Distinct EH domains of the endocytic TPLATE complex confer lipid and protein binding

Abstract

Clathrin-mediated endocytosis (CME) is the gatekeeper of the plasma membrane. In contrast to animals and yeasts, CME in plants depends on the TPLATE complex (TPC), an evolutionary ancient adaptor complex. However, the mechanistic contribution of the individual TPC subunits to plant CME remains elusive. In this study, we used a multidisciplinary approach to elucidate the structural and functional roles of the evolutionary conserved N-terminal Eps15 homology (EH) domains of the TPC subunit AtEH1/Pan1. By integrating high-resolution structural information obtained by X-ray crystallography and NMR spectroscopy with all-atom molecular dynamics simulations, we provide structural insight into the function of both EH domains. Both domains bind phosphatidic acid with a different strength, and only the second domain binds phosphatidylinositol 4,5-bisphosphate. Unbiased peptidome profiling by mass-spectrometry revealed that the first EH domain preferentially interacts with the double N-terminal NPF motif of a previously unidentified TPC interactor, the integral membrane protein Secretory Carrier Membrane Protein 5 (SCAMP5). Furthermore, we show that AtEH/Pan1 proteins control the internalization of SCAMP5 via this double NPF peptide interaction motif. Collectively, our structural and functional studies reveal distinct but complementary roles of the EH domains of AtEH/Pan1 in plant CME and connect the internalization of SCAMP5 to the TPLATE complex.

Fig. 1. EH domains of AtEH1/Pan1 differ in their Ca²⁺-binding capacities.

a Domain organization of AtEH1/Pan1 and AtEH2/Pan1. Both proteins contain two Eps15 homology domains (EH), a coiled-coil domain (CC), and an acidic (A)-motif. A schematic representation of a multiple sequence alignment (MSA), shows strong conservation of the EH domains (blue lines) across the plant kingdom. Percentages indicate the relative number of identical amino acids. b–g Cartoon representation of the X-ray structure of EH1.1 and NMR/all-atom molecular dynamics structure of EH1.2. Ions are shown as orange (Ca²⁺) or grey (Na⁺) spheres. Insets show the ion coordination in each EF-hand loop. Ca²⁺ coordinating residues and water molecules (W) are indicated in (c, d) and (f, g). h Total reflection X-ray fluorescence (TXRF) intensities of Ca²⁺ normalized to Cl⁻ of samples containing 2 mM of each EH domain in the presence of 0.5 mM free Ca²⁺. The mean (n = 5) is indicated as a pink line. Statistical analysis was performed using a two-tailed Welch’s t-test (p = 1.056 × 10⁻⁴). i Ca²⁺ concentration relative to the amount of protein as measured by inductively coupled plasma mass spectrometry (ICP-MS) after subtraction of the amount of free Ca²⁺ in the medium. The mean (n = 3) is indicated as a pink line. Statistical analysis was performed using a two-tailed Welch’s t-test (p = 1.046 × 10⁻⁵). j ¹⁵N-¹H HSQC spectra of each EH domain before (black) and after (red) Ca²⁺ chelation by 10 mM EDTA.

Fig. 2. Differential binding of EH domains of AtEH1/Pan1 to anionic phospholipids.

a Coomassie-blue stained SDS-PAGE analysis of liposome binding comparing the binding of EH domains between PC (an equimolar mixture of PE and PC) and 10% PIP₂ containing liposomes in the presence of 10 µM Ca²⁺. S = supernatant, P = pellet. b Quantification of lipid binding (ratio of cumulative grey values between pellet and supernatant) as shown in (a). Different letters indicate significant differences between samples using a one-sided Kruskal–Wallis test (p ≤ 0.05). n = 3 independent experiments. c Localization of triple EH domains fused C-terminally to GFP in N. benthamiana epidermal leaf cells. Cells were triggered to divide by overexpression of the Cyclin D protein. The early and late stages of cell division are depicted (arrowheads). In contrast to EH1.2, EH1.1 is retained more prominent at the late cell plate (arrowhead) and also labels the nuclear envelope (asterisk). Scale bar indicates 10 µm. The experiment was repeated twice independently with similar results. d Coomassie-blue stained SDS-PAGE analysis of PA liposome binding of both EH domains as well EH1.2 mutated in its predicted PIP₂-binding site (EH1.2 R/K>E). S = supernatant, P = pellet. e Quantification of PA lipid binding (pellet versus supernatant) as shown in (d). Different letters indicate significant differences between samples using a one-sided Kruskal–Wallis test (p ≤ 0.05). n = 4 or 5 independent experiments. f, g CG-MD simulations of EH1.1 (f) and EH1.2 (g) with a lipid bilayer containing 20% PA. On the left, the mean number of contacts with PA for each domain are shown. The contacts were defined as the number of PA phosphate groups within 0.8 nm of protein atoms calculated through the whole simulation time and averaged over all CG-MD replicas. On the right, a cartoon representation of each EH domain is shown. The color gradient (yellow to blue) indicates the extent of interaction with PA. Residues with the highest contact number are shown as sticks. Ca²⁺ is shown as an orange sphere.

