Crystal structure at 1.9 A resolution of the apoovotransferrin N-lobe bound by sulfate anions: implications for the domain opening and iron release mechanism.
Crystal structure at 1.9 A resolution of the apoovotransferrin N-lobe bound by sulfate anions: implications for the domain opening and iron release mechanism.
复制标题
硫酸根阴离子结合的脱辅铁转铁蛋白 N 叶的 1.9 A 晶体结构：对结构域开放和铁释放机制的影响。
作者：
K. Mizutani;H. Yamashita;B. Mikami;M. Hirose
一些功能性证据表明，阴离子结合到非协同位点是阴离子介导的转铁蛋白铁释放机制的先决条件。我们在这里报告的硫酸根阴离子结合位点的卵转铁蛋白N-叶通过1.90 A分辨率载脂蛋白晶体结构的位置的结构证据。晶体在硫酸铵溶液中生长，属于空间群P6（3）22，晶胞参数为：a = B = 125.17 A，c = 87.26 A。通过同晶置换，使用Pt和Au衍生物进行结构测定。对于最终模型，结构精修在7.0-1.90 A的分辨率范围内给出了0.187的R因子。从电子密度图上可以看出，存在四个SO（4）（2）（-）键合阴离子.其中三个表现出相当低的B因子都位于开放的域间裂隙（网站1-3）。在位点1，结合阴离子直接与Fe（3+）配位配体相互作用; SO（4）O 1和SO（4）O3与His 250 NE 2形成氢键。相同硫酸根阴离子的氧原子O3与铰链链中的Ser 91 OG形成氢键。SO（4）O2和SO（4）O3分别与Arg 121 NE和NH 2形成氢键，这两个基团都是全转铁蛋白中CO（3）（2）（-）阴离子的共有锚基. SO（4）（2）（-）的前一个氧原子也与Ser 122 N形成氢键，而Ser 122 N与铁离子配体Asp 60 OD 2形成氢键。位点1和2中的一些SO（4）（2）（-）氧原子通过H（2）O分子与功能重要的蛋白质基团间接相互作用，例如其他Fe（3+）-配位配体Tyr 92 OH和Tyr 191 OH，以及二赖氨酸触发基团Lys 209 NZ。在位点3，SO（4）O 1和SO（4）O 4分别与Ser 192 OG和Tyr 191 N形成氢键，SO（4）O2与Ser 192 N和Ser 192 OG形成氢键.这些结构数据是一致的，认为阴离子结合的域间裂缝，特别是网站1和2，发挥关键作用的域开放和协同碳酸根阴离子释放的铁释放机制的卵转铁蛋白N-叶。
Several lines of functional evidence have shown that anion binding to a nonsynergistic site is a prerequisite for the anion-mediated iron release mechanism of transferrins. We report here structural evidence of the location of sulfate anion binding sites of the ovotransferrin N-lobe via the 1.90 A resolution apo crystal structure. The crystals were grown in an ammonium sulfate solution and belonged to space group P6(3)22 with the following unit cell dimensions: a = b = 125.17 A and c = 87.26 A. The structural determination was performed by isomorphous replacement, using Pt and Au derivatives. The structure refinement gave an R-factor of 0.187 in the resolution range of 7.0-1.90 A for the final model. From the electron density map, the existence of four bound SO(4)(2)(-) anions was detected. Three of them that exhibited reasonably low B-factors were all located in the opened interdomain cleft (sites 1-3). In site 1, the bound anion directly interacts with an Fe(3+)-coordinating ligand; SO(4) O1 and SO(4) O3 form hydrogen bonds with His250 NE2. Oxygen atom O3 of the same sulfate anion makes a hydrogen bond with Ser91 OG in a hinge strand. The sulfate anion in site 2 partially occupies the synergistic anion binding sites; SO(4) O2 and SO(4) O3 are hydrogen bonded to Arg121 NE and NH2, respectively, both of which are consensus anchor groups for CO(3)(2)(-) anion in holotransferrins. The former oxygen atom of SO(4)(2)(-) is also hydrogen bonded to Ser122 N, which forms a hydrogen bond with Fe(3+)-coordinating ligand Asp60 OD2 in holotransferrins. Some of the SO(4)(2)(-) oxygen atoms in sites 1 and 2 interact indirectly through H(2)O molecules with functionally important protein groups, such as the other Fe(3+)-coordinating ligands, Tyr92 OH and Tyr191 OH, and a dilysine trigger group, Lys209 NZ. In site 3, SO(4) O1 and SO(4) O4 form hydrogen bonds with Ser192 OG and Tyr191 N, respectively, and SO(4) O2 forms hydrogen bonds with Ser192 N and Ser192 OG. These structural data are consistent with the view that the anion bindings to the interdomain cleft, especially to sites 1 and 2, play crucial roles in the domain opening and synergistic carbonate anion release in the iron release mechanism of the ovotransferrin N-lobe.