Download 1. The system: peptide class I MHC complexes

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:
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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…