Yao Huili, Rui Huan, Kumar Ritesh, Eshelman Kate, Lovell Scott, Battaile Kevin P, Im Wonpil, Rivera Mario
Department of Chemistry, ‡Del Shankel Structural Biology Center, and §Department of Molecular Biosciences and Center for Bioinformatics, University of Kansas , Multidisciplinary Research Building, 2030 Becker Drive, Lawrence, Kansas 66047, United States.
Biochemistry. 2015 Mar 3;54(8):1611-27. doi: 10.1021/bi501255r. Epub 2015 Feb 17.
X-ray crystallography, molecular dynamics (MD) simulations, and biochemistry were utilized to investigate the effect of introducing hydrophobic interactions in the 4-fold (N148L and Q151L) and B-pores (D34F) of Pseudomonas aeruginosa bacterioferritin B (BfrB) on BfrB function. The structures show only local structural perturbations and confirm the anticipated hydrophobic interactions. Surprisingly, structures obtained after soaking crystals in Fe2+-containing crystallization solution revealed that although iron loads into the ferroxidase centers of the mutants, the side chains of ferroxidase ligands E51 and H130 do not reorganize to bind the iron ions, as is seen in the wt BfrB structures. Similar experiments with a double mutant (C89S/K96C) prepared to introduce changes outside the pores show competent ferroxidase centers that function akin to those in wt BfrB. MD simulations comparing wt BfrB with the D34F and N148L mutants show that the mutants exhibit significantly reduced flexibility and reveal a network of concerted motions linking ferroxidase centers and 4-fold and B-pores, which are important for imparting ferroxidase centers in BfrB with the required flexibility to function efficiently. In agreement, the efficiency of Fe2+ oxidation and uptake of the 4-fold and B-pore mutants in solution is significantly compromised relative to wt or C89S/K96C BfrB. Finally, our structures show a large number of previously unknown iron binding sites in the interior cavity and B-pores of BfrB, which reveal in unprecedented detail conduits followed by iron and phosphate ions across the BfrB shell, as well as paths in the interior cavity that may facilitate nucleation of the iron phosphate mineral.
利用X射线晶体学、分子动力学（MD）模拟和生物化学方法，研究了在铜绿假单胞菌细菌铁蛋白B（BfrB）的4倍对称轴（N148L和Q151L）和B孔（D34F）中引入疏水相互作用对BfrB功能的影响。结构显示仅存在局部结构扰动，并证实了预期的疏水相互作用。令人惊讶的是，将晶体浸泡在含Fe2+的结晶溶液后获得的结构表明，尽管铁加载到突变体的铁氧化酶中心，但铁氧化酶配体E51和H130的侧链并未像野生型BfrB结构那样重新组织以结合铁离子。对准备在孔外引入变化的双突变体（C89S/K96C）进行的类似实验表明，其铁氧化酶中心功能正常，与野生型BfrB中的中心功能相似。将野生型BfrB与D34F和N148L突变体进行比较的MD模拟表明，突变体的灵活性显著降低，并揭示了连接铁氧化酶中心与4倍对称轴和B孔的协同运动网络，这对于赋予BfrB中的铁氧化酶中心有效发挥功能所需的灵活性很重要。与此一致的是，相对于野生型或C89S/K96C BfrB，溶液中4倍对称轴和B孔突变体的Fe2+氧化效率和摄取量显著受损。最后，我们的结构显示在BfrB的内腔和B孔中有大量以前未知的铁结合位点，这以前所未有的细节揭示了铁和磷酸根离子穿过BfrB外壳所遵循的通道，以及内腔中可能促进磷酸铁矿物成核的路径。
