Additional file 1: of Thermodynamic and kinetic stability of the Josephin Domain closed arrangement: evidences from replica exchange molecular dynamics

Supporting Information to Thermodynamic and Kinetic Stability of the Josephin Domain Closed Arrangement: Evidences from Replica Exchange Molecular Dynamics. Additional Information on Replica Exchange applied approach, kinetic estimation. (PDF 1183 kb

JD configuration in the open-like shape (top).

<p>The more compact (bottom) JD globular shape is the consequence of the hairpin closure dynamics. Several possible configurations of open/close hairpin are superimposed.</p

The visual inspection of MD trajectories reveals Leu84-Trp87 conformational change and solvent exposure after the JD-JD binding.

<p>Four snapshots throughout the MD are shown (red, green, yellow, and brown); the 1YZB conformation of Leu84-Trp87 is shown in black. The JD structure is transparent and grey. Hairpin region and Arg101/Arg103 residues are labeled.</p

α4-Protein/centre of mass (COM) distribution.

<p>Similar behavior (Gaussian-like) is shown for JD^Wat (light grey curve) and JD^A101/A103 (gray curve). A subset of sampled JD-JD configurations (black curve) reveals α4 moving far from the protein centre of mass.</p

Secondary structure percentage, calculated over the available NMR configurations contained in 1YZB (a), the configurations are taken every 50 ps from JD^Wat(b), JD-JD (c), and JD^A101/A103 (d) in the last 50 ns of the MD trajectories.

<p>Secondary structures are indicated by different colours in the figure: green (coil), blue (helix), red (β-sheet).</p

Radius of Gyration (RG) distribution of the single JD, from trajectories of JD^Wat(a), JD-JD (b), and JD^A101/A103 (c) simulations.

<p>The experimental RG value of 1YZB and 2AGA (open/closed hairpin, respectively) are indicated for comparison.</p

Single JD root-mean-square fluctuations (RMSF).

<p>Fluctuations of α4 are zoomed in the top-right panel where it is worth noticing that residues Leu84-Trp87 increase the RMSF as a consequence of JD dimerization (black curve). After Ala-mutation (gray curve) on Arg101-/Arg103 the RMSF value decrease to the value achieved by JD^Wat (light gray curve).</p

Self-Assembled Ligands Targeting TLR7: A Molecular Level Investigation

Toll-like receptors (TLRs) are pattern recognition transmembrane proteins that play an important role in innate immunity. In particular, TLR7 plays a role in detecting nucleic acids derived from viruses and bacteria. The huge number of pathologies in which TLR7 is involved has led to an increasing interest in developing new compounds targeting this protein. Several conjugation strategies were proposed for TLR7 agonists to increase the potency while maintaining a low toxicity. In this work, we focus the attention on two promising classes of TLR7 compounds derived from the same pharmacophore conjugated with phospholipid and polyethylene glycol (PEG). A multidisciplinary investigation has been carried out by molecular dynamics (MD), dynamic light scattering (DLS), electron paramagnetic resonance (EPR), and cytotoxicity assessment. DLS and MD indicated how only the phospholipid conjugation provides the compound abilities to self-assemble in an orderly fashion with a maximal pharmacophore exposition to the solvent. Further EPR and cytotoxicity experiments highlighted that phospholipid compounds organize in stable aggregates and well interact with TLR7, whereas PEG conjugation was characterized by poorly stable aggregates at the cells surface. The methodological framework proposed in this study may be used to investigate, at a molecular level, the interactions generally occurring between aggregated ligands, to be used as drugs, and protein receptors