Oligomerization of the Tetraspanin CD81 via the Flexibility of Its δ-Loop

Abstract

Tetraspanins are master organizers in the plasma membrane, forming tetraspanin-enriched microdomains with one another and other surface molecules. Their rod-shaped structure includes a large extracellular loop (LEL) that plays a pivotal role in tetraspanin network formation. We performed comparative atomistic and coarse-grain molecular-dynamics simulations of the LEL in isolation and full-length CD81, and reproduced LEL flexibility patterns known from wet-lab experiments in which the LEL δ-loop region showed a pronounced flexibility. In a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine lipid bilayer and a plasma membrane environment, the conformational flexibility of the δ-loop initiates CD81-CD81 contacts for oligomerization. Furthermore, in the plasma membrane, CD81-ganglioside bridges arising from preformed glycolipid patches cross-link the complexes. The data suggest that exposing a flexible domain enables binding to interaction partners by circumventing the restriction of orientation and conformational freedom of membrane proteins.

Figure 1

Helical stability and backbone flexibility of the isolated LEL of CD81. (a) Left: CD81_LEL initial conformation (chain A of the x-ray structure (20) (PDB: 1G8Q)). Right: after a 1 μs unbiased, atomistic MD simulation (run B; see also Fig. S1b). (b) Top: occurrence of helical structure per residue (based on the ϕ/ψ dihedral angles) over time, averaged over four runs. Bottom: Cα RMSF as a measure of the local protein backbone flexibility. The highest flexibility of the fluctuating termini is due to the lack of anchoring to the TM segments present in the full-length protein (compare with Fig. 2b, which shows the flexibility of this region in the full-length protein). Colored lines show the flexibility profiles from the individual MD runs; average (n = 4) is shown in black. The pictogram shows the CD81 LEL domain structure, including cysteine disulfide bridges indicated by additional black lines connecting residues 157 with 175, and 156 with 190. To see this figure in color, go online.

Figure 2

Helical stability and backbone flexibility of CD81_FL. (a) Left: conformation of the full-length protein embedded in a POPC bilayer after a 1 μs unbiased, atomistic MD simulation (run D; see also Fig. S2b). TM1–4 in dark yellow; helices α, β, and ε in gray; loops γ and δ in orange and red, respectively. Red, blue, and tan atoms of the lipid bilayer represent oxygen, nitrogen, and phosphate, respectively. Right: enlarged view of the SEL and LEL. (b) Helical structure per residue (top) and Cα RMSF (bottom). Note that a direct comparison of the average RMSFs in (b) (shown in black; n = 4) and Fig. 1b suggests that the LEL domain flexibility increases in CD81_FL; however, this is a result of least-square fitting to all Cα atoms of the full-length protein (for comparison of exclusively LEL segments, see Fig. 3a). To see this figure in color, go online.

Figure 3

LEL segmental flexibility enables the space exploration activity of the δ-loop. (a) Comparison of the average Cα RMSFs of CD81_LEL (n = 4) (same as in Fig. 1b) and CD81_FL (n = 4), with only its LEL section included for analysis. The shape of the blue trace is slightly different from the respective section in Fig. 2b (in particular at the termini) due to the protein segment considered for fitting. (b) Left: superimposed protein backbone traces obtained from the four MD trajectories (shown are 4 × 98 snapshots from 10 ns intervals, excluding the first 20 ns) of the isolated LEL. Right: the LEL in CD81_FL. For superimposition, Cα atoms that exhibited an average RMSF value smaller than 0.15 nm were used for least-square fitting. Protein segments are colored as in the pictogram in (a). (c) Increase of the SASA of the individual LEL segments, referring to the respective MD initial structure (gray bars, CD81_LEL; blue bars, CD81_FL) in percent (averaged over the four MD runs; n = 4, values are given as means ± SD). For clarity, the extreme value obtained for the ε-helix in CD81_LEL is not fully illustrated because it does not result from relevant dynamics (see text). To see this figure in color, go online.

Figure 4

Structural flexibility and interactions of CD81 in a CG POPC bilayer system. (a) Left: mapping of the atomistic structure of the full-length CD81 onto the CG scale (gray, backbone particles; orange, side-chain particles). Right: top and side views of the MD initial configuration, containing 49 copies of CG CD81 embedded into a preequilibrated POPC lipid bilayer model (≈70 × 70 nm along the bilayer plane). The aqueous phase is shown as a semitransparent volume surface (for clarity, ions are not shown). (b) System configuration after a 2.5 μs unbiased MD simulation. (c) The black trace indicates the mean Cα RMSF, including all individual CD81 chains (values are given as means ± SD; n = 49, the range of the SD is indicated in gray). (d) Protein-protein RDF. The red area indicates the range used to define direct CD81-CD81 interactions (distance threshold = 0.6 nm). (e) CD81 oligomerization over time. (f) Illustration of a CD81 dimer (the protein surface is semitransparent and the CD81 domain is colored according to the topology diagram in c). (g) CD81-CD81 contacts normalized per residue (the sum exceeds 100%, as one residue can be involved in more than one contact). To see this figure in color, go online.

Figure 5

CD81 oligomerization in a CG model of the plasma membrane. (a) Top view of the MD initial configuration, containing 49 copies of CG CD81 embedded into a preequilibrated plasma membrane model (≈70 × 70 nm along the bilayer plane). (b) System configuration after a 7.5 μs unbiased MD simulation. (c and d) CD81 oligomerization over time (c) and CD81-CD81 contacts per residue (d) analyzed as in Fig. 4. To see this figure in color, go online.

Figure 6

CD81-GM contacts over time and interaction sites. (a) Snapshot of the simulation system after a 7.5 μs MD simulation. Gangliosides are colored in blue and proteins are in yellow/gray (side-chain/backbone particles) or, in the case of the two example states in (b), in orange/gray. (b) Left: configuration in which gangliosides form glycolipid bridges among three nondirectly interacting CD81 molecules. Right: direct CD81-CD81 interaction. Protein surfaces are shown as semitransparent. A gray-to-red color gradient indicates protein interactions per residue within 0.6 nm. (c) Protein-lipid RDF. The red area indicates the range of CD81-lipid distances used for contact definition (distance threshold = 0.6 nm). (d) Number of protein-lipid interactions as a function of time (black, CD81-PC interactions; blue, CD81-GM interactions). (e) CD81-GM contacts per residue scaled to the residue distance to the hydrophobic belt center of the protein (see Fig. S6 for original data and residue distances). To see this figure in color, go online.