Asymmetry as the key to clathrin cage assembly - PubMed (original) (raw)

Asymmetry as the key to clathrin cage assembly

Wouter K den Otter et al. Biophys J. 2010.

Abstract

The self-assembly of clathrin proteins into polyhedral cages is simulated for the first time (to our knowledge) by introducing a coarse-grain triskelion particle modeled after clathrin's characteristic shape. The simulations indicate that neither this shape, nor the antiparallel binding of four legs along the lattice edges, is sufficient to induce cage formation from a random solution. Asymmetric intersegmental interactions, which probably result from a patchy distribution of interactions along the legs' surfaces, prove to be crucial for the efficient self-assembly of cages.

2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1

The triskelion simulation model, shown here in top view (left) and side view (right), is based on the idealized structure of clathrin. Three proximal legs run from the central hub (red) to a knee (green), under a pucker angle χ relative to the threefold rotational symmetry axis (_C_3), followed by three distal legs running from the knees to the ankles (yellow). In all simulations, the pucker angle was set equal to 101°. The particle is simulated as a rigid unit with proximal and distal legs of equal length.

Figure 2

Figure 2

Triskelia of the first simulation model, at segmental binding energies ε = 7 k_B_T (A) and ε = 8 k_B_T (B), have a tendency to self-assemble into chainlike structures. The number of branch points increases with the segmental binding energy, and thereby changes the shape of the aggregates.

Figure 3

Figure 3

Experimentally resolved structure of a lattice edge in a hexagonal barrel viewed (A) from the top, (B) from the side, and (C) in cross section. The two antiparallel proximal leg-segments (red and green) are in the front of the top view and on top in the side view and cross section, the two antiparallel distal leg-segments (blue and amber) are in the back of the top view and at the bottom of the side view and cross section. Proximal and distal legs are similar in structure and are clearly asymmetric, with two faces made of _α_-helices and two faces made of connecting loops. A gradual right-handed twist along the leg has rotated the elliptic cross sections of the distal legs by ∼90° relative to those of the proximal legs. The cartoons in Figs. 4 and 7 are based on this structure. (Reproduced with permission from the authors and Macmillan Publishers from the supplementary material to Fotin et al. (8).)

Figure 4

Figure 4

Schematic of the probable edge structure in a clathrin cage, based on the experimental structure (5–8) provided in Fig. 3. An edge (marked by an arrow in panel A) combines two antiparallel proximal legs (dark blue) of the two clathrins centered at the flanking vertices with two antiparallel distal legs (light green) of two clathrins centered at next-nearest vertices. In the cross section (B), the hub-to-knee and knee-to-ankle directions are indicated by the symbols ⊙ and ⊗ for segments pointing out of and into the paper, respectively. The diagram illustrates that the asymmetric segments are preferentially oriented with the shaded regions (schematically representing the segmental interactions) oriented toward the center of the edge. Rotating the upper-right dark blue clathrin by 180° around the direction of the marked edge yields the upside-down red clathrin (C), whose poor binding is explained in cross section (D) by a mismatch of the shaded areas. The latter configuration is very common, however, in the simulations with the nondirectional model, where it gives rise to the typical stringlike structures of Fig. 2 with alternating up-down orientations for consecutive pairs of particles.

Figure 5

Figure 5

Snapshots after ∼5,000,000 Monte Carlo trial moves per particle, starting from a random initial configuration, for the interaction models with nonpatchy (A) and patchy (B) segmental interactions at a segmental interaction energy of ε = 8 k_B_T. Whereas the model with nondirectional interactions yields chainlike aggregates of various shapes (two isolated clusters are shown in Fig. 2), the model with directional interactions readily self-assembles into near-spherical cages of fairly uniform size (two isolated cages are shown in Fig. 6). The actual simulation boxes are approximately four times as large, containing 104 particles at a density of 10−3_σ_−3 corresponding to the critical assembly concentration of ∼100 _μ_g/ml.

Figure 6

Figure 6

Two typical self-assembled cages grown at ε = 8 k_B_T with the patchy model. Closed polyhedral cages formed by triskelia with a pucker χ = 101° contain ∼60–70 particles, forming exactly 12 pentagonal faces and ∼20 hexagonal faces. The cage on the left is rotated to create a coalescence of the edges in the front with those in the rear.

Figure 7

Figure 7

Cross section of a cage edge showing the preferred orientations of the m^-vectors for maximum binding between the leg segments. The two proximal-proximal m^-vectors are antiparallel, as are the two distal-distal m^-vectors, whereas combinations of a proximal and a distal segment can yield both parallel and antiparallel m^-vectors depending on the segments involved.

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