Hydroxamic Acid-Modified Peptide Library Provides Insights into the Molecular Basis for the Substrate Selectivity of HDAC Corepressor Complexes.

Targeting the lysine deacetylase activity of class I histone deacetylases (HDACs) is potentially beneficial for the treatment of several diseases including human immunodeficiency virus (HIV) infection, Alzheimer's disease, and various cancers. It is therefore important to understand the function and mechanism of action of these enzymes. Class I HDACs act as catalytic components of seven large, multiprotein corepressor complexes. Different HDAC corepressor complexes have specific, nonredundant roles in the cell. It is likely that their specific functions are at least partly influenced by the substrate specificity of the complexes. To address this, we developed chemical tools to probe the specificity of HDAC complexes. We assessed a library of acetyl-lysine-containing substrate peptides and hydroxamic acid-containing inhibitor peptides against the full range of class I HDAC corepressor complexes. The results suggest that site-specific HDAC corepressor complex activity is driven in part by the recognition of the primary amino acid sequence surrounding a particular lysine position in the histone tail.

In vitro evolution predicts emerging SARS-CoV-2 mutations with high affinity for ACE2 and cross-species binding.

Emerging SARS-CoV-2 variants are creating major challenges in the ongoing COVID-19 pandemic. Being able to predict mutations that could arise in SARS-CoV-2 leading to increased transmissibility or immune evasion would be extremely valuable in development of broad-acting therapeutics and vaccines, and prioritising viral monitoring and containment. Here we use in vitro evolution to seek mutations in SARS-CoV-2 receptor binding domain (RBD) that would substantially increase binding to ACE2. We find a double mutation, S477N and Q498H, that increases affinity of RBD for ACE2 by 6.5-fold. This affinity gain is largely driven by the Q498H mutation. We determine the structure of the mutant-RBD:ACE2 complex by cryo-electron microscopy to reveal the mechanism for increased affinity. Addition of Q498H to SARS-CoV-2 RBD variants is found to boost binding affinity of the variants for human ACE2 and confer a new ability to bind rat ACE2 with high affinity. Surprisingly however, in the presence of the common N501Y mutation, Q498H inhibits binding, due to a clash between H498 and Y501 side chains. To achieve an intermolecular bonding network, affinity gain and cross-species binding similar to Q498H alone, RBD variants with the N501Y mutation must acquire instead the related Q498R mutation. Thus, SARS-CoV-2 RBD can access large affinity gains and cross-species binding via two alternative mutational routes involving Q498, with route selection determined by whether a variant already has the N501Y mutation. These mutations are now appearing in emerging SARS-CoV-2 variants where they have the potential to influence human-to-human and cross-species transmission.

Histone H2B Deacylation Selectivity: Exploring Chromatin's Dark Matter with an Engineered Sortase.

We describe a new method to produce histone H2B by semisynthesis with an engineered sortase transpeptidase. N-Terminal tail site-specifically modified acetylated, lactylated, and ß-hydroxybutyrylated histone H2Bs were incorporated into nucleosomes and investigated as substrates of histone deacetylase (HDAC) complexes and sirtuins. A wide range of rates and site-specificities were observed by these enzyme forms suggesting distinct biological roles in regulating chromatin structure and epigenetics.