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