Dynamics of the antigen-binding grooves in CD1 proteins: reversible hydrophobic collapse in the lipid-free state

Abstract

CD1 proteins mediate the presentation of endogenous and foreign lipids on the cell surface for recognition by T cell receptors. To sample a diverse antigen pool, CD1 proteins are repeatedly internalized and recycled, assisted, in some cases, by lipid transfer proteins such as saposins. The specificity of each CD1 isoform is, therefore, conferred in part by its intracellular pathway but also by distinct structural features of the antigen-binding domain. Crystal structures of CD1-lipid complexes reveal hydrophobic grooves and pockets within these binding domains that appear to be specialized for different lipids. However, the mechanism of lipid loading and release remains to be characterized. Here we gain insights into this mechanism through a meta-analysis of the five human CD1 isoforms, in the lipid-bound and lipid-free states, using all-atom molecular dynamics simulations. Strikingly, for isoforms CD1b through CD1e, our simulations show the near-complete collapse of the hydrophobic cavities in the absence of the antigen. This event results from the spontaneous closure of the binding domain entrance, flanked by two α-helices. Accordingly, we show that the anatomy of the binding cavities is restored if these α-helices are repositioned extrinsically, suggesting that helper proteins encountered during recycling facilitate lipid exchange allosterically. By contrast, we show that the binding cavity of CD1a is largely preserved in the unliganded state because of persistent electrostatic interactions that keep the portal α-helices at a constant separation. The robustness of this binding groove is consistent with the observation that lipid exchange in CD1a is not dependent on cellular internalization.

Keywords: Adaptive Immunity; Allosteric Regulation; Antigen Presentation; Cellular Immune Response; Hydrophobic Collapse; Lipid Binding Protein; Molecular Dynamics.

FIGURE 1.

Displacement of the portal helices (α1 and α2) flanking the entrance of the antigen binding grooves in CD1a through CD1e, relative to the experimental crystal structures, in simulations of the lipid-bound (black lines) and lipid-free (red lines) states of each binding domain. A, the displacement of the helices is quantified by monitoring the distance between pairs of residues facing each other along the length of the helices. For CD1b, data are also shown for the complete α1–3/β₂m complex in the lipid-free state. For CD1e, only the lipid-free state was simulated because no lipids were resolved in the available crystal structure. The values plotted are time averages over the final 50 ns of the simulations. For the lipid-free states, global averages are obtained from three independent simulations. Error bars show mean ± S.D. The gray band indicates variations of ±1 Å to be expected at room temperature. B, protein surface area buried between α1 and α2, as a function of simulation time, for the lipid-bound (black) and lipid-free states (colors).

FIGURE 2.

Persistence or collapse of the antigen binding grooves in simulations of lipid-bound and lipid-free CD1 isoforms relative to the experimental crystal structures. The anatomy of the binding grooves are depicted at 20-ns intervals throughout the simulations. This anatomy is represented graphically by the surface encompassing the volume of the various pockets and tunnels, as calculated with a grid-based search method. The simulation systems analyzed are the same as in Fig. 1.

FIGURE 3.

Configuration of the portal helices α1 and α2 in the x-ray structures of CD1a through CD1e compared with the lipid-free simulations. The plots show a two-dimensional projection of the Cα-trace of the helices viewed perpendicularly to the floor of the binding cavity. In each case, the atomic coordinates plotted are time averages over the last 50 ns of each repeat simulation. The S.D. around these averages are omitted because these are typically smaller than the differences between repeats.

FIGURE 4.

Side chain interactions sustain the entrance of CD1a but not CD1b–e in the absence of a lipid antigen. A, final simulation snapshot of each CD1 binding domain in the lipid-free state. Each domain is represented as a schematic and colored according to the secondary structure. The configurations of helices α1 and α2 in the respective crystal structures are overlaid (fitted to the α2 helix backbone) and shown in transparent gray. Side chains of key residues in α1 and α2 are labeled. The surrounding solvent molecules included in the simulation are omitted for clarity. B, persistence of the electrostatic interactions across the center of the entrance to the binding groove in CD1a in the lipid-bound and lipid-free simulations.

FIGURE 5.

Near-complete recovery of the anatomy of the CD1b binding groove upon repositioning of the portal helices α1 and α2 to the lipid-bound configuration, starting from the collapsed state depicted in Fig. 4. The displacement of the helices is induced progressively over a 50-ns targeted simulation (A) in which biasing forces act exclusively on the backbone atoms of the helices (k = 600 kcal mol/Ų). The resulting configuration was then sustained for another 50 ns (B). The hydrophobic groove is represented graphically by the surface encompassing the volume of the various pockets and tunnels therein, calculated as in Fig. 2. The recovery is shown in 10-ns intervals. The anatomy of the binding groove is largely restored, despite the fact that the simulation does not impose a specific configuration on the protein side chains.