Structure-based drug design has often been restricted by the rather static picture of protein–ligand complexes presented by crystal structures, despite the widely accepted importance of protein flexibility in biomolecular recognition. Here we report a detailed experimental and computational study of the drug target, human heat shock protein 90, to explore the contribution of protein dynamics to the binding thermodynamics and kinetics of drug-like compounds. We observe that their binding properties depend on whether the protein has a loop or a helical conformation in the binding site of the ligand-bound state. Compounds bound to the helical conformation display slow association and dissociation rates, high-affinity and high cellular efficacy, and predominantly entropically driven binding. An important entropic contribution comes from the greater flexibility of the helical relative to the loop conformation in the ligand-bound state. This unusual mechanism suggests increasing target flexibility in the bound state by ligand design as a new strategy for drug discovery.
Tail-anchored (TA) proteins are involved in cellular processes including trafficking, degradation, and apoptosis. They contain a C-terminal membrane anchor and are posttranslationally delivered to the endoplasmic reticulum (ER) membrane by the Get3 adenosine triphosphatase interacting with the hetero-oligomeric Get1/2 receptor. We have determined crystal structures of Get3 in complex with the cytosolic domains of Get1 and Get2 in different functional states at 3.0, 3.2, and 4.6 angstrom resolution. The structural data, together with biochemical experiments, show that Get1 and Get2 use adjacent, partially overlapping binding sites and that both can bind simultaneously to Get3. Docking to the Get1/2 complex allows for conformational changes in Get3 that are required for TA protein insertion. These data suggest a molecular mechanism for nucleotide-regulated delivery of TA proteins.
Connexin43 is degraded by the proteasomal as well as the lysosomal pathway with ubiquitin playing a role in both degradation pathways. So far, no ubiquitin protein ligase has been identified for any of the connexins. By using pull-down assays, here we show binding of a ubiquitin protein ligase, Nedd4, to the C-terminus of connexin43. This observation was confirmed in vivo by coimmunoprecipitation and immunofluorescence, showing colocalization of Nedd4 and connexin43. Binding of Nedd4 to its interaction partners is generally carried out by its WW domains. Our results indicate that the interaction with connexin43 occurs through all three WW domains of Nedd4. Furthermore, whereas WW1 and WW2 domains mainly interact with the unphosphorylated form of connexin43, WW3 binds phosphorylated and unphosphorylated forms equally. In addition, using the surface plasmon resonance approach we show that only the WW2 domain binds to the PY motif located at the C-terminus of connexin43. Suppression of Nedd4 expression with siRNA resulted in an accumulation of gap junction plaques at the plasma membrane, suggesting an involvement of the ubiquitin protein ligase Nedd4 in gap junction internalization.
We here report on non-equilibrium targeted Molecular Dynamics simulations as tool for the estimation of protein-ligand unbinding kinetics. With this method, we furthermore investigate the molecular basis determining unbinding rates, correlating simulations with experimental data from SPR kinetics measurements and X-ray crystallography on two small molecule compound libraries bound to the N-terminal domain of the chaperone Hsp90. Within the investigated libraries, we find ligand conformational changes and protein-ligand nonbonded interactions as discriminators for unbinding rates. Ligands with flexible chemical scaffold may remain longer at the protein target if they need to pass through extended conformations upon unbinding, or if they exhibit strong electrostatic and/or van der Waals interactions with the target. Ligands with rigid chemical scaffold can exhibit longer residence times if they need to perform any kind of conformational change for unbinding, while electrostatic interactions with the protein can facilitate unbinding. Our resultsshow that understanding the unbinding pathway and the protein-ligand interactions along this path is crucial for the prediction of small molecule ligands with defined unbinding kinetics. Supporting InformationFour supporting tables, nine supporting figures and additional references (PDF) SMILES annotations (CSV) Accession CodesThe crystallographic coordinates of novel compounds are deposited in the Protein Data Bank under the accession codes 5LRL (2d) and 5LO1 (2j). Authors will release the atomic coordinates and experimental data upon article publication.
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