PKR is a member of the eIF2α family of protein kinases that inhibit translational initiation in response to stress stimuli and functions as a key mediator of the interferon-induced antiviral response. PKR contains a dsRNA binding domain that bind to duplex regions present in viral RNAs, resulting in kinase activation and autophosphorylation. An emerging theme in the regulation of protein kinases is the allosteric linkage of dimerization and activation. The PKR kinase domain forms a back-to-back parallel dimer which is implicated in activation. We have developed a sensitive homo-FRET assay for kinase domain dimerization to directly probe the relationship between RNA binding, activation, and dimerization. In the case of perfect duplex RNAs, dimerization is correlated with activation and dsRNAs containing 30 bp or more efficiently induce kinase domain dimerization and activation. However, more complex duplex RNAs containing a 10–15 bp 2'-O-methyl RNA barrier produce kinase dimers but do not activate. Similarly, inactivating mutations within the PKR dimer interface that disrupt key electrostatic and hydrogen binding interactions fail to abolish dimerization. Our data support a model where activating RNAs induce formation of a back-to-back parallel PKR kinase dimer whereas nonactivating RNAs either fail to induce dimerization or produce an alternative, inactive dimer configuration, providing an additional mechanism for distinguishing between host and pathogen RNA.

a b s t r a c tThe selective deamination of adenosines (A) to inosines (I) in messenger RNAs (mRNAs) can alter the encoded protein's amino acid sequence, with often critical consequences on protein stability, localization, and/or function. Insulin-like growth factor-binding protein 7 (IGFBP7) supports cell-adhesion and stimulates fibroblast proliferation with IGF and insulin. It exists in both proteolytically processed and unprocessed forms with altered cell-extracellular matrix interactions. Here we show that editing of IGFBP7 transcripts impacts the protein's susceptibility to proteolytic cleavage, thus providing a means for a cell to modulate its functionality through A-to-I RNA editing. Structured summary of protein interactions:MT-SP1 cleaves IGFBP7 by protease assay (View interaction).

both side-chain rotamer preferences and for optimal amino acid sequence. These results pose a series of questions not just about fixed-charge, pairwise decomposable sequence design models, but also our novel polarizable, many-body optimization algorithms and how to use both approaches synergistically. 1705-PlatDe Novo Design and Biophysical Characterization of an Affinity-Enhanced Protein Displaying the Structure of the Broadly Neutralizing HIV-1 2F5

Protein kinase R (PKR) is a key pattern recognition receptor of the innate immune pathway. PKR is activated by double-stranded RNA (dsRNA) that is often produced during viral genome replication and transcription. PKR contains two tandem double-stranded RNA binding domains at the N-terminus, dsRBD1 and dsRBD2, and a C-terminal kinase domain. In the canonical model for activation, RNAs that bind multiple PKRs induce dimerization of the kinase domain that promotes an active conformation. However, there is evidence that dimerization of the kinase domain is not sufficient to mediate activation and PKR activation is modulated by the RNA-binding mode. dsRBD2 lacks most of the consensus RNA-binding residues, and it has been suggested to function as a modulator of PKR activation. Here, we demonstrate that dsRBD2 regulates PKR activation and identify the N-terminal helix as a critical region for modulating kinase activity. Mutations in dsRBD2 that have minor effects on overall dsRNA-binding affinity strongly inhibit the activation of PKR by dsRNA. These mutations also inhibit RNA-independent PKR activation. These data support a model where dsRBD2 has evolved to function as a regulator of the kinase.

The ubiquitin specific protease 7 (USP7) is considered a highly promising well-validated target for various malignancies including multiple myeloma, neuroblastoma and ovarian cancer. The aim of this study was to identify potent and safe USP7 inhibitors as potential cancer drugs via an integrated computational approach combining pharmacophore screening, QSAR models, MD simulations and MM/GBSA free energy analysis. In this study, the crystal structure of USP7 was comprehensively explored using a combination of fragment-based and macromoleculeligand-complex-based e-pharmacophore as well as ligand-based pharmacophore modelling. We used three fragment libraries as well as 46 experimentally known USP7 inhibitors to develop these pharmacophores. The fragment libraries were docked via a virtual screening protocol using Glide-HTVS, -SP and -XP, and the top sequential 20% fragments were used to develop the pharmacophore models. We then screened the SPECS library ($220,000 drug-like molecules), and the top ligands were docked by Glide-SP. The lead ligands from each pharmacophore were further examined by 1 ns MD simulations and MM/GBSA free energy analysis. The most energtically stable ligands advanced to 50 ns MD simulations and MM/GBSA free energy analysis. In total, we ran 1 ns MD simulations for 1137 ligands. Based on their MM/GBSA analysis, 18 ligands were selected for 50 ns MD simulations along with one USP7 inhibitors (HBX19818). We hereby propose 18 ligands as potential USP7 inhibitors that showed conformational stability and high binding site affinity with nearly no predicted toxicities, suggesting more energetically favorable protein-ligand interactions.The identification of potent and safe ubiquitin specific protease 7 small molecules serves an important contribution to the field of cancer drug design and development, enabling further research in this promising field.