Two novel compounds were identified as Naa50 binders/inhibitors using DNA-encoded technology screening. Biophysical and biochemical data as well as cocrystal structures were obtained for both compounds (3a and 4a) to understand their mechanism of action. These data were also used to rationalize the binding affinity differences observed between the two compounds and a MLGP peptide-containing substrate. Cellular target engagement experiments further confirm the Naa50 binding of 4a and demonstrate its selectivity toward related enzymes (Naa10 and Naa60). Additional analogs of inhibitor 4a were also evaluated to study the binding mode observed in the cocrystal structures.

No abstract

A detailed analysis is carried out on both published experimental results and new experiments for the methylation kinetics of two-site DNA substrates (with site separations between 100 and 800 bp) catalyzed by bacterial DNA adenine methyltransferase (Dam). A previously reported rate enhancement for the second methylation event (relative to that of the first methylation) is shown to result from elevated substrate specificity for singly methylated DNA over that of unmethylated DNA and not processive turnover of both sites by the same copy of Dam. An elementary model is suggested that cleanly fits the experimental data over a broad range of intersite separations. The model hypothesizes a looping mediated interference between competing unmethylated Dam sites on the same DNA strand.

DYT1 dystonia is a debilitating neurological movement disorder that arises upon Torsin ATPase deficiency. Nuclear envelope (NE) blebs that contain FG-nucleoporins (FG-Nups) and K48-linked ubiquitin are the hallmark phenotype of Torsin manipulation across disease models of DYT1 dystonia. While the aberrant deposition of FG-Nups is caused by defective nuclear pore complex assembly, the source of K48-ubiquitylated proteins inside NE blebs is not known. Here, we demonstrate that the characteristic K48-ubiquitin accumulation inside blebs requires p97 activity. This activity is highly dependent on the p97 adaptor UBXD1. We show that p97 does not significantly depend on the Ufd1/Npl4 heterodimer to generate the K48-ubiquitylated proteins inside blebs, nor does inhibiting translation affect the ubiquitin sequestration in blebs. However, stimulating global ubiquitylation by heat shock greatly increases the amount of K48-ubiquitin sequestered inside blebs. These results suggest that blebs have an extraordinarily high capacity for sequestering ubiquitylated protein generated in a p97-dependent manner. The p97/UBXD1 axis is thus a major factor contributing to cellular DYT1 dystonia pathology and its modulation represents an unexplored potential for therapeutic development.

The role of hydration water on protein and DNA surfaces is fundamental to their interaction and processes, yet experimentally challenging to study. We recently applied a novel Overhauser dynamic nuclear polarization‐enhanced NMR method to the study of biological hydration water which provides unprecedented insights into ps to sub‐ns water dynamics within 5‐10 Angstrom of molecular surfaces: waters loosely associated with DNA have surprisingly high degrees of mobility, nearly unperturbed, while waters surrounding protein surfaces are significantly fortified, compared to the dynamics of bulk water. Here we investigated how water mobility contributes to the site location mechanisms of several DNA binding proteins. Dimethyl sulfoxide increases this water mobility on molecular surfaces (DNA or protein) and is predicted to increase the site location efficiency of proteins that rely extensively on a sliding mechanism; we confirmed this to be the case with EcoRI endonuclease when adding 1% DMSO. In contrast, glycerol is predicted to have a retarding (or sometimes neutral) effect on surface water mobility and thus sliding translocation kinetics, which we again confirm to be the case with EcoRI endonuclease when adding 1% glycerol. In contrast, the translocation efficiency of DNA adenine methyltransferase (Dam) that does not rely extensively on sliding is not altered with either osmolyte addition. The observed effects on processivity have been corrected for minor changes in catalytic efficiency in the presence of osmolytes. Our results reveal that water’s fast moving properties around DNA are important for protein‐DNA interactions and in particular, the translocation along the double helix, whose efficiency can be slowed or enhanced when the water barrier on the DNA or protein surfaces is strengthened or weakened by the addition of glycerol or DMSO.