The first structures of α-synuclein (αSyn) fibrils have recently been solved. Here, we use a unique combination of molecular dynamics simulation strategies to address the minimal nucleation size of the 11-amino acid NAC protofibril solved by X-ray and to interrogate the dynamic behavior of unexpected crystal waters in the steric zipper. We found that protofibrils of >8 chains are thermodynamically stabilized due to protection of the fibril core from solvent influx and ordering of the end strands by the fibril core. In these stable oligomers, water molecules resolved in the crystal structure freely exchange with bulk solvent but are, on average, stably coordinated along the β-sheet by inward-facing Thr72 and Thr75. We confirm the persistence of this water coordination via simulations of the full-length Greek-key structure solved by NMR and speculate that these Thr-water networks are important in the context of enhanced fibril nucleation in the familial A53T mutation.
Tumor necrosis factor receptor 1 (TNFR1) is a member of the TNFR superfamily which can be activated by binding to one of two cognate ligands, tumor necrosis factor (TNF‐α) or lymphotoxin‐α (LT‐α), via its extracellular domain. Ligand binding leads to network formation and signaling cascade of IκBα degradation and NF‐κB activation, resulting in inflammatory and autoimmune diseases. Current treatments, such as etanercept and infliximab, reduce symptoms through sequestration of free ligands. However, these treatments are expensive and induce dangerous side‐effects due to off‐target inhibition of other related TNF receptors that are not involved in the disease condition. As a consequence, there is a desperate need for receptor‐specific treatments, which hold the promise of overcoming the limitations of conventional therapeutics. It has been proposed that TNFR1 pre‐ligand assembly domain (PLAD), a portion of the extracellular region of TNFR1 that mediates receptor‐chain association essential for signaling, is an important therapeutic target in inflammatory arthritis. This motivates the discovery of small‐molecule inhibitors that could bind to TNFR1 PLAD to disrupt receptor‐receptor interactions and inhibit downstream signaling. In this study, we have engineered a biosensor exhibiting fluorescence resonance energy transfer (FRET) by attaching fluorophores to the intracellular domain of TNFR1. By using a fluorescence lifetime screening platform, two hit compounds were identified to inhibit TNFR1 function. These small‐molecule inhibitors reduce FRET and disrupt PLAD‐PLAD interaction without ablating ligand binding. The biosensor and the fluorescence lifetime screening approach reported here provide a very promising high‐throughput drug discovery platform for TNF receptors, as well as oligomeric receptors in general. Support or Funding Information This work is supported by National Institutes of Health Grant R01 GM107175 and R42 DA037622
The widely accepted model for tumor necrosis factor 1 (TNFR1) signaling is that ligand binding causes receptor trimerization, which triggers a reorganization of cytosolic domains and thus initiates intracellular signaling. This model of stoichiometrically driven receptor activation does not account for the occurrence of ligand independent signaling in overexpressed systems, nor does it explain the constitutive activity of the R92Q mutant associated with TRAPS. More recently, ligand binding has been shown to result in the formation of high molecular weight, oligomeric networks. Although the dimer, shown to be the preligand structure, is thought to remain present within ligand–receptor networks, it is unknown whether network formation or ligand-induced structural change to the dimer itself is the trigger for TNFR1 signaling. In the present study, we investigate the available crystal structures of TNFR1 to explore backbone dynamics and infer conformational transitions associated with ligand binding. Using normal-mode analysis, we characterize the dynamic coupling between the TNFR1 ligand binding and membrane proximal domains and suggest a mechanism for ligand-induced activation. Furthermore, our data are supported experimentally by FRET showing that the constitutively active R92Q mutant adopts an altered conformation compared to wild-type. Collectively, our results suggest that the signaling competent architecture is the receptor dimer and that ligand binding modifies domain mobilities intrinsic to the receptor structure, allowing it to sample a separate, active conformation mediated by network formation.
University of Minnesota Ph.D. dissertation. December 2015. Major: Biomedical Engineering. Advisor: Jonathan Sachs. 1 computer file (PDF); xiv, 179 pages.
