The ETS family members display specific DNA binding
site preferences. As an example, PU.1 and ETS-1 recognize
different DNA sequences with a core element centered over
5′-GGAA-3′ and 5′-GGAA/T-3′, respectively.
To understand the molecular basis of this recognition,
we carried out site-directed mutagenesis experiments followed
by DNA binding studies that use electrophoretic mobility
shift assay (EMSA) and surface plasmon resonance methods.
EMSA experiments identified amino acid changes A231S and/or
N236Y as being important for PU.1 recognition of both 5′-GGAA-3′
and 5′-GGAT-3′ containing oligonucleotides.
To confirm these data and obtain accurate binding parameters,
we performed kinetic studies using surface plasmon resonance
on these mutants. The N236Y substitution revealed a weak
protein-DNA interaction with the 5′-GGAA-3′
containing oligonucleotide caused by a faster release of
the protein from the DNA (koff tenfold
higher than the wild-type protein). With the double mutant
A231S-N236Y, we obtained an increase in binding affinity
and stability toward both 5′-GGAA-3′ and 5′-GGAT-3′
containing oligonucleotides. We propose that substitution
of alanine for serine introduces an oxygen atom that can
accept hydrogen and interact with potential water molecules
or other atoms to make an energetically favorable hydrogen
bond with both 5′-GGAA-3′ and 5′-GGAT-3′
oligonucleotides. The free energy of dissociation for the
double mutant A231S-N236Y with 5′-GGAA-3′ (ΔΔG((A231S-N236Y)
− (N236Y)) = −1.2 kcal mol−1)
confirm the stabilizing effect of this mutant in the protein-DNA
complex formation. We conclude that N236Y mutation relaxes
the specificity toward 5′-GGAA-3′ and 5′-GGAT-3′
sequences, while A231S mutation modulates the degree of
specificity toward 5′-GGAA-3′ and 5′GGAT-3′
sequences. This study explains why wild-type PU.1 does
not recognize 5′-GGAT-3′ sequences and in addition
broadens our understanding of 5′-GGAA/T-3′
recognition by ETS protein family members.
