1 research outputs found
Peptide Library Approach to Uncover Phosphomimetic Inhibitors of the BRCA1 C‑Terminal Domain
Many
intracellular protein–protein interactions are mediated
by the phosphorylation of serine, and phosphoserine-containing peptides
can inhibit these interactions. However, hydrolysis of the phosphate
by phosphatases, and the poor cell permeability associated with phosphorylated
peptides has limited their utility in cellular and <i>in vivo</i> contexts. Compounding the problem, strategies to replace phosphoserine
in peptide inhibitors with easily accessible mimetics (such as Glu
or Asp) routinely fail. Here, we present an <i>in vitro </i>selection strategy for replacement of phosphoserine. Using mRNA display,
we created a 10 trillion member structurally diverse unnatural peptide
library. From this library, we found a peptide that specifically binds
to the C-terminal domain (BRCT)<sub>2</sub> of breast cancer associated
protein 1 (BRCA1) with an affinity comparable to phosphorylated peptides.
A crystal structure of the peptide bound reveals that the pSer-x-x-Phe
motif normally found in BRCA1 (BRCT)<sub>2</sub> binding partners
is replaced by a Glu-x-x-4-fluoroPhe and that the peptide picks up
additional contacts on the protein surface not observed in cognate
phosphopeptide binding. Expression of the peptide in human cells led
to defects in DNA repair by homologous recombination, a process BRCA1
is known to coordinate. Overall, this work validates a new <i>in vitro</i> selection approach for the development of inhibitors
of protein–protein interactions mediated by serine phosphorylation