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1. The system: peptide class I MHC complexes The class I major histocompatibility complex (MHC) molecule is a non-covalently linked heterotrimeric complex consisting of a heavy chain, the small subunit 2-microglobulin (2m), and a peptide typically 8-11 amino acids in length. Class I MHC molecules bind and present antigenic peptides to cell surface T cell receptors; binding of a T cell receptor to a class I peptide/MHC complex is required for initiation and propagation of a cellular immune response. Peptides are bound by the MHC in a groove formed by the heavy chain 1 and 2 domains, resulting in a relatively flat surface available for recognition by T cell receptors. Antigenic peptides tend to be bound very stably, with affinities typically in the nanomolar range and halflives of an hour or more. A. peptide B. Heavy chain (1 & 2) 2m Heavy chain (3) Figure 1. Crystallographic structure of a representative class I peptide/MHC complex. A) Side view of the complex, showing the heavy chain, peptide, and 2m. B) Top view, showing only the peptide and the peptide binding domain. 2. A peptide conformational change upon receptor binding The crystallographic structure of the complex of the Tax peptide (sequence LLFGYPVYV) with the class I MHC HLA-A2 shows the center of the peptide looping up and out of the peptide binding groove (pdb entry 1DUZ). The structure is at a reasonably high resolution of 1.8 Å, and the peptide is completely visible in the electron density. In the structure of the same peptide/MHC complex bound to two different T cell receptors, the peptide is in a different conformation: it is “squished” into the binding groove (see pdb entry 1AO7). The RMSD of all atoms of the two peptides is 1.62 Å, including only the backbone it is 1.02 Å. Although this may seem small, consider it is over 154 atoms in the all atom case or 72 for the backbone. Figure 2. “Side view” of the Tax/HLA-A2 structure, showing the conformation of the peptide without the TCR bound (green) and the conformation when the TCR is bound (blue). The MHC molecule is rendered transparent to visualize the entire peptide. The conformational change is centered around the side chain of Phe 3 and the backbones of proline 6 and valine 7. 3. Implication of the peptide conformational change 3.a. Induced fit vs. conformational ensembles The issue of induced-fit versus conformational dynamics remains controversial. While some accept the concept of induced fit as a shift in an ensemble of populations towards a particular conformation, many continue to view induced fit in the traditional single molecule limit: one protein bumps into another and squishes it into some different conformation. Two recent, high-profile publications in Science have demonstrated the role of conformational ensembles in protein function (signal transduction by Dorothy Kern and antibody binding by Dan Tawfik). The fact that this work was published in Science highlights how well the traditional single-molecule limit remains entrenched. With the Tax/HLA-A2 system, we can address the following questions about induced fit vs. conformational ensembles: With what frequency does the peptide sample conformations close to the “squished” state in the unliganded Tax/HLA-A2 structure (or does it do it all)? What is the most likely pathway for moving to the squished state? What is the energetic cost for moving to the squished state? Just how dynamic is the peptide in the binding groove? 3.b. Peptide dynamics and immune reactivity While addressing the role of conformational ensembles vs. induced fit is important, the issue of peptide dynamics in the MHC binding groove also speaks to the function of the peptide/MHC complex. If the peptide populates different conformations, it could broaden reactivity towards T cell receptors. Consider the peptide/MHC surface: different peptide conformations means surfaces with different chemical properties. As T cell receptors resemble antibodies with randomly generated binding loops, there is a strong likelihood that a peptide populating different conformations will be recognized by a greater number of receptors than a peptide that only populates one major conformation. Thus there is an opportunity to investigate the potential role of peptide dynamics in broadening immune reactivity (as of yet, no dynamic studies of peptides bound to class I MHC molecules have been performed). 4. Correlating indications of peptide dynamics with binding thermodynamics We have recently measured binding thermodynamics (G, H, and S) for a labeled derivative of the Tax peptide binding to HLA-A2. To be honest, our measurements probably aren’t that accurate as they require some fishy assumptions. However, our assumptions are less problematic for relative measurements, i.e. the difference in binding thermodynamics between two peptides (G, H, S). We could enhance this study by performing measurements with peptides designed to increase or restrict dynamics in the binding groove, say glycine or proline substitutions. Measurements of S could be correlated with NMR measurements of dynamics, coupled with simulations revealing exact molecular motions. 5. Things to consider Is this the best system for this work? We only have one observation of a peptide conformational change, and to be honest, it’s really not that big. How much of a stretch is it to tie into the immune system? Ideally, we could actually demonstrate that peptide dynamics actually does broaden reactivity, but then we would have to find a T cell receptor that recognizes the up conformation. That’s basically a fishing expedition. Is this a methods proposal or a research proposal? If it’s a methods proposal, again, is this the right system? If it’s a research proposal we need a “hook” – the peptide binding experiments add something, but it will be a tough sell if we don’t have some tangible goal, i.e. designing tight binding peptides, etc. If we make it a research proposal, we would probably need to study a whole range of peptides, not just derivatives of the Tax peptide, but others, maybe even some with some great biological interest. That’s a real possibility – I know of one tumor derived peptide that is proposed to be inactive because it is too dynamic. We could propose using dynamics simulations, NMR, and binding measurements to redesign this peptide to make it less dynamic, tighter binding, and more active… Hmm…