The RERE Deacetylase Complex

Histone Deacetylases (HDACs) are important transcriptional regulators which remove acetyl groups from histone tails. Removal of acetyl groups affects gene expression. HDACs are part of large co-regulatory complexes. One of these complexes is called RERE and the core is comprised of: RERE, ATN1 and HDAC1. The RERE protein is a member of the atrophin family and is associated with developmental defects and neurological diseases. The aim of this project was to use structural and biochemical approaches to further our understanding of this poorly characterised complex. Using peptide binding arrays and fluorescence anisotropy data, the active transcription mark, H3R26me2 was identified as a binding target for the RERE BAH domain. ChIP-sequencing data showed RERE aligned with other active histone marks, which supports the role of RERE as a transcriptional activator. BioID and pulldown approaches suggest RERE forms a complex with SFPQ and NONO. These are co-regulator and splicing factors suggesting that RERE may regulate multiple stages of gene transcription. Electron microscopy and structural prediction techniques were used to propose a model of the RERE complex. The model shows that RERE does not dimerise through the ELM2 or SANT domains, unlike the homologous MTA1 protein. The RERE zinc finger folds back and interacts with the BAH domain, suggesting that they may work in tandem to interact with a nucleosome. The NONO RRM domains were also shown to form an interaction with the variable region of the BAH domain. Finally, histone tail peptide inhibitors, Apicidin analogues, and a potent HDAC3 specific inhibitor, were tested against the seven HDAC complexes. HDAC inhibitors are important therapeutic drugs however, they target multiple HDAC complexes which lead to severe side effects. The HDAC complexes exhibited different inhibition profiles against these inhibitors, even with the same HDAC. This suggests that it is possible to target specific HDAC complex.</p

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

SARS-CoV-2 RBDs with Q498H mutations have high affinity for ACE2.

(a) Soluble forms of HuACE2, WH-RBD and mutant RBD were expressed in HEK 293 cells and purified on nickel affinity columns. Purified proteins were resolved by SDS/PAGE and detected by Coomassie staining. Molecular masses are shown in kDa. (b) Biolayer interferometry plots of kinetics of binding RBD to HuACE2. WH-RBD, or the indicated RBD, was in solution and Hu-ACE2 immobilized on sensors. Binding was measured with RBD concentrations of 12, 30, 60, 120nM for WH-RBD and 6, 12, 30, 60nM for the mutants. Fitted curves are in red. Data are shown for single experiments representative of at least three independent experiments for each RBD. (c) Kinetic binding constants for RBD binding to HuACE2 measured using biolayer interferometry. Data are shown as means and SEM for at least three independent experiments. (d) Cellular binding of RBD and S477N/Q498H-RBD. Vero-E6 cells were incubated without RBD (C) or with a range of concentrations of WH-RBD (circles) or S477N/Q498H-RBD (triangles) as indicated, at 37°C for 15 mins before washing, antibody staining of bound RBD with fluorescently conjugated antibody, and flow cytometry. Data are shown as mean fluorescence intensity vs RBD concentration (nM) for a single experiment representative of three.</p

Cryo-EM data collection, refinement and validation statistics.

(DOC)</p

The Q498H mutation enables binding of SARS-CoV-2 RBD to rat ACE2.

(a) Kinetic binding constants for RBD binding to RaACE2 measured using biolayer interferometry. Binding was measured with RBD concentrations of 48, 90, 150, 300 and 600nM. Where no binding was detected an additional 1200nM RBD concentration was also tested for binding. RBD with the Q498H mutation binds rat receptor. Data are shown as means and SEM for at least three independent experiments. (b) Rat (RaACE2) and mouse ACE2 (MoACE2) differ from human ACE2 (HuACE2) in key residues contributing to SARS-CoV-2 RBD binding. Residues in human ACE2 at the RBD binding interface are aligned with the corresponding residues in rat and mouse ACE2. Residues that form hydrogen bonds or salt bridges with RBD are in bold and those that differ in rodent are in red. (c) Bonding interactions between Q24 and N487 (d) D30 and K417 (e) Y83 and N487 plus Y489, and (f) K353 and G496, in HuACE2 (yellow) and RBD (green).</p

Q498H and Q498R mutations modify binding of B.1.351 and B.1.617.1/3 variant RBD to HuACE2.

(a) Biolayer interferometry was performed with the indicated RBD in solution and HuACE2 immobilise on the sensor. Binding was measured with RBD concentrations of 12, 30, 60, 120nM, and an additional 6nM concentration in some cases. Curves were fitted and used to calculate Kon, Koff and KD. Data are shown as means and SEM for at least three independent experiments. (b) H498 and Y501 in SARS-CoV-2 RBD compete for interaction with Y41 in HuACE2 and the clash between residues is indicated by the overlapping Van der Waals surfaces. The proximity of the H498 side chain is shown with respect to the side chain of a Y501 inserted into our RBD:ACE2 complex structure. (c) R498 and Y501 side chain positions shown along with D38, Y41 and Q42 (PDB accession number 7BH9 [14]).</p

Example of multiple conformations of Q498 suggested in crystal structures.

The 2Fo-Fc electron density map (in blue, contoured at 1.5σ) and Fo-Fc difference map (contoured at -3σ (red) and green (+3σ)) are shown for the WT crystal structure of ACE2-RBD (PDB ID 6M0J [13]). The positive (green) density adjacent to the side chain of Q498 suggests this can exist in different conformations, thus weakening the interaction with neighbouring residues. Examination of the region of Q498 in PDB entries 7WQB, 7RPV, 7EFR, 7EFP, 7NXC, 7L0N, 7DMU and 6VW1 all show difference electron density adjacent to the side chain. PDB entries 7EKE, 7EKY, 7EKH, 7EKF and 6LZG all have the residue modelled in dual conformations. Only 7LO4 does not show disorder in Q498. (DOCX)</p

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

Effects of Gln498His and Gln498Arg mutations on binding of B.1.617.1/3 and B.1.351 variant RBD to RaACE2.

Effects of Gln498His and Gln498Arg mutations on binding of B.1.617.1/3 and B.1.351 variant RBD to RaACE2.</p

Structure of S477N/Q498H-RBD in complex with HuACE2.

Cryo-EM structure and model of the ACE2-RBD complex. Sharpened Cryo-EM map (a) of the HuACE2-S477N/Q498H-RBD complex with ACE2 coloured yellow and RBD in green. The refined coordinates shown in cartoon representation (b) and coloured as above. In panel (c) H498 can be seen in proximity to ACE2 Y41 forming a non-planar π-interaction while ACE2 residues K353 and D38 are within hydrogen bonding distance to H498 and could contribute to the tighter interaction formed by this RBD mutant, whereas Panel (d) shows Q498 in WH-RBD (PDB accession number 6M0J [13]) in proximity to ACE-2 Q42. Panel (e) indicates that the S477N mutation places this longer side-chain closer to S19 in ACE2 and within hydrogen bonding distance thus enhancing the binding between HuACE2 and S477N/Q498H-RBD. Panel (f) shows positioning of S477 in WH-RBD and ACE2 S19 (PDB accession number 6M0J [13]).</p