Characterization of Cullin-box sequences that direct recruitment of Cul2-Rbx1 and Cul5-Rbx2 modules to elongin BC-based ubiquitin ligases
JOURNAL OF BIOLOGICAL CHEMISTRY
Authors: Mahrour, Nawel; Redwine, William B.; Florens, Laurence; Swanson, Selene K.; Martin-Brown, Skylar; Bradford, William D.; Staehling-Hampton, Karen; Washburn, Michael P.; Conaway, Ronald C.; Conaway, Joan W.
The Elongin BC-box protein family includes the von Hippel-Lindau tumor suppressor and suppressor of cytokine signaling proteins, which are substrate recognition subunits of structurally related classes of E3 ubiquitin ligases composed of Elongin C-Elongin B-Cullin 2-Rbx1 (Cul2 ubiquitin ligases) or of Elongin C-Elongin B-Cullin 5-Rbx2 (Cul5 ubiquitin ligases). The Elongin BC complex acts as an adaptor that links a substrate recognition subunit to heterodimers of either Cullin 2 (Cul2) and RING finger protein Rbx1 or Cullin 5 (Cul5) and Rbx2. It has been shown (Kamura, T., Maenaka, K., Kotoshiba, S., Matsumoto, M., Kohda, D., Conaway, R. C., Conaway, J. W., and Nakayama, K. I. (2004) Genes Dev. 18, 3055 - 3065) that interaction of BC-box proteins with their cognate Cul-Rbx module is determined by specific regions, called Cul2- or Cul5-boxes, located immediately downstream of their BC-boxes. Here, we investigate further the mechanisms governing assembly of BC-box proteins with their specific Cul-Rbx modules. Through purification and characterization of a larger collection of BC-box proteins that serve as substrate recognition subunits of Cul2 and Cul5 ubiquitin ligases and through structure-function studies, we define Cul2- and Cul5-boxes in greater detail. Although it previously appeared that there was little sequence similarity between Cul5- and Cul2-box motifs, analyses of newly identified BC-box proteins reveal that residues conserved in the Cul2-box represent a subset of those conserved in the Cul5-box. The sequence motif LP phi P, which is conserved in most Cul5-boxes and has been suggested to specify assembly of Cul5 ligases, is compatible with Cul2 interaction. Finally, the spacing between BC- and Cullin- boxes is much more flexible than has been appreciated and can vary from as few as 3 and as many as similar to 80 amino acids. Taken together, our findings shed new light on the mechanisms by which BC- box proteins direct recruitment of Cullin-Rbx modules during reconstitution of ubiquitin ligases.
Synthesis, structures and Helicobacter pylori urease inhibitory activity of copper(II) complexes with tridentate aroylhydrazone ligands
JOURNAL OF INORGANIC BIOCHEMISTRY
Authors: Pan, Lin; Wang, Cunfang; Yan, Kai; Zhao, Kedong; Sheng, Guihua; Zhu, Hailiang; Zhao, Xinlu; Qu, Dan; Niu, Fang; You, Zhonglu
A series of new copper(II) complexes were prepared. They are [CuL1 (NCS)] (1), [CuCILl]center dot CH3OH (2), [CuCIL2]center dot CH3OH (3), [CuL3(NCS)]center dot CH3OH (4), [CuL4(NCS)]center dot 0.4H(2)O (5), and [CuL5(bipy)] (6), where L-1, L-2, L-3 and L-4 are the deprotonated form of N'-(2-hydroxybenzylidene)-3-methylbenzohydrazide, 4-bromo-N'-(2-hydroxy-5-methoxybenzylidene)benzohydrazide, N'-(2-hydroxy-5-methoxybenzylidene)-3-methylbenzohydrazide and 2chloro-N'-(2-hydroxy-5-methoxybenzylidene)benzohydrazide, respectively, L-5 is the dianionic form of N'-(2-hydroxybenzylidene)-3-methylbenzohydrazide, and bipy is 2,2'-bipyridine. The complexes were characterized by infrared and UV-Vis spectra and single crystal X-ray diffraction. The Cu atoms in complexes 1, 2, 3, 4 and 5 are coordinated by the NOO donor set of the aroylhydrazone ligands, and one Cl or thiocyanate N atom, forming square planar coordination. The Cu atom in complex 6 is in a square pyramidal coordination, with the NOD donor set of L-1, and one N atom of bipy defining the basal plane, and with the other N atom of bipy occupying the apical position. Complexes 1, 2, 3, 4 and 5 show effective urease inhibitory activities, with IC50 values of 5.14, 0.20, 4.06, 5.52 and 0.26 mu M, respectively. Complex 6 has very weak activity against urease, with IC50 value over 100 mu M. Molecular docking study of the complexes with the Helicobacter pylori urease was performed. The relationship between structures and urease inhibitory activities indicated that copper complexes with square planar coordination are better models for urease inhibition. (C) 2016 Elsevier Inc. All rights reserved.