Won Hyung-Sik, Lee Yeon-Hee, Kim Ji-Hun, Shin In Seon, Lee Mann Hyung, Lee Bong-Jin
National Research Laboratory for Membrane Protein Structure, College of Pharmacy, Seoul National University, Seoul 151-742, Korea.
J Biol Chem. 2004 Apr 23;279(17):17466-72. doi: 10.1074/jbc.M308390200. Epub 2004 Feb 9.
Urease activation is critical to the virulence of many human and animal pathogens. Urease possesses multiple, nickel-containing active sites, and UreE, the only nickel-binding protein among the urease accessory proteins, activates urease by transporting nickel ions. We performed NMR experiments to investigate the solution structure and the nickel-binding properties of Bacillus pasteurii (Bp) UreE. The secondary structures and global folds of BpUreE were determined for its metal-free and nickel-bound forms. The results indicated that no major structural change of BpUreE arises from the nickel binding. In addition to the previously identified nickel-binding site (Gly(97)-Cys(103)), the C-terminal tail region (Lys(141)-His(147)) was confirmed for the first time to be involved in the nickel binding. The C-terminally conserved sequence ((144)GHQH(147)) was confirmed to have an inherent nickel-binding ability. Nickel addition to 1.6 mm subunit, a concentration where BpUreE predominantly forms a tetramer upon the nickel binding, induced a biphasic spectral change consistent with binding of up to at least three nickel ions per tetrameric unit. In contrast, nickel addition to 0.1 mm subunit, a concentration at which the protein is primarily a dimer, caused a monophasic spectral change consistent with more than 1 equivalent per dimeric unit. Combined with the equilibrium dialysis results, which indicated 2.5 nickel equivalents binding per dimer at a micromolar protein concentration, the nickel-binding stoichiometry of BpUreE at a physiological concentration could be three nickel ions per dimer. Altogether, the present results provide the first detailed structural data concerning the nickel-binding properties of intact, wild-type BpUreE in solution.
脲酶激活对许多人类和动物病原体的毒力至关重要。脲酶具有多个含镍活性位点，而脲酶辅助蛋白中唯一的镍结合蛋白UreE通过转运镍离子来激活脲酶。我们进行了核磁共振实验，以研究巴氏芽孢杆菌（Bp）UreE的溶液结构和镍结合特性。确定了BpUreE无金属形式和镍结合形式的二级结构和整体折叠。结果表明，镍结合并未引起BpUreE的主要结构变化。除了先前确定的镍结合位点（Gly(97)-Cys(103)）外，首次证实C末端尾部区域（Lys(141)-His(147)）参与镍结合。C末端保守序列（(144)GHQH(147)）被证实具有固有的镍结合能力。向1.6 mM亚基添加镍，在该浓度下BpUreE在镍结合后主要形成四聚体，诱导了双相光谱变化，这与每个四聚体单元至少结合三个镍离子一致。相比之下，向0.1 mM亚基添加镍，在该浓度下蛋白质主要是二聚体，导致单相光谱变化，与每个二聚体单元结合超过1当量一致。结合平衡透析结果，即在微摩尔蛋白质浓度下每个二聚体结合2.5个镍当量，生理浓度下BpUreE的镍结合化学计量可能是每个二聚体三个镍离子。总之，目前的结果提供了关于溶液中完整野生型BpUreE镍结合特性的首个详细结构数据。
