NMR study on ubiquitin-like domain of hHR23B

Abstract

The ubiqutin (Ub)-like domain of hHR23B is known to specifically interact with S5a, a subunit of 26S proteasome complex. Since hHR23B is one of necessary factor in DNA repair systems, such an interaction might play a role in the regulation of the DNA repair process. The structural studies of the ub-like domain of hHR23B using nuclear magnetic resonance (NMR) spectroscopy was attempted in this study to delineate the specific interaction between the Ub-like domain of hHR23B and S5a. Ub-like domain of hHR23B with a N-terminal six histidine tag overexpressed as a soluble form in E.coli. Binding assay confirmed the interaction between GST-S5a (residues of 263 to 342). Protein backbone resonances of $C^\alpha$, $C^\beta$, CO, $N^H$, and $H^N$ were fully assigned using several hetero-nuclear NMR spectroscopy ; CBCA(CO)NH, HNCACB, $1^H-{15}^N HSQC$, $1^H-{13}^C$ HSQC and HN(CO). Unlike the ubiquitin protein family, featuring the $\beta \beta \alpha \beta \beta \alpha \beta$ fold of ubiquitin, Ub-like domain of hHR23B has $\beta \beta \alpha \beta \alpha \beta \beta$. Furthermore, Ub-like domain in hHR23B has additional eight amino acids to ubiquitin at C-terminus. These characteristics seem to explain the cause that Ub-like domain of hHR23B has specific interaction to S5a.