Dynamic interactions between clathrin and locally structured elements in a disordered protein mediate clathrin lattice assembly

Abstract

Assembly of clathrin lattices is mediated by assembly/adaptor proteins that contain domains that bind lipids or membrane-bound cargo proteins and clathrin binding domains (CBDs) that recruit clathrin. Here, we characterize the interaction between clathrin and a large fragment of the CBD of the clathrin assembly protein AP180. Mutational, NMR chemical shift, and analytical ultracentrifugation analyses allowed us to precisely define two clathrin binding sites within this fragment, each of which is found to bind weakly to the N-terminal domain of the clathrin heavy chain (TD). The locations of the two clathrin binding sites are consistent with predictions from sequence alignments of previously identified clathrin binding elements and, by extension, indicate that the complete AP180 CBD contains ∼12 degenerate repeats, each containing a single clathrin binding site. Sequence and circular dichroism analyses have indicated that the AP180 CBD is predominantly unstructured and our NMR analyses confirm that this is largely the case for the AP180 fragment characterized here. Unexpectedly, unlike the many proteins that undergo binding-coupled folding upon interaction with their binding partners, the AP180 fragment is similarly unstructured in its bound and free states. Instead, we find that this fragment exhibits localized β-turn-like structures at the two clathrin binding sites both when free and when bound to clathrin. These observations are incorporated into a model in which weak binding by multiple, pre-structured clathrin binding elements regularly dispersed throughout a largely unstructured CBD allows efficient recruitment of clathrin to endocytic sites and dynamic assembly of the clathrin lattice.

Figure 1

The domain structure of AP180. AP180 is a 92kD clathrin assembly protein with a structured N-terminal ANTH domain which interacts with membrane phospholipids, and a disordered clathrin binding domain that interacts with the N-terminal domain of the clathrin heavy chain (TD). The putative clathrin binding motifs are shown in red. The ANTH domain bound to PIP₂ was modeled using the coordinates in PDB file 1hfa. Indicated is the location and sequence of AP180 M5, the recombinant fragment of AP180 containing 2 putative clathrin binding sites that was used in this study.

Figure 2

Backbone sequential resonance assignments for AP180 M5. Two-dimensional ¹H-¹⁵N HSQC spectrum of AP180 M5 showing the backbone assignments. Peaks representing the side chain signals are not shown.

Figure 3

AP180 M5 undergoes chemical shift perturbations in the presence of clathrin TD. A. An HSQC spectrum of 500 μM ¹⁵N-¹³C labeled AP180 M5 is shown in black while a spectrum of 500 μM ¹⁵N-¹³C labeled AP180 M5 with 500 μM unlabeled clathrin TD is shown in red. B. Chemical shift differences between AP180 M5 in the free and TD bound states. The composite absolute shift perturbations shown in blue include the backbone H^N, H^α, C^O and N^H shift perturbations. The regions showing significant chemical shift changes are consistent with the putative clathrin binding sites identified by our previous studies.

Figure 4

Analysis of the chemical shift changes in ¹⁵N-AP180 M5 upon titration with unlabeled clathrin TD. A,E: HSQC spectra of two representative amino acid residues within clathrin binding site 1 (A) and clathrin binding site 2 (E). As the [clathrin TD]_total/[AP180 M5]_total ratio increased, the peak positions shifted as indicated by progression from black to red to green to magenta colored peaks. The asterisks below the sequences indicate the residues used for the subsequent K_D determination. Peaks that broadened extensively were omitted from the K_D analysis. B,C. Determination of the K_D of clathrin binding site 1 in the WT AP180 M5 (B), and in a single-site AP180 M5 in which binding site 2 was mutated (C). F,G: Determination of the K_D of clathrin binding site 2 in WT AP180 M5 (F), and in a single-site AP180 M5 in which binding site 1 was mutated (G). Plotted (B,C,F,G) are the weighted average chemical shift changes of the ¹H and ¹⁵N resonance of the amino acid residues. The data shown in each panel was globally fit to a hyperbolic equation, and fits are indicated with red traces. D,H: Determination of the dissociation rate constant k_off of clathrin binding site 1 (D) and site 2 (H) in the WT AP180 M5. The line width analysis was performed as described in materials and methods, and global fits are indicated with red traces.

