Ash1L (absent, small, or homeotic-like 1) is a histone methyltransferase (HMT) that mono- and dimethylates lysine 36 of histone H3 (H3K36). It contains the evolutionarily conserved SET domain which catalyzes histone lysine methylation and plays a fundamental role in epigenetic regulation. We previously described crystal structures and backbone assignment of Ash1L SET domain. Crystal structures showed that the substrate binding site is blocked by an autoinhibitory loop. This loop has the highest crystallographic B factors in the SET domain, suggesting that it undergoes conformational dynamics. Analysis of the assignment validated observation from crystal structures, as backbone resonances of residues in the autoinhibitory loop are largely missing in the NMR spectrum. Recent studies reported that histone binding protein Mrg15 activates Ash1L and destabilizes the autoinhibitory loop. However, the dynamics of Ash1L SET, especially during enzyme activation and inhibition, is still poorly understood.<br/>We developed small molecule inhibitors that bind to Ash1L SET. Structural studies revealed that these compounds bind to a pocket close to the autoinhibitory loop. We performed NMR experiments to investigate inhibitor binding in solution. Analysis of these experiments showed that inhibitor binding resulted in a significant number of chemical shift perturbations, and we observed 199 resonances out of 213 non-proline residues, comparing to 181 resonances for Ash1L SET alone. The emergence of new resonances indicates that compound binding rigidifies conformation of the SET domain. This is consistent with unfavorable changes in entropy observed from isothermal titration calorimetry (ITC) experiments. To understand the effect of inhibitor binding on the dynamics of Ash1L SET domain, we assigned the NMR spectra of Ash1L-inhibitor complex. To date we have assigned 88% observed resonances in the SET domain. The assignment demonstrates that inhibitor reduces flexibility of the SET domain in solution. We were able to identify residues that are critical for inhibitor binding, and this will lead us to design compounds with more potent inhibitory activities.

Dynamics of Ash1L SET domain in the presence of inhibitors

2019 Fall National Meeting

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.