|
Figure 4.
Figure 4. PCAF Bromodomain Competing against TAR RNA for
Binding to Tat AcK50 Peptide(A) Superimposition of a selected
region of 2D ^1H-^15N HSQC spectra of a ^13C/^15N-labeled PCAF
bromodomain protein in the free form (black), in the presence of
Tat AcK50 peptide (molar ratio of 1:1.2) (red), and in the
presence of Tat AcK50 peptide and TAR RNA (molar ratio of
1:1.2:1 for protein:peptide:RNA) (blue). The spectra show
chemical shift changes of the backbone amide resonances of
protein residues due to peptide binding.(B) Superimposition of
2D ^1H-^13C HSQC spectra of the PCAF bromodomain collected under
the same conditions as described in (A). The NMR spectra exhibit
chemical shift changes of the side chain methyl group resonances
of protein residues due to peptide binding. The same
color-coding scheme was used as in (A). Arrows indicate chemical
shift changes of protein NMR resonances from the free form
(black) to the Tat AcK50 peptide-bound form (red). Note that
only the bromodomain residues (i.e., A757 and V752) that
directly interact with the Tat peptide, as shown in the
three-dimensional structure, exhibited major chemical shift
changes upon peptide binding or in competing against TAR RNA for
binding to the Tat peptide. More importantly, addition of TAR
RNA causes only small shifts of the protein signals from the Tat
peptide-bound position toward the free form position, suggesting
that the PCAF bromodomain competes effectively against TAR RNA
for binding to the lysine-acetylated Tat peptide. We observed by
NMR no significant nonspecific interactions between the protein
and TAR RNA under these conditions.
|