Figure 5

AUC reveals 1:1 binding of AP180 M5 to clathrin TD and confirms weak binding measured by NMR. 1 μM fluorescently labeled AP180 M5 was incubated with a concentration series of unlabeled clathrin TD. After the reactions were allowed to come to equilibrium, free AP180 M5 was separated from bound by AUC. A. Enhanced van Holde-Weischet integral S-distribution plots for a titration of 1 μM fluorescently labeled AP180 M5, with increasing concentrations of clathrin TD (purple: 50 μM; blue: 110 μM; turquoise: 170 μM; green: 230 μM; yellow: 290 μM; orange: 350 μM). Only AP180 M5 is detectable in the titration experiment, since it is labeled with Alexa 488 and fluorescence emission at 500 nm is followed. Free AP180 M5 (black curve) and free TD (red curve, measured with interference optics) are also shown as controls. AP180 M5 sediments with an S-value of ~1S, clathrin TD sediments with ~2.9S, and the AP180 M5:clathrin TD complex with ~3S. B. Genetic algorithm analysis combined with 50 iterations of a Monte Carlo analysis of a mixture of 1 μM AP180 M5 with 230 μM TD reveals the more extended (high f/f_o) structure for free AP180 M5 and the more compact AP180 M5:clathrin TD complex. The molecular weight for both species agrees well with the molecular weight predicted from sequence, and indicates that one clathrin TD is binding per AP180 M5. Also visible is a signal from a minor species which represents the free fluorescent dye. The relative signal of each species is shown as a color gradient in units of counts of fluorescence emission. C. Using the analysis method described in panel B to quantify the amount of bound AP180 M5, plots of binding as a function of clathrin TD concentration were generated for experiments carried out with either WT AP180 M5, the single-site AP180 site 1 mutant, or the AP180 M5 site 2 mutant. K_Ds were determined by fitting the data to a hyperbolic equation, and are in good agreement with the K_Ds determined by the NMR analysis.

Figure 6

Analysis of secondary chemical shifts indicates that AP180 M5 contains no regions of α helical or β street structure, in either the free or bound state. It has been reported that for residues in stable α helices, the average secondary chemical shifts are 2.5 ppm for ¹³C^α and −0.38 ppm for ¹H^α, with near random coil values for ¹³C^β and positive values for ¹³C^o. In stable β sheets, the average secondary chemical shifts are −2.0 ppm for ¹³C^α, 2.5 ppm for ¹³C^β and 0.38 ppm for ¹H^α, with negative values for ¹³C^o. A. Secondary chemical shifts of free 500 μM ¹⁵N-¹³C labeled AP180 M5 for ¹³C^α, ¹³C^β, ¹³C^o and ¹H^α were calculated by subtracting random coil shifts corrected for sequence-dependent variations from the experimental chemical shifts. No regions with patterns indicative of either α helix or β sheet were identified. B. Secondary chemical shifts of 500 μM ¹⁵N-¹³C labeled AP180 M5 with 500 μM unlabeled clathrin TD for ¹³C^o and ¹H^α were calculated in the same way. No regions with patterns indicative of either α helix or β sheet were identified.

Figure 7

NOESY experiments reveal local β turn-like structures in AP180 M5 at the clathrin binding sites. A. Summary of ¹H-¹H NOEs of 500 μM ¹⁵N labeled AP180 M5 (top) and 500 μM ¹⁵N labeled AP180 M5 with 500 μM unlabeled clathrin TD (bottom). The different thicknesses of the lines indicate the NOE signal intensities. B. A prediction of the ¹H-¹H NOEs of the AP3 β3 clathrin box peptide bound to TD.

Figure 8

The AP180 M5 polypeptide chain is less flexible at the clathrin binding sites whether free or bound to clathrin TD. A. The T₂ relaxation times and ¹H-¹⁵N NOEs of 500 μM ¹⁵N-AP180 M5 in the free state. B. The T₂ relaxation times and ¹H-¹⁵N NOEs of 500 μM ¹⁵N-AP180 M5 with 500 μM unlabeled clathrin TD. Regions showing shorter T₂ relaxation times and higher ¹H-¹⁵N NOEs values represent less flexibility in the polypeptide chain.

Figure 9

The line fishing model for assembling the endocytic apparatus. After docking to the plasma membrane via interactions between the N-terminal ANTH domain of AP180 (yellow) and membrane bound PIP₂, the long and flexible C-terminal domain of AP180 (red) can bind and recruit clathrin (black) from a large volume of cytosol to initiate the formation of a clathrin coated pit. The large number of clathrin binding sites (green) recruit multiple clathrin heavy chains together to form the vertexes of the clathrin lattice (adapted from with permission from the Journal of Biological Chemistry).