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Structural Studies of Cell Surface Molecules Involved in Recognition by the Immune System
Summary: Pamela Björkman is interested in the structure and function of molecules mediating cell surface recognition.
My laboratory is interested in protein-protein interactions, particularly those mediating immune recognition. We use x-ray crystallography and biochemistry to study purified proteins, and confocal and electron microscopy to examine protein complexes in cells. Some of our work focuses upon homologs and mimics of class I major histocompatibility complex (MHC) proteins. Classical class I MHC proteins present peptides derived from self and nonself proteins to T cells during immune surveillance. MHC homologs share similar three-dimensional structures with classical MHC molecules but have different functions, including immune functions (IgG transport by FcRn, the neonatal Fc receptor, and evasion of the immune response by viral MHC mimics) and nonimmune functions (regulation of iron or lipid metabolism by HFE and ZAG, and serving as a chaperone for pheromone receptors in the case of the M10 proteins). We are also comparing the structures and functions of host and viral Fc receptors with FcRn.
Transfer of maternal IgG (immunoglobulin G) molecules to the fetus or infant is a mechanism by which a mammalian neonate acquires 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. We are now using information obtained from our crystallographic and biochemical studies to determine how FcRn-IgG complexes are transported across polarized epithelial cells. We are using a combination of confocal and electron microscopy to study the itineraries of FcRn-containing endosomes in transfected epithelial cells and in the proximal small intestine of neonatal rats. Using electron tomography and a new ligand-labeling/identification protocol, we derived a three-dimensional map of transport vesicles in neonatal intestinal epithelial cells at a resolution of 4–6 nm. We are also doing structure/function studies of other Fc receptors that are not MHC homologs: gE-gI, a viral Fc receptor for IgG; FcαRI, a host receptor for IgA; and the polymeric immunoglobulin receptor (pIgR), which transports dimeric IgA and polymeric IgM into secretions. Both FcRn and gE-gI exhibit strongly pH-dependent binding to IgG, but in opposite directions (FcRn binds IgG at acidic, but not basic, pH, and gE-gI binds IgG at basic, but not acidic, pH). pH-dependent ligand binding is critical for FcRn's function in IgG transport; thus we believe it is also critical for the function of gE-gI. (The studies of Fc receptors are funded by the National Institutes of Health.)
HFE, a recently discovered class I MHC homolog, 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. With the demonstration that it binds to transferrin receptor (TfR), the receptor by which cells acquire iron-loaded transferrin, HFE has been linked to iron metabolism. We have solved crystal structures of HFE alone and HFE bound to TfR. The interaction of HFE with TfR is a fascinating system to study because we can use crystal structures to determine 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 TfR, and how the HFE interaction with TfR compares with interactions of ligands with MHC and MHC-like (e.g., FcRn) proteins. We are using confocal microscopy and other imaging techniques to investigate HFE and TfR intracellular trafficking in transfected cell lines. In addition, we are exploring the roles of other molecules, such as ferroportin and hemojuvelin, in the regulation of iron metabolism. (Some of this work is funded by the National Institutes of Health.)
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. We recently solved the crystal structure of the HCMV homolog bound to a host receptor protein, which we are comparing to our structure of the same receptor bound to a human MHC protein. Surprisingly, the viral MHC homolog contains a peptide-binding groove that includes virtually all the features of grooves in classical host class I MHC molecules and is occupied by a peptide. Our HCMV homolog/receptor cocrystal structure does not answer the question of why the viral MHC homolog binds peptides, however, because the receptor binding site on the homolog is distant from its peptide-binding groove. We hope to use this structural information to understand how peptide binding by a class I MHC mimic is used in viral evasion of the host immune response.
Recent studies from Catherine Dulac's laboratory (HHMI, Harvard University) have revealed expression of a family of class Ib MHC proteins (M10s) that interact with putative pheromone receptors in the rodent vomeronasal organ. This interaction may play a direct role in the detection of pheromonal cues that initiate reproductive and territorial behaviors. Our crystal structure of M10.5 shows that M10 proteins fold into a structure similar to that of a bona fide class I MHC molecule. Unexpectedly, however, the M10.5 counterpart of the MHC peptide-binding groove is open and unoccupied, revealing the first structure of an empty class I MHC molecule. Our biochemical data suggest that M10.5 associates with some sort of groove occupant, most likely nonpeptidic. The challenge now is to discover the physiological ligands of M10 proteins and understand how they associate with pheromone receptors to influence mating behaviors.
Our structural work on class I MHC homologs has elucidated unexpected recognition properties of the MHC fold. For FcRn and HFE, our 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. We have elucidated the structural basis of FcRn's pH-dependent interaction with IgG and are now focusing on cell biological studies of intracellular trafficking, for which the pH-dependent interaction is critical. The pH dependence of the HFE-TfR and gE-gI/IgG interactions suggested that intracellular trafficking studies would be interesting, so much of our future efforts on FcRn, HFE, and gE-gI systems will involve imaging techniques to probe their functions in a cellular context.
Last updated: August 28, 2007
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