Mass Spectrometry-Based Protein Footprinting Defines the Binding Pocket of Crotonylated H3K14 in the PHD1 Domain of BAF45D within the BAF Chromatin Remodeling Complex

The BRG-/BRM-associated
factor (BAF) chromatin remodeling complex
is a central actor in transcription. One mechanism by which BAF affects
gene expression is via its various histone mark readers, including
double plant homeodomains (DPF), located in the BAF45D subunit. DPF
domains recognize lysine acetyl and acylations, including crotonylation,
localized at promoters and enhancers. Despite a significant degree
of conservation between DPF domains, attempts to crystallize BAF45D
with a crotonylated histone 3 peptide (H3K14Cr) were unsuccessful.
In addition, recent cryoEM and modeled structures failed to define
the Req domain of BAF45D, which is responsible for reading lysine
modifications. Thus, the precise mechanism of crotonyl group recognition
and binding by BAF45D within the BAF complex remains unclear. We turned
to protein footprinting mass spectrometry to map the binding interface
between H3K14Cr and BAF45D. This technique is able to demarcate protein-binding
interfaces by modifying surface-accessible residues and is not limited
by protein size or composition. Experiments performed in the isolated
DPF domain of BAF45D (BAF45DDPF)-delineated H3K14Cr peptide
binding across the PHD1 and PHD2 pockets. We observed markedly similar
effects on the BAF45D subunit when assessing H3K14Cr binding in the
purified full BAF complex. The ATPase motor, BRM, also displayed H3K14Cr-protected
peptides in two separate domains that were subsequently evaluated
in direct binding assays. These data confirm the BAF45D–crotonylamide
interaction within its obligate complex and are the first to demonstrate
H3K14Cr direct binding to BRM.