Abstract 1546 Use of 2D-NMR to Determine Specific Contributions of Ubiquitin Binding Sites to Polyubiquitin Chain Recognition Within ASCC2 Subunit of DNA Alkylation Damage Complex ALKBH3-ASCC

DNA alkylation damage can be a cause of disease when it is improperly repaired and is also selectively used to treat diseases, such as its use in chemotherapeutics. Consequently, it is critical to understand the mechanisms by which DNA alkylation damage is repaired. It was recently discovered that K63-polyubiquitin chains recruit the ALKBH3-ASCC DNA repair complex to alkylation damage sites. ASCC2 is the protein subunit of the ALKBH3-ASCC complex that is responsible for binding polyubiquitin chains, though the basis for ASCC2's specificity for K63-linked polyubiquitin chains is unknown. ASCC2's ubiquitin-binding CUE domain is hypothesized to contain two binding sites; a conserved binding site present in all CUE domains, and a novel second ubiquitin-binding site that may impart selectivity for K63-linked chains. To test if both putative binding sites contribute to ASCC2's affinity for K63-linked polyubiquitin chains, 2D-NMR experiments were utilized to determine the individual binding affinities (Kd values) for the distal and proximal ubiquitin within K63Ub2. Chemical shift perturbation (CSP) analysis for residues from both the distal and proximal ubiquitin have shown that the individual Kd value for the proximal ubiquitin is around 2-fold higher, which suggests a stronger binding affinity for the distal ubiquitin when interacting with polyubiquitin chains. Our laboratory is currently conducting PRE-NMR, NOE-NMR, and cross-linking experiments to further characterize the intermolecular interactions responsible for mediating the binding of the ASCC2 CUE domain to proximal ubiquitin. This will aid us in elucidating the structural basis of ASCC2's selectivity for K63-linked polyubiquitin chains and subsequently rationalize the enhanced binding affinity for the distal ubiquitin compared to the proximal. Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award number R15GM140410. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.