BRCT domains contain an intrinsic post‐translational modification (PTM) recognition code that affects it stability

Abstract

BRCA1 C‐terminal (BRCT) domains are protein binding modules that relay signals throughout various cellular pathways. The human genome encodes 23 BRCT‐domain containing proteins that are commonly found in tandem and have been characterized as phosphoprotein binding domains with functions throughout the DNA damage response. Emerging evidence suggests these domains bind other post‐translationally modified (PTM) substrates although the details are not defined. Recently, the BRCT domains from BRCA1 were shown to bind an asymmetrically dimethylated arginine (ADMA) p300 substrate to regulate the transcription of the p21 in response to DNA damage. Based on this observation, we hypothesize that the methyl‐specific binding site for the ADMA interaction is distinct from the phospho‐substrate recognition interface. To test this hypothesis, we conducted peptide binding and thermal stability experiments to demonstrate the change in stability upon binding phospho‐versus methylated substrates. Our results show that phosphorylated substrates increase the stability of BRCA1's BRCT domains. Doubly phosphorylated substrates showed the greatest increase in stability while the methylated substrate demonstrated a decrease in stability. To define the BRCT‐ADMA binding interface, we set out to determine the three‐dimensional structure. High‐throughput co‐crystallization of the BRCA1 BRCT protein with a ADMA peptide derived from the p300 substrate identified one condition for crystal growth optimization. In silico modeling of the BRCA1 BRCT structure with the ADMA molecule has highlighted two potential binding sites for the ADMA substrate, which is not conserved throughout the BRCT domain family. Our results indicate that while BRCT domains share a conserved mode of binding phosphorylated substrates, which increases their stability, and their ability to binding methylated substrates may not be conserved. These finding will be critical in understanding how BRCT domains can accommodate binding various modified substrates and regulating signals throughout the DNA damage response.Support or Funding InformationNCI K22 CA166544‐03This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.