Abstract 2099 Using Nuclear Overhauser Effect (NOE) 3-Dimensional NMR to characterize the binding interactions of the ASCC2 CUE Domain and the Proximal Ubiquitin of K63 Diubiquitin

Alkylation damage, a lesion to DNA or RNA that can be toxic, occurs to DNA thousands of times a day for every cell. Alkylation damage can be caused by a number of endogenous factors, including metabolism and external factors such as environmental agents and alkylating drugs. Alkylation damage is repaired by the ALKBH3-ASCC complex, which is recruited by K63-linked polyubiquitin. Within the ALKBH3-ASCC repair complex, there are four proteins, which include ALKBH3, ASCC1, ASCC2, and ASCC3. ASCC2 is directly involved in binding the K63-linked polyubiquitin, which are chains that recruit the ALKBH3-ASCC complex to sites of alkylation damage. The CUE domain is the part of ASCC2 that binds to K63-linked polyubiquitin. A CUE domain consists of three alpha helices and contains a hydrophobic patch, which is what binds to the ubiquitin's hydrophobic patch. Typically, CUE domains are not selective for specific types of polyubiquitin chains, however, the ASCC2 CUE domain is unique because it was found to have a strong binding affinity for K63-linked polyubiquitin chains. Evidence shows that ASCC2 binds to diubiquitin at the distal ubiquitin (linked through C-terminus) using the conserved hydrophobic patch found in all CUE domains, but the details of the binding between the ASCC2 CUE domain and the proximal ubiquitin (linked through lysine) are still unknown. NOE 3-D NMR can be used to determine the intermolecular interactions between the CUE domain and the proximal ubiquitin. The goal is to determine the distances between the atoms at the binding interface and to create a model of the interactions between ASCC2 and the proximal ubiquitin. NOE NMR is a process that requires compounds to be selectively isotopically labeled before testing. The specific labeling for the experiment is 2H-15N ASCC2, 13C distal ubiquitin, and 2H proximal ubiquitin. The labeling with deuterium, purification, and concentration of the proximal ubiquitin has been completed and a similar process for the distal ubiquitin and ASCC2 CUE domain is expected to be completed in the coming months. This will be followed by the performance of a ubiquitin conjugation reaction and NMR data collection at The Johns Hopkins University Biomolecular NMR Center. Once the data are obtained, the intermolecular distances of atoms at the binding interface will be determined and used to create a model of the interaction between the ASCC2 CUE domain and K63-linked diubiquitin. Funding through NIH grant.