Two Loops Undergoing Concerted Dynamics Regulate the Activity of the ASH1L Histone Methyltransferase

Abstract

ASH1L (Absent, Small, or Homeotic-like 1) is a histone methyltransferase (HMTase) involved in gene activation that is overexpressed in multiple forms of cancer. Previous studies of ASH1L’s catalytic SET domain identified an autoinhibitory loop that blocks access of histone substrate to the enzyme active site. Here, we used both NMR and X-ray crystallography to identify conformational dynamics in the ASH1L autoinhibitory loop. Using site-directed mutagenesis we found that point mutations in the autoinhibitory loop that perturb the structure of the SET domain result in decreased enzyme activity, indicating that the autoinhibitory loop is not a simple gate to the active site but is rather a key feature critical to ASH1L function. We also identified a second loop in the SET-I subdomain of ASH1L that experiences conformational dynamics, and we trapped two different conformations of this loop using crystallographic studies. Mutation of the SET-I loop led to a large decrease in ASH1L enzymatic activity in addition to a significant conformational change in the SET-I loop, demonstrating the importance of the structure and dynamics of the SET-I loop to ASH1L function. Furthermore, we found that three C-terminal chromatin-interacting domains greatly enhance ASH1L enzymatic activity and that ASH1L requires native nucleosome substrate for robust activity. Our study illuminates the role of concerted conformational dynamics in ASH1L function and identifies structural features important for ASH1L enzymatic activity.

H3K36 Methyltransferases as Cancer Drug Targets: Rationale and Perspectives for Inhibitor Development

“…Molecular dynamics simulations, crystallographic and NMR studies have shown that the NSD1 and ASH1L autoinhibitory loops experience conformational heterogeneity in the absence of nucleosome substrate [127,128]. In none of the observed conformations does the autoinhibitory loop completely open to allow nucleosome binding, suggesting that interaction with nucleosome is required to induce loop opening.…”

“…In none of the observed conformations does the autoinhibitory loop completely open to allow nucleosome binding, suggesting that interaction with nucleosome is required to induce loop opening. Nevertheless, conformational dynamics of the loop may be important for facilitating the larger conformational change that must occur upon nucleosome binding [127,128]. Interestingly, the autoinhibitory loop in the crystal structure of the related NSD3 SET domain is disordered [129], further suggesting that autoinhibitory loop dynamics are characteristic of the NSD and related SET domains.…”

“…In NSD1, the autoinhibitory loop is stabilized by a β-turn within the loop as well as hydrophobic contacts with SET-I [121]. In contrast, no β-turn exists in the ASH1L autoinhibitory loop, and there are longer range contacts to SET-I [120,127]. In the SETD2 autoinhibitory loop, there is also no β-turn to stabilize the loop, but the interaction of Arg1670 at the beginning of the loop with the substrate lysine-binding channel pulls the loop into contact with SET-I [122] ( Figure 3B).…”

“…First, developing in vitro methylation assays for H3K36 KMTases can be challenging because the SET domain may require chromatin-interacting domains and/or cofactor proteins for optimal activity on nucleosome substrate. For example, the isolated ASH1L SET domain has weak KMTase activity on nucleosomes, but constructs including the PHD, Bromo and BAH domains have increased activity [127]. An additional obstacle is that inhibitors of KMTases appear to be rare in current high-throughput screening libraries, as three different pharmaceutical companies published SAM-competitive inhibitors of the EZH2 KMTase with similar pyridone-containing scaffolds [4].…”

Dynamic behavior of the post‐SET loop region of NSD1: Implications for histone binding and drug development

“…The dynamics of the post‐SET loop are significant, as the loop must move prior to peptide binding or its movement must be induced upon interaction with the substrate. In addition, mutagenesis studies on the corresponding region of ASH1L have suggested that this loop region is not merely a blockade to peptide binding, but rather plays a more complex role in enzymatic activity . As ligands targeting this region of NSD1 would have to bind prior to the peptide binding in order to be effective, it is necessary to understand the dynamics of this loop region.…”