Introduction
Major histocompatibility complex (MHC) molecules present oligopeptides peptide antigens from immunogenic proteins to activate T-cell responses. A critical aspect of antigen presentation is peptide binding to MHC molecules. Only 
1% of peptides of sufficient length are able to bind to any given MHC class I or class II gene product. Though numerous methods exist for quantitating peptide binding to MHC molecules, none detects binding in real time—until now, that is. In this issue of Nature Chemical Biology, Venkatraman et al.1 report that upon binding to the human MHC class II molecule DR1, peptides modified with environmentally sensitive fluorophores demonstrate large increases in fluorescence, an increased Stokes shift in emission, and increased fluorescence lifetime, all of which can be exploited to monitor peptide binding in real time.
MHC molecules are divided into two classes: class I molecules, which typically present peptides of 8–11 amino acids to CD8+ T cells, and class II molecules, which typically present peptides of 13–25 amino acids to CD4+ T cells. Genes encoding MHC class I and class II molecules are the most polymorphic genes known in vertebrates, and in most species hundreds to thousands of alleles exist at appreciable frequencies. Though exceptions abound, in general, class I–associated peptides are generated from endogenously synthesized proteins via proteolysis in the cytosol and endoplasmic reticulum (where loading occurs), whereas class II–associated peptides are generated from extracellular proteins and loaded in endosomal compartments2, 3. CD8+ T cells play critical roles in immunity to intracellular pathogens and tumors, whereas CD4+ T cells orchestrate CD8+ and antibody responses to foreign and self antigens. Both T cell types are believed to be important in autoimmune diseases such as type I diabetes and multiple sclerosis.
Although mechanisms exist to ensure that nascent MHC molecules are loaded with peptides in the appropriate intracellular compartment, a substantial fraction of MHC molecules exist in a peptide-receptive state throughout the secretory pathway, including the cell surface. Though the physiological significance of peptide-receptive MHC molecules is uncertain, their existence is a boon to immunologists because incubation of living cells with synthetic peptides loads MHC molecules and has become a standard technique for activating T cells in vitro.
Fluorescent peptides have been previously used to study peptide association with class I molecules in living cells4, however this approach was limited by the inability to distinguish bound versus free peptide. To overcome this limitation, Venkatraman et al. designed a peptide modified with environmentally sensitive fluorophores, which change their fluorescent properties based on the polarity of the surroundings. Using the known crystal structure of a peptide–DR1 complex as a guide, fluorogenic groups were designed to reside in a nonpolar pocket on the DR1 binding groove (Fig. 1a). When these fluorogenic peptides bound to MHC molecules, the authors observed a 1,000-fold increase in fluorescence. The spectral properties of the bound fluorophore were unchanged between pH 7 and pH 5. This is particularly important for class II–binding peptides, given that the pH of physiological endosomal loading compartments extends to as low as 5. Venkatraman et al.1 show that fluorogenic peptide binding to DR1 on live cells can be measured by flow cytometry, and they use this direct method to demonstrate that the fraction of peptide-receptive molecules on the surface of dendritic cells (an immune cell that functions to activate T-cell responses in vivo) varies with the differentiation status of the cell (Fig. 1b).
Figure 1: Location, location, location: self-reporting peptides identify peptide-receptive MHC molecules.
(a) MHC molecules bind optimal synthetic peptides with relatively high affinity (Kd of 10 nM). Venkatraman et al.1 used structure-based design to create an MHC-binding synthetic peptide that demonstrates increased fluorescence when the fluorophore-modified side chain binds a hydrophobic pocket that provides a significant fraction of the binding energy of the peptide-MHC interaction. (b) Peptide-receptive MHC molecules exist on the surface of all cell types that express MHC molecules. The self-reporting fluorescent peptides provide a simple and direct measure of the number of peptide-receptive molecules, and were used to demonstrate that the fraction of peptide-receptive DR1 molecules decreases upon maturation of dendritic cells (DCs).
Katie Ris
Full size image (39 KB)This approach opens numerous possibilities for increased understanding of peptide binding to MHC molecules and the trafficking and loading of MHC in cellular subcompartments. For instance, real-time peptide binding can now be measured using either purified MHC molecules or MHC molecules on living cells, and changes in the spectral properties of bound peptide may reveal dynamic conformational alterations in the complex that are induced by its interaction with the T-cell receptor. Visualization of complex formation by fluorescence microscopy should reveal the intracellular sites of exogenous peptide loading in cultured cells, and perhaps even in living animals through the use of multiphoton microscopy. By combining this approach with high-throughput screening, small molecules can be identified that block peptide binding in vitro and in vivo. Local delivery of such compounds might be of use in blocking self-peptide presentation to autoimmune T cells. More generally, using appropriately designed peptides that fit into nonpolar pockets of target proteins, this approach should be adaptable to creating self-reporting probes that enable visualization of their targets in living cells via fluorescence microscopy.