Fig. 3. The first EH domain of AtEH1/Pan1 interacts with the N-terminal double NPF motif of SCAMP5.

a Scheme of the peptidome profiling experiment. b Graphical representation of SCAMP5, the double N-terminal NPF motif is indicated in yellow. c BLI steady-state kinetics of the binding of EH1.1 of AtEH1/Pan1 and NPF peptides with or without mutations. WT Protein but not the W49A mutant binds the double NPF peptide with a measurable affinity. Mutation of any of the NPF motifs abrogates binding. Data are presented as mean values ± SD. n = 3 independent experiments. d ¹H-¹⁵N-HSQC spectra of EH1.1 titrated with increasing amounts of the SCAMP5 double NPF peptide (grey to red). Peak trajectories of selected residues are indicated by arrows. Their weighted chemical shift perturbations were used to obtain binding isotherms and derive an apparent dissociation constant of the interaction (Supplementary Fig. 5). e Cartoon representation of EH1.1 (6-105) colored with a gradient (pink to purple) indicate the extent of chemical shift perturbations induced by the SCAMP5 double NPF peptide binding. Residues showing large chemical shift perturbations (>0.3 ppm) are shown as sticks. f Similar as in panel d but the EH1.1 structure is colored according to ConSurf colors denoting evolutionary conservation (blue to white to purple). Most residues affected by peptide binding are well-conserved. g Ratiometric bimolecular fluorescence complementation (rBiFC) analysis showing interaction between AtEH1/Pan1 and SCAMP5. Both proteins specifically interact when tagged at the N-terminus. Mutating the SCAMP5-binding site in the first EH domain of AtEH1/Pan1 (W49A) drastically reduces the interaction. Scale bar is 50 µm.  h Quantification of the YFP/RFP fluorescence ratios from the experiment in (g). The black lines represent the median and the red circles represent the mean. The amount of quantified cells is indicated below each boxplot. Letters above the plots indicate statistically significant differences analyzed by one-sided Welch’s ANOVA post hoc pairwise comparison was performed with the package multcomp utilizing the Tukey contrasts (p ≤ 0.001). The boxplot extends from the 25th to 75th percentiles. The line inside the box marks the median. The whiskers go down and up to the 95% percentile. i Co-localization of SCAMP5-GFP and AtEH1/Pan1-mRuby3 at the plasma membrane and the cell plate in Arabidopsis root cells. Cells in different phases of cytokinesis (early, late and post) are depicted. SCAMP5 recruitment to the cell plate precedes AtEH1/Pan1 whereas the presence of the latter at the newly formed cross wall exceeds SCAMP5 following completion of cytokinesis. The results shown were observed in two independent lines. Scale bar indicates 5 µm. j Confocal analysis of SCAMP5-GFP vs ΔN-SCAMP5-GFP. An increased plasma membrane localization was observed in the absence of the double NPF motif. Colors are shown according to signal intensity (red to green to blue). The images are representative of two independent lines. Scale bar indicates 10 µm. k Quantification of the plasma membrane vs the cytoplasm of two independent lines for both constructs as shown in (j). The mean is shown as a pink dot. The amount of quantified cells is indicated below each boxplot. Different letters indicate significant differences between samples analyzed by one-sided Welch’s ANOVA. Post hoc pairwise comparison was performed with the package multcomp utilizing the Tukey contrasts (p ≤ 0.001). The boxplot extends from the 25th to 75th percentiles. The line inside the box marks the median. The whiskers go down and up to the 95% percentile.