Structural studies of hepatitis B virus X protein, HBx-interacting protein, and antifungal protein tenecin 3간염 B 바이러스 X 단백질과 X 결합 단백질 및 항진균 단백질 테네신 3의 구조 연구
The hepatitis B virus X protein (HBx) is highly linked with liver diseases and the development of hepatocellular carcinoma. A novel protein, designated as HBx-interacting protein (XIP), has been shown to abolish the transactivation functions of HBx. Under physiological conditions, XIP was composed mainly of random-coils; however, in the presence of sodium dodecyl sulfate (SDS) the helical population increased up to 40 %. The secondary structure of XIP was determined with the chemical shift index method using heteronuclear multidimensional nuclear magnetic resonance spectroscopy in the presence of SDS. Analysis identified cx-helices that included amino acid 8 to 12, 32 to 38, 42 to 54 and 82 to 91. XIP deletion mutants were synthesized and used in in vitro binding assays. The results of these experiments indicated that nearly the entire sequence of XIP is required for binding to HBx and that truncation of the cc-helices in XIP reduced the overall helicity of the protein more than expected by the extent of the truncation. These results imply that the deletion of a portion of a-helix affects the formation of other helical segment in the presence of SDS and that the compact folding of XIP may be necessary to generate functional protein-protein interaction surfaces.
Preparation of HBx was attempted. Several versions of fusion proteins were constructed such as polyhistidine-tag, glutathione S transferase, thioredoxin and maltose binding protein (MBP) fusions. Except MBP-X, all of fusion proteins were overexpressed as inclusion bodies. Histagged HBx was refolded in acidic pH. However, ATPase assay proved that the refolded protein did not have a functional activity. Although MBP-X was soluble, light scattering measurement indicated that the protein existed as soluble aggregates. Moreover, MBP-X was not found to interact with XIP. The forced monomerization of MBP-X was not sufficient to recover the activity of HBx. Oxidative refolding was performed on MBP-X, and a monom...