Crystallization and stoichiometry of binding of a complex between a rat intestinal Fc receptor and Fc.
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.
Huber
A H
AH
Division of Biology, California Institute of Technology, Pasadena 91125.
Kelley
R F
RF
Gastinel
L N
LN
Bjorkman
P J
PJ
D000818:Animals; D002151:Calorimetry; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry* / Q000378:metabolism; D007422:Intestines / Q000737:chemistry*; D051381:Rats; D011961:Receptors, Fc / Q000737:chemistry* / Q000378:metabolism; D014961:X-Ray Diffraction
D007141:Immunoglobulin Fc Fragments; D011961:Receptors, Fc