Trikha J, Theil E C, Allewell N M
Department of Biochemistry, University of Minnesota, St. Paul 55108, USA.
J Mol Biol. 1995 May 19;248(5):949-67. doi: 10.1006/jmbi.1995.0274.
Ferritin is a highly conserved multisubunit protein in animals, plants and microbes which assembles with cubic symmetry and transports hydrated iron ions and protons to and from a mineralized core in the protein interior. We report here the high resolution structures of recombinant amphibian red-cell L ferritin and two mutants solved under two sets of conditions. In one mutant, Glu56, 57, 58 and 60 were replaced with Ala, producing a lag phase in the kinetics of iron uptake. In the second mutant, His25 was replaced with Tyr with, at most, subtle effects on function. A molecule of betaine, used in the purification, is bound in all structures at the 2-fold axis near the recently identified heme binding site of bacterioferritin and horse spleen L ferritin. Comparisons of the five amphibian structures identify two regions of the molecule in which conformational flexibility may be related to function. The positions and interactions of a set of 10 to 18 side-chains, most of which are on the inner surface of the protein, are sensitive both to solution conditions and to the Glu-->Ala mutation. A subset of these side-chains and a chain of ordered solvent molecules extends from the vicinity of Glu56 to 58 and Glu60 to the 3-fold channel in the wild type protein and may be involved in the transport of either iron or protons. The "spine of hydration" is disrupted in the Glu-->Ala mutant. In contrast, H25Y mutation shifts the positions of backbone atoms between the site of the mutation and the 4-fold axis and side-chain positions throughout the structure; the largest changes in the position of backbone atoms are in the DE loop and E helix, approximately 10 A from the mutation site. In combination, these results indicate that solvation, structural plasticity and cooperative structural changes may play a role in ferritin function. Analogies with the structure and function of ion channel proteins such as annexins are noted.
铁蛋白是动物、植物和微生物中一种高度保守的多亚基蛋白，它以立方对称形式组装，并在蛋白质内部的矿化核心与外界之间运输水合铁离子和质子。我们在此报告了重组两栖类红细胞L型铁蛋白及其两个突变体在两组条件下解析出的高分辨率结构。在一个突变体中，Glu56、57、58和60被替换为Ala，导致铁摄取动力学出现滞后阶段。在第二个突变体中，His25被替换为Tyr，对功能的影响至多很细微。在所有结构中，用于纯化的甜菜碱分子结合在2重轴附近，靠近最近确定的细菌铁蛋白和马脾L型铁蛋白的血红素结合位点。对五个两栖类结构的比较确定了分子中的两个区域，其中构象灵活性可能与功能相关。一组10至18个侧链的位置和相互作用，其中大多数位于蛋白质的内表面，对溶液条件和Glu→Ala突变均敏感。这些侧链的一个子集和一串有序的溶剂分子从Glu56至58和Glu60附近延伸至野生型蛋白质中的3重通道，可能参与铁或质子的运输。“水合脊柱”在Glu→Ala突变体中被破坏。相比之下，H25Y突变使突变位点与4重轴之间的主链原子位置以及整个结构中的侧链位置发生移动；主链原子位置变化最大的是DE环和E螺旋，距突变位点约10埃。综合来看，这些结果表明溶剂化、结构可塑性和协同结构变化可能在铁蛋白功能中发挥作用。文中还提到了与诸如膜联蛋白等离子通道蛋白的结构和功能的类比。
