(A) Model of the hub-LCb complex determined by X-ray crystallography with 2F_o-F_c electron density (0.06e/A³, 1.2σ, blue mesh). Details are shown in numbered boxes indicating the regions. Regions 1 and 2 show CHC straight knee conformations (brown ribbons) and region 3 shows the CHC bent knee conformation (blue ribbons), N-terminal to the proximal (prox) leg. CLC residues are shown as yellow ribbons and highlighted by arrows in region 1. Asterisks in region 5 (trimerization domain) indicate helices from the C-terminus of CLC that make contacts with two different CHCs. (B) The high resolution structure of the clathrin heavy chain proximal leg (1B89, green ribbon, Ybe et al., 1999) fitted into the 2F_o-F_c electron density from the proximal leg region. Arrows point to density corresponding to CLC. (C) Straight (brown ribbons) or bent (blue ribbons) legs from the 7.9 Å, spacegroup I4₁22 structure were aligned with each other in PYMOL. Boxes show contacts between crystal symmetry-mates (green) surrounding the N-terminus of straight or bent legs, colored as above. Arrows point to areas of symmetry-mate contact closest to the N-terminus of each leg. (D) Structural model of the clathrin hub-LCb complex. CHC helices are in brown (straight conformation) and blue (bent conformation) and CLC is in yellow. CLC extends from the trimerization domain to the knee region on two legs with straight knees (arrows) while on the bent leg CLC forms a more compact structure in the N-terminal region (arrowheads). (E) Diagram indicating the possible FRET interactions and the locations of the fluorescent dyes in blue and green. Orange arrows indicate distances favorable for FRET between two clathrin light chains (background at the trimerization domain or between termini of clathrin light chains). Dashed black arrow indicates a distance unfavorable for FRET. (F) CLC labeled randomly with two different fluorophores was bound to clathrin hub and these complexes were mixed with complexes of hub with unlabeled CLC to prevent intermolecular energy transfer (as determined in Figure S1D). FRET was measured in either the unassembled or the assembled state. Each bar represents the mean ±SEM (n=3).

(A) Expanded boxed region shows the position of the KR loop (blue spheres, residues 1161–1165 in CHC) relative to CLC. The straight knee conformation (brown) associated with the extended form of CLC (yellow) and the bent knee conformation (cyan) associated with the compact form of CLC (not shown) are overlaid. The KR loop is indicated by red arrowheads. Major deviations between the overlaid structures occur distal to the KR loop. (B) Expanded box region of a, rotated 90° (straight knee only). (C) Alignment of various CHC protein sequences around the KR loop (bold, yellow highlight). Red highlights show differences that alter the charge distribution. Residues mutated to alter the KR loop charge indicated by asterisks (K1163E and R1165D). (D) Surface plasmon resonance (SPR) analysis of clathrin light chain interactions with wild type clathrin hub or KR loop mutant (mut) hub. Human neuronal LCb was immobilized on the SPR chip surface and wild type or KR loop mutant hub flowed over at different concentrations. The fitted binding affinity for wild type was 25 nM while KR loop mutant was 59.1 nM. The difference in affinity for CLC was due to a slowed on-rate for the KR loop mutant likely due to decreased electrostatic attraction. (E) Purified hubs (wild-type, WT or mutant, KR) were saturated with CLC (wild type neuronal LCb (WT CLC) or neuronal LCb with E₂₀ED₂₂ mutated to K₂₀KK₂₂ (MUT CLC)) or left unoccupied and assayed for assembly at pH 6.7 by light scattering. The ratio of assembly signal for hub with light chain to assembly signal for hub without light chain for is plotted for each reaction (mean±SEM). (F) Representative kinetic assembly data for WT or KR loop mutant (MUT) hub with or without CLC bound, as indicated. Assembly is detected photometrically by an increase in light scatter at OD₃₂₀.

(A) Clathrin heavy chain triskelia with all straight knees or all bent knees were modeled by alignment of distal legs (residues 1–1130) from the cryoEM structure of Fotin et al. (2004) with the hub structures determined here. Straight conformation (brown) and bent conformation (cyan) of knee regions are shifted ~30° starting from the KR loop hinge point. (B and C) Alignment of two modeled hubs, both with a single full length bent knee leg (B) or straight knee leg (C), with two assembled triskelia in the cryoEM structure (Fotin et al., 2004)(1XI4). Black bars indicate regions used to align cryoEM and hub crystal structures and the clathrin light chains are shown in yellow in their relative positions in the hub-LCb complex. Arrows indicated point of steric clashes in C. Insets indicate the geometric alignment of the bent or straight leg triskelia as modeled in (A) along edges of the clathrin basket.