Conformation of a Clathrin Triskelion in Solution

Abstract

A principal component in the protein coats of certain post-golgi and endocytic vesicles is clathrin, which appears as a three-legged heteropolymer (known as a triskelion) that assembles into polyhedral cages principally made up of pentagonal and hexagonal faces. In vitro, this assembly depends upon the pH, with cages forming more readily at low pH and less readily at high pH. We have developed procedures, on the basis of static and dynamic light scattering, to determine the radius of gyration, R g , and hydrodynamic radius, R H , of isolated triskelia, under conditions where cage assembly occurs. Calculations based on rigid molecular bead models of a triskelion show that the measured values can be accounted for by bending the legs and a puckering at the vertex. We also show that the values of R g and R H measured for clathrin triskelia in solution are qualitatively consistent with the conformation of a triskelion in a "D 6 barrel" cage assembly measured by cryoelectron microscopy.A major component of the protein coats of certain endocytic vesicles is clathrin, a heteropolymer composed of a 192 kDa heavy chain and a variable, ca. 23-27 kDa, associated light chain (1). Three clathrin molecules join at a common hub to form a threelegged "triskelion", which is the basic building block of the coats. Each leg is approximately 3.0 nm thick and 52.0 nm in length and ends in a globular "terminal domain" of radius 5.0 nm. By convention, a leg is divided into a proximal segment adjoining the central hub, a linker region, and a distal segment that connects with the terminal domain. A clathrin heavy chain runs the entire length of the leg, with a light chain attached near the common hub (see Figure 1). In vitro, the triskelia assemble into polyhedral cages (or "baskets") composed of pentagonal and hexagonal faces (2), mimicking the structures seen on the outside of endocytic vesicles. One can dissociate baskets or uncoat vesicles by changing the properties † This work was supported by extramural grants from the National Institutes of Health and intramural funds from the National Institute HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript of the solution in which they are suspended (e.g., by changing pH and ionic strength) (3) or by adding an uncoating ATPase (4).Triskelia assemble at low pH to form baskets (3) or at higher pH, when clathrin assembly proteins (e.g., AP-2, AP-180, etc.) are added (5). It is not currently known why these conditions favor assembly. Possible mechanisms supporting assembly at low pH include conformational changes in the tertiary structure of the triskelia, which might relieve steric hindrances to basket assembly, and enhanced interactions between the intertwined triskelia that form the basket struts. The role of APs as assembly proteins may be to increase mechanical linkages between the triskelia (5-7).In this paper, we report light-scattering measurements on clathrin in solution, including conditions where assembly occurs. From static light scatteri...

Clathrin Coat Disassembly by the Yeast Hsc70/Ssa1p and Auxilin/Swa2p Proteins Observed by Single-particle Burst Analysis Spectroscopy

“…3d). These conditions have been shown to disassemble mammalian clathrin baskets into free triskelia (30). Although we cannot be sure that the artificial disassembly of yeast clathrin baskets yields a homogeneous population of triskelia, it does confirm that baskets made with GFP-labeled clathrin can be disassembled to products of a size consistent with individual triskelia: the dramatic shift in the mean diffusion time and observed fluorescent burst height of the GFPlabeled material are both consistent with more extensive disassembly of the clathrin baskets.…”

“…As a reference, triskelia were isolated nonenzymatically ("artificially disassembled"), by adding 2 M Tris, pH 7.0, to the clathrin basket-enriched fraction to a final concentration of 0.5 M (30). The sample was incubated 1 h at 4°C and then centrifuged for 1 h at 168,000 ϫ g. The supernatant fraction was analyzed using BAS and FCS with the laser power set to 150 W.…”

Chameleon behaviour of a-synuclein : brownian dynamics simulations of protein aggregation

“…. These values agree with the previous calculations [61,223], and the corresponding average translational diffusion coefficient is in good agreement with experimental data [223].…”

“…Ferguson et al [61] and Yoshimura et al [223]: in their evaluation of clathrin's mobility matrices a single clathrin is approximated as an array of 52 spheres inspired by the real dimensions (leg segment length and diameter) and overall shape of the protein. At this point the orientation of the triskelion must be specified: the center of mass is at the origin of the body-fixed coordinate system, the third axis of this orthonormal system runs along the three-fold rotational symmetry axis, the particle's first proximal leg lies in the plane spanned by the first and third axis, and the second axis follows from the cross product of the other two axes.…”

Impact of defect creation and motion on the thermodynamics and large-scale reorganization of self-assembled clathrin lattices

“…10 These cage-like lattices contain a clathrin subunit at each vertex, and the three triskelion arms associate with neighboring triskelia via favorable heavy-and light-chain interactions. 8,9 Thus, the resulting lattice has three bonds at each vertex when fully satisfied, resulting in a honeycomb structure with prevalent five-membered and seven-membered ring defects. As in any closed polyhedral lattice, such ring defects permit closure of the lattice into a three-dimensional shell.…”