My laboratory is interested in the structure and function of molecules involved in cell surface recognition, particularly those mediating recognition in the immune system. We use a combined approach of x-ray crystallography to determine structures, molecular biological techniques to produce proteins for crystallization and to modify them, and biochemistry to study the properties of the proteins we make. Much of our work has focused upon homologs of class I MHC proteins, which function in many ways that are distinct from their immunological role in peptide presentation to T cells. Our interest in these types of proteins has grown since we solved the structure of the neonatal Fc receptor (FcRn), an MHC homolog that transports immunoglobulin G (IgG). In the FcRn system, we are using structural information to address cell biological issues involving intracellular receptor-ligand trafficking. Our studies of other MHC homologs have expanded our interests to include aspects of iron metabolism (for our work on HFE), cancer (for our work on Zn-a2-glycoprotein; ZAG), and virology (for our work on viral MHC homologs).

Transfer of maternal IgG molecules to the fetus or infant is a mechanism by which mammalian neonates acquire humoral immunity to antigens encountered by the mother. The protein responsible for the transfer of IgG is the MHC class I-related receptor FcRn. MHC class I molecules have no reported function as immunoglobulin receptors; instead they bind and present short peptides to T cells as part of immune surveillance to detect intracellular pathogens. We solved the crystal structures of rat FcRn both alone and complexed with Fc. Our crystallographic and biochemical studies suggest that FcRn dimerizes in response to IgG binding, which may serve as a component of a signal to initiate internalization of FcRn/IgG complexes. We also hypothesize that formation of an oligomeric ribbon of FcRn dimers on adjacent membranes bridged by IgG molecules is required for FcRn function. We are now interested in understanding the roles of the FcRn dimer and the oligomeric ribbon in IgG transport. We have developed assays to investigate the processes that must both occur for the formation of oligomeric ribbons: (i) dimerization of FcRn, and (ii) bridging of FcRn dimers by Fc. We are in the process of characterizing oligomeric ribbon formation in vitro using biophysical assays, and have extended these studies to real time confocal imaging of cells undergoing FcRn-mediated transcytosis of IgG. We are also beginning structure/function studies of two other Fc receptors that are not MHC homologs: gE/gI, a viral Fc receptor for IgG, and FcaR, a host receptor for IgA.

HFE is a recently discovered class I MHC homolog that is involved in the regulation of iron metabolism, an unexpected function for an MHC-related protein. HFE was discovered when its gene was found to be mutated in patients with the iron overload disease hereditary hemochromatosis. HFE has been linked to iron metabolism with the demonstration that it binds to transferrin receptor, the receptor by which cells acquire iron-loaded transferrin. We solved crystal structures of HFE alone and HFE bound to transferrin receptor. The interaction of HFE with transferrin receptor is a fascinating system to study because we can use crystal structures to answer biochemical, functional, and evolutionary questions that address how binding of HFE interferes with transferrin binding, if conformational changes in the receptor are involved in the binding of either transferrin or HFE, which part of the MHC-like HFE structure binds transferrin receptor, and how the HFE interaction with the receptor compares with interactions of ligands with MHC and MHC-like (e.g., FcRn) proteins. We are expanding our studies to include cell biological investigations of HFE and transferrin receptor intracellular trafficking in transfected cell lines using confocal microscopy and other imaging techniques.

We also study Zn-a2-glycoprotein (ZAG), a soluble MHC class I homolog present in low concentrations in most bodily fluids. ZAG was isolated from blood more than 30 years ago, but it's been a molecule in search of a function for a long time. Recently researchers at Aston University in the U.K. discovered that ZAG is involved in cachexia, a wasting syndrome that can affect people with terminal illnesses. ZAG is responsible for the fat-depletion component of cachexia, since it stimulates lipid breakdown in adipocytes and reduces fat stores in laboratory animals. We have purified ZAG from serum and completed its crystal structure, which revealed an as yet unidentified non-peptide compound in the ZAG counterpart of the MHC peptide binding site. A combination of structural studies, computational analysis, and ligand binding experiments will be used for analyzing the function and potential roles of ZAG in lipid catabolism under normal and pathological conditions.
We are also interested in other MHC homologs, including proteins encoded by viruses. Both human and murine cytomegalovirus (HCMV, MCMV) express a relative of MHC class I heavy chains, probably as part of the viral defense mechanism against the mammalian immune system. Our biochemical studies show that the HCMV homolog associates with endogenous peptides resembling those that bind to class I MHC molecules. Current efforts forcus upon defining the structure and function of these homologs in order to understand why viruses make them and how they interfere with the host immune system.

Our structural work on class I MHC homologs has elucidated new and unexpected recognition properties of the MHC fold. For FcRn and HFE, the structural and biochemical studies have revealed a similar fold and some common properties, including the assumption that both receptors "lie down" parallel to the membrane when binding ligand, and a sharp pH dependent affinity transition near neutral pH. In the case of FcRn, we have elucidated the structural basis of its pH dependent interaction with IgG and will now focus upon cell biological studies of intracellular trafficking, for which the pH dependent interaction is critical. The pH dependence of the HFE-TfR interaction suggested to us that intracellular trafficking studies of HFE would be interesting, so much of our future efforts on both the FcRn and HFE systems will center around probing their function in a cellular context using imaging techniques. Our functional studies of ZAG are at an earlier stage, since a receptor for ZAG has not been identified and the mechanism by which ZAG promotes lipid degradation is unknown. We are at an even earlier stage in our studies of the viral MHC homologs, in which our primary goal will be to solve crystal structures of one or both viral homologs alone and complexed with their cellular receptors.
In addition to the studies of MHC homologs, we are interested in using structure/function studies to understand bacterial pathogenesis and the innate immune response to bacterial infection. Our efforts in these areas involve the study of the Yersinia pseudotuberculosis protein invasin, the insect immune response protein hemolin, and the mammalian mannose receptor.
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