NMR study on hHR23B-S5a complex

Abstract

Covalent modification of proteins by the small, evolutionary conserved protein ubiquitin plays a central role in a variety of cellular processes, including bulk protein degradation, cell-cycle control, DNA repair, signal transduction, and transcriptional regulation. Cellular proteins to be degraded are covalently tagged with multiubiquitin chains that are recognized by the 26S proteasome. Although the 20S core performs the degradation, the 19S regulatory cap complex is responsible for the recognition of polyubiquitinated substrates. One subunit of the 19S component, S5a, has been shown to bind polyubiquitin chains as well as the ubiquitin-like domain of hHR23B, the human homologue of the yeast Rad23 protein. We have fosused on hHR23B-S5a complex using nulear magnetic resonance (NMR) spectroscopy. This study elucidates the specific interaction between the ubl domains of hHR23B and S5a. Ubl domain of hHR23B (1-81) with a N-terminal six histidine tag overexpressed as a soluble form in E.coli. And PUBS2 domain of S5a with GST tag overexpressed as a soluble form in E.coli. Several domains of GST fusion proteins were constructed such as 263 to 307, 263 to 315, and 263 to 342, respectively. However, GST-S5a (253-342 ) forms dimmer that comfirmed by HPLC and corss-linking experiments. Also GST-S5a (263-307) is less overexpressed than GST-S5a (263-315) so that we prepared the GST-S5a (263-315) for further NMR study. Binding assay confirmed the interaction between hHR23B and GST-S5a (form 263 to 342). To characterize the interaction with S5a, a NMR study was performed where the $^{15}N-^1H$ HSQC spectrum of ubl domains of hHR23B was monitored upon addition of excess of S5a. Concentrations were calculated by using extinction coefficients based on amino acid composition. Reversely, to characterize the interaction with hHR23B, a NMR study was performed where the $^{15}N-^1H$ HSQC spectrum of S5a was monitored upon addition of excess of hHR23B. We confirmed the stable co...