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The fragment docking program solvation energy for exhaustive docking (SEED) is evaluated on 15 different protein targets, with a focus on enrichment and the hit rate. It is shown that SEED allows for consistent computational enrichment of fragment libraries, independent of the effective hit rate. Depending on the actual target protein, true positive rates ranging up to 27% are observed at a cutoff value corresponding to the experimental hit rate. The impact of variations in docking protocols and energy filters is discussed in detail. Remaining issues, limitations, and use cases of SEED are also discussed. Our results show that fragment library selection or enhancement for a particular target is likely to benefit from docking with SEED, suggesting that SEED is a useful resource for fragment screening campaigns. A workflow is presented for the use of the program in virtual screening, including filtering and postprocessing to optimize hit rates.

The ATPase family, AAA domain-containing protein 2 (ATAD2) has a C-terminal bromodomain, which functions as a chromatin reader domain recognizing acetylated lysine on the histone tails within the nucleosome. ATAD2 is overexpressed in many cancers and its expression is correlated with poor patient outcomes, making it an attractive therapeutic target and potential biomarker. We solved the crystal structure of the ATAD2 bromodomain and found that it contains a disulfide bridge near the base of the acetyllysine binding pocket (Cys1057-Cys1079). Sitedirected mutagenesis revealed that removal of a free C-terminal cysteine (C1101) residue greatly improved the solubility of the ATAD2 bromodomain in vitro. Isothermal titration calorimetry experiments in combination with the Ellman's assay demonstrated that formation of an intramolecular disulfide bridge negatively impacts the ligand binding affinities and alters the thermodynamic parameters of the ATAD2 bromodomain interaction with a histone H4K5ac peptide as well as a small molecule bromodomain ligand. Molecular dynamics simulations indicate that the formation of the disulfide bridge in the ATAD2 bromodomain does not alter the structure of the folded state or flexibility of the acetyllysine binding pocket. However, consideration of this unique structural feature should be taken into account when examining ligandbinding affinity, or in the design of new bromodomain inhibitor compounds that interact with this acetyllysine reader module. K E Y W O R D S acetyllysine, ATAD2, bromodomain inhibitor, chromatin reader domain, disulfide bridge, epigenetics, histone, post-translational modification

A detailed understanding of the conformational dynamics of biological molecules is difficult to obtain by experimental techniques due to resolution limitations in both time and space. Computer simulations avoid these in theory but are often too short to sample rare events reliably. Here we show that the progress index-guided sampling (PIGS) protocol can be used to enhance the sampling of rare events in selected parts of biomolecules without perturbing the remainder of the system. The method is very easy to use as it only requires as essential input a set of several features representing the parts of interest sufficiently. In this feature space, new states are discovered by spontaneous fluctuations alone and in unsupervised fashion. Because there are no energetic biases acting on phase space variables or projections thereof, the trajectories PIGS generates can be analyzed directly in the framework of transition networks. We demonstrate the possibility and usefulness of such focused explorations of biomolecules with two loops that are part of the binding sites of bromodomains, a family of epigenetic "reader" modules. This real-life application uncovers states that are structurally and kinetically far away from the initial crystallographic structures and are also metastable. Representative conformations are intended to be used in future high-throughput virtual screening campaigns.