Huber A H, Kelley R F, Gastinel L N, Bjorkman P J
Division of Biology, California Institute of Technology, Pasadena 91125.
J Mol Biol. 1993 Apr 5;230(3):1077-83. doi: 10.1006/jmbi.1993.1220.
Fc receptors expressed in the gut of newborn rodents bind to maternal immunoglobulin in milk at pH 6.5, and transport it to the bloodstream of the neonate, where it dissociates at pH 7.4. The rat intestinal Fc receptor (FcRn) consists of a heavy chain, with significant sequence similarity to the heavy chain of class I MHC molecules, complexed to the class I light chain, beta 2-microglobulin. Although FcRn is predicted to contain a groove analogous to that which serves as the MHC peptide-binding site, the immunoglobulin ligand of FcRn is a macromolecule instead of a peptide. We have expressed and crystallized a secreted form of FcRn, and here report the crystallization of a complex between FcRn and its Fc ligand. Isolated FcRn-Fc complexes crystallize in space group I222 or I2(1)2(1)2(1) with unit cell dimensions a = 125 A, b = 152 A and c = 216 A. The crystals diffract to 5.5 A resolution with anisotropic diffraction to 3.5 A. Data collection from cryopreserved crystals may allow the resolution limit to be extended, since the major reason for the poor resolution appears to be radiation decay. Even a low-resolution view of how FcRn binds Fc would be of interest to see if the binding site corresponds to the functional part of an MHC molecule. Since the structure of Fc is known, and a structure determination of FcRn is underway, it may be possible to locate the Fc binding site on FcRn at low resolution. As an initial characterization of the FcRn-Fc mode of interaction, and to facilitate the structure determination, we have determined the stoichiometry of binding of FcRn to Fc. We show that two FcRn molecules bind per Fc, as determined by analysis of gels of washed crystals, a column binding assay, and isothermal titration calorimetry.
新生啮齿动物肠道中表达的Fc受体在pH 6.5时与乳汁中的母体免疫球蛋白结合，并将其转运至新生儿的血液中，在pH 7.4时发生解离。大鼠肠道Fc受体（FcRn）由一条重链和与I类MHC分子重链具有显著序列相似性的轻链组成，该轻链与I类轻链β2-微球蛋白形成复合体。尽管预计FcRn含有一个类似于作为MHC肽结合位点的凹槽，但FcRn的免疫球蛋白配体是一个大分子而非肽。我们已经表达并结晶了一种分泌形式的FcRn，在此报告FcRn与其Fc配体之间复合物的结晶情况。分离得到的FcRn-Fc复合物在空间群I222或I2(1)2(1)2(1)中结晶，晶胞参数为a = 125 Å，b = 152 Å，c = 216 Å。晶体衍射分辨率为5.5 Å，各向异性衍射分辨率为3.5 Å。对冷冻保存晶体的数据收集可能会使分辨率极限得以提高，因为分辨率较差的主要原因似乎是辐射损伤。即使是FcRn与Fc结合方式的低分辨率视图，也会让人感兴趣，看看结合位点是否与MHC分子的功能部分相对应。由于Fc的结构已知，并且FcRn的结构测定正在进行中，因此有可能在低分辨率下确定FcRn上的Fc结合位点。作为对FcRn-Fc相互作用模式的初步表征，并为促进结构测定，我们已经确定了FcRn与Fc结合的化学计量比。我们通过对洗涤后晶体的凝胶分析、柱结合测定和等温滴定量热法表明，每个Fc结合两个FcRn分子。
