The Interaction Between Ubquitin-binding Domains And Its Structure

Part IUbiquitination is an important signal that targets proteins for degradation or regulates their functions. To date, there are over 15 different ubiquitin-binding domains identified. The previously identified BMSC-UbP protein derived from bone marrow stromals cells contains an ubiquitin-associated (UBA) domain at the C terminus that has been implicated in linking the ubiquitin system and cellular processes. We find it can bind with the ubiquitin-like domain (UBQ) of BAT-3 by GST pull-down. Then, we use the NMR titration to confirm the binding site of BMSC-UbP-UBA domain with BAT-3 UBQ domain. The chemical shift perturbation studies revealed that the UBA domain binds with the conserved five-strandedβ-sheet of UBQ via hydrophobic interactions. The structural model of BMSC-UbP-UBA domain complexed with BAT-3 UBQ domain was constructed by chemical shift mapping combined with the docking program HADDOCK, which is in agreement with the result from mutagenesis studies. In the complex structure, five residues (Met76, Ile78, Leu99, Leu101 and Phe103) of BMSC-UbP UBA form a trident anchoring the domain to the hydrophobic concave surface of BAT3 UBQ domain.Part II1. In the present paper we report the results obtained with the photosystemâ…¡(PSâ…¡) isolated from spinach and treated by MgCl₂, and studied the effect of Mg²⁺ on spectral characteristics and photosynthetic functions of PSâ…¡. The results showed that Mg²⁺ treatment at a suitable concentration could significantly increase the absorption intensity of PSâ…¡and the intensity ratio of Soret band to Q band of chlorophyll-a, elevate the excited peak intensity at 230, 278 and 343 nm, and the emitted peak intensity at 304 and 682 nm, and the ratio of F278/F230, respectively. The results implied that Mg²⁺ increased absorbance for visible light, improved energy transfer among amino acids within PSâ…¡protein complex and accelerate energy transport from tyrosine residue to chlorophyll-a. And the photochemical activity and oxygen evolving rate of PSâ…¡were enhanced by Mg²⁺. This is viewed as evidence that Mg²⁺ can promote energy transfer and oxygen evolution in PSâ…¡o f spinach.2. Mg²⁺ in various concentrations was added to purified Rubisco in vitro to gain insight into the mechanism of molecular interactions between Mg²⁺ and Rubisco; this greatly increased the enzyme activity. The enzyme activity essays showed that the reaction between Rubisco and Mg²⁺ was two-order, which means that the enhancement of Rubisco activity was accelerated by low concentration of Mg²⁺ and slowed by high concentration of Mg²⁺. The kinetics constant (Km) and Vmax was 1.91μM and 1.13μmol CO2·mg-1 protein·min-1, respectively, at a low concentration of Mg²⁺, and 3.45μM and 0.32μmol CO2·mg-1 protein·min-1, respectively, at a high concentration of Mg²⁺. By UV absorption and fluorescence spectroscopy assays, the Mg²⁺ was determined to be directly bound to Rubisco; the binding site of Mg²⁺ to Rubisco was 0.275. The binding constants (KA) of the binding site were 6.33×10⁴ and 5.5×10⁴L·mol^-1. Based on the analysis of CD spectra, it was concluded that the binding of Mg²⁺ in the range of 5–75μM did not alter the secondary structure of Rubisco, suggesting that the observed enhancement of Rubisco carboxylase activity was caused by a subtle structural change of the active site through the formation of the complex with Mg²⁺. But high concentration of Mg²⁺ altered the secondary structure of Rubisco, led to the reduction in Rubisco carboxylase activity.