular hydrogen bonds. The <i>folded</i> conformations show multiple turns stabilized by highly populated transannular hydrogen bonds comprising centers F⁶W⁷K⁸ or W⁷K⁸Y⁹. Some of these conformations have not been characterized previously. The equilibrium populations that are experimentally difficult to access were estimated by replica-exchange MD simulations and validated by comparison of experimental NMR data with chemical shifts calculated with density-functional theory. UII exhibits approximately 72% <i>open</i>:28% <i>folded</i> conformations in aqueous solution. URP shows very similar ring conformations as UII but differs in an <i>open:folded</i> equilibrium shifted further toward <i>open</i> conformations (86:14) possibly arising from the absence of folded N-terminal tail-ring interaction. The results suggest that the different biological effects of UII and URP are not caused by differences in ring conformations but rather by different interactions with UTR

Exploring conformation of human fatty acid synthase inhibitors using Replica Exchange Molecular Dynamics

Human fatty acid synthase (hFAS) is a homodimer multienzyme complex involved in the lipogenesis and catalysis of long-chain fatty acids. hFAS is overexpressed in cancer cells and enhances tumor growth. A recent study reported a new potent and selective inhibitor of the β-ketoacyl reductase (KR) domain of hFAS, GSK2194693. An x-ray crystal structure of this inhibitor bound to the KR domain provides binding mode information regarding the druggable pocket. In this thesis simulations and analysis of the solution-phase conformational ensembles of four inhibitors of the human fatty acid synthase are required. The ensembles are generated using replica exchange enhanced sampling molecular dynamics approaches for two force fields, and analysed using a combination of dihedral and Cartesian space clustering, and principal components analysis. These ensembles are compared to experimental data derived using nuclear magnetic resonance from C4X Discovery to evaluate the convergence of our data and to analyze the influence of the force field on the quality of the sampling. We find that while the simulations are able to identify all the conformations found by NMR, their relative populations are in less satisfactory agreement. The ligandreceptor complex binding modes were also investigated by first identifying conformations of the four compounds with shape and chemical group similarity using clustering and superimposition methodologies. Then, in a ligand preorganization approach to identify if the solution phase conformations obtained from NMR and REMD bind favourably to the receptor binding pocket, the interactions made with hFAS were evaluated keeping the conformations and the receptor rigid. Potential binding modes for the compounds were generated with consistent interactions. Contacts found in the x-ray structure GSK2194069 were highly conserved in the compounds and additional hydrogen bonds were identified. Thus, this study offers valuable information for future drug development and optimization

Performance evaluation of molecular docking and free energy calculations protocols using the D3R Grand Challenge 4 dataset

International audienceUsing the D3R Grand Challenge 4 dataset containing Beta-secretase 1 (BACE) and Cathepsin S (CatS) inhibitors, we have evaluated the performance of our in-house docking workflow that involves in the first step the selection of the most suitable docking software for the system of interest based on structural and functional information available in public databases, followed by the docking of the dataset to predict the binding modes and ranking of ligands. The macrocyclic nature of the BACE ligands brought additional challenges, which were dealt with by a careful preparation of the three-dimensional input structures for ligands. This provided top-performing predictions for BACE, in contrast with CatS, where the predictions in the absence of guiding constraints provided poor results. These results highlight the importance of previous structural knowledge that is needed for correct predictions on some challenging targets. After the end of the challenge, we also carried out free energy calculations (i.e. in a non-blinded manner) for CatS using the pmx software and several force fields (AMBER, Charmm). Using knowledge-based starting pose construction allowed reaching remarkable accuracy for the CatS free energy estimates. Interestingly, we show that the use of a consensus result, by averaging the results from different force fields, increases the prediction accuracy

All docking results for possible unit cells composed of previously determined trimers.

<p>For both HFBI and HFBII pentamers composed of trimers A and B, in each case the two trimers sharing a protein, could be constructed. These pentamers could not be docked to themselves thus they can not form a unit cell. For both HFBI and HFBII, hexamers could be constructed from two identical trimers, for HFBI from both trimer A and D (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003745#pcbi-1003745-g002" target="_blank">figure 2</a>), to form structures <i>α</i> and <i>β</i>, and for HFBII, from trimer A. In all three of these cases the hexamers could be successfully docked to themselves, as can be seen from the “18mers”, composed of three hexamers docked in a ring.</p

Graphical demonstration of our reasoning regarding possible structures.

<p>a) given that hydrophobin protein on hydrophobic surface has a diameter of ∼20 Å, for proteins to be in contact two possible lattice vectors with triangular symmetry can be seen, length ∼35 Å and ∼53 Å. Since experimental results show for HFBI and HFBII the lattice vectors are ∼54 Å and ∼55 Å respectively, this precludes the first lattice vector (∼35 Å). If we constrain the proteins to be in contact with a neighbor, then there exist only three possible structures that will possess this symmetry, b) c) and d).</p

Comparison of the 3 protein and 6 protein unit cell structures for HFBII, the 6 protein unit cell is the better fit to the experimentally observed lattice parameter and provides greater coverage of the surface.

<p>Schematics of the P3 and P6 unit cells of the respective structures are shown at the bottom.</p

Results demonstrating verification of the 3 protein unit cell structure with P3 symmetry, protein-protein docking matches symmetric creation from the 5 protein structure.

<p>The results were successful, docking both the A and B trimers for both HFBI and HFBII.</p

Docking results for HFBI and HFBII fitting three protein unit (trimer).

<p>All structures within the top 1% scored are included; four different structures were found (A–D) for HFB I and 5 different structures were found (A–E), for HFB II.</p

A) Schematic showing the construction of our spin model that allows for both P6 and P3 symmetry ordering on a triangular lattice as a simplified model of the hydrophobin surface.

<p>B) Plots of the largest cluster size vs. Monte Carlo time for spin model where both P3 and P6 symmetry are permitted, and one where only P6 is permitted. We see that the size of the largest cluster increases linearly for the system with only P6 symmetry and exponentially for the system where both the P3 and P6 symmetries are permitted. C) Visualization of both systems where only P6 ordering is allowed and where both P6 and P3 ordering are allowed, at 60, 80, 100, and 120 Monte Carlo steps. Protein positions on the triangular lattice are red and the largest cluster is shown in yellow. The much faster exponential, as opposed to linear, growth in the cluster size for the system with both P6 and P3 ordering can clearly be seen; the largest cluster percolates the 40×40 unit cell system at 120 Monte Carlo steps.</p