Abstract 1454 Structural Elucidation of the ASCC2 CUE Domain Binding Interface with K63-Linked Polyubiquitin through Hydrogen-Deuterium Exchange NMR Analysis

Alkylation-induced DNA damage is a potentially toxic lesion that can disrupt vital cellular processes and, in some cases, even trigger cell death. In response to alkylation damage, the ALKBH3-ASCC DNA alkylation repair complex localizes to sites of damage through the selective recognition of K63-linked polyubiquitin (K63polyUb) chains. This recognition is facilitated by the ASCC2 protein subunit within the complex via its CUE domain, a ubiquitin-binding region encompassing approximately 50 amino acids. The molecular basis for this polyubiquitin-binding specificity was previously unclear, as CUE domains in other ubiquitin-binding proteins typically only bind to a single ubiquitin and do not differentiate between different polyubiquitin linkages. It has recently been revealed that the ASCC2 CUE domain possesses two distinct ubiquitin-binding interfaces, allowing it to simultaneously interact with adjacent ubiquitin moieties within K63polyUb chains. While the distal (ubiquitin bonded through its C-terminus) binding interface has been extensively characterized, the constituent residues of the proximal (ubiquitin bonded through its lysine side chain) interface remain elusive. Here, we have utilized nuclear magnetic resonance (NMR) spectroscopy to elucidate the composition of this unique proximal binding interface by detecting disparities in amide proton hydrogen–deuterium exchange (HDX) rates between the free ASCC2 CUE domain and its complexed form in solution. Exchange rates were monitored using band-selective optimized flip-angle short transient (SOFAST) heteronuclear multiple quantum coherence (HMQC) experiments. We observed that the residues 23lle, 50Leu, and 6Lys of the proximal ubiquitin displayed significant changes in exchange rate upon binding to the ASCC2 CUE domain, suggesting potential involvement of these residues at the binding interface. In conclusion, our findings support the potential of HDX as a tool for probing the binding interface of the ASCC2:K63polyUb interaction. The residues identified through HDX will be incorporated into the Xplor-NIH docking software to enhance the existing model of the interaction, utilizing the established structures of ubiquitin and the ASCC2 CUE domain. These refined models will not only enable the formulation of novel hypotheses regarding the ASCC2 CUE domain's unique specificity for K63-linked chains but also provide insight into the intricate mechanisms governing ubiquitin signaling. Characterizing the binding interface between the ASCC2 CUE domain and K63polyUb chains is of paramount importance within the realm of ubiquitin signaling, as it sheds light on the intricate mechanisms by which ubiquitin-binding proteins distinguish among diverse polyubiquitin modifications abundantly present in the cell. Furthermore, these findings will expand our understanding of ubiquitin-mediated repair mechanisms and hold promise for the development of targeted therapeutic interventions in DNA damage-related disorders. This research was made possible through the support of the National Institute of General Medical Sciences of the National Institutes of Health under Award Number 2R15GM140410-02.