Proline-rich domain of human ALIX contains multiple TSG101-UEV interaction sites and forms phosphorylation-mediated reversible amyloids

Abstract

Proline-rich domains (PRDs) are among the most prevalent signaling modules of eukaryotes but often unexplored by biophysical techniques as their heterologous recombinant expression poses significant difficulties. Using a "divide-and-conquer" approach, we present a detailed investigation of a PRD (166 residues; ∼30% prolines) belonging to a human protein ALIX, a versatile adaptor protein involved in essential cellular processes including ESCRT-mediated membrane remodeling, cell adhesion, and apoptosis. In solution, the N-terminal fragment of ALIX-PRD is dynamically disordered. It contains three tandem sequentially similar proline-rich motifs that compete for a single binding site on its signaling partner, TSG101-UEV, as evidenced by heteronuclear NMR spectroscopy. Global fitting of relaxation dispersion data, measured as a function of TSG101-UEV concentration, allowed precise quantitation of these interactions. In contrast to the soluble N-terminal portion, the C-terminal tyrosine-rich fragment of ALIX-PRD forms amyloid fibrils and viscous gels validated using dye-binding assays with amyloid-specific probes, congo red and thioflavin T (ThT), and visualized by transmission electron microscopy. Remarkably, fibrils dissolve at low temperatures (2 to 6 °C) or upon hyperphosphorylation with Src kinase. Aggregation kinetics monitored by ThT fluorescence shows that charge repulsion dictates phosphorylation-mediated fibril dissolution and that the hydrophobic effect drives fibril formation. These data illuminate the mechanistic interplay between interactions of ALIX-PRD with TSG101-UEV and polymerization of ALIX-PRD and its central role in regulating ALIX function. This study also demonstrates the broad functional repertoires of PRDs and uncovers the impact of posttranslational modifications in the modulation of reversible amyloids.

Keywords: NMR; amyloids; intrinsically disordered protein; posttranslational modifications; signal transduction.

Fig. 1.

ALIX domain organization and summary of ALIX-PRD constructs used in the current work. (A) Schematic of ALIX organization. Primary sequence of PRD is shown with prolines (∼30%) and tyrosines (∼9%) labeled in purple and red, respectively. (B) Recombinant PRD constructs, namely GB1–${\text{PRD}}_{703-868}^{\text{Strep}},$ GB1–${\text{PRD}}_{703-815}^{\text{Strep}},$ GB1–${\text{PRD}}_{703-800}^{\text{Strep}},$ and GB1–PRD_800–868. The positions of purification tags, 6×His and strep, are marked (primary sequence of strep tag is shown). TEV protease cutting sites are shown in gray and marked with dashed lines and scissors. Recombinant expression of GB1–${\text{PRD}}_{703-868}^{\text{Strep}}$ and GB1–${\text{PRD}}_{703-815}^{\text{Strep}}$ resulted in truncated fragments because of ribosomal stalling induced by polyproline stretches, especially at residue P₈₀₁, marked by a red circle and vertical red line. (C) SDS-PAGE analysis of purified PRD constructs [16% wt/vol tris(hydroxymethyl)aminomethane–glycine gel]; the order of GB1–PRD fusion constructs is the same as the one depicted in B. TEV-cleaved products, namely ${\text{PRD}}_{703-800}^{\text{Strep}}$ and PRD_800–868, are marked with arrows. (D) Amino acid composition of ${\text{PRD}}_{703-800}^{\text{Strep}}$ (Top) and PRD_800–868 (Bottom). Vertical bars marked with orange and gray asterisks denote contributions from nonnative strep tag and remnant-glycine residues of TEV cleavage sites, respectively.

Fig. 2.

NMR analyses of interactions of ${\text{PRD}}_{703-800}^{\text{Strep}}$ with TSG101-UEV. (A and B) Overlay of expanded regions of the ¹H-¹⁵N TROSY (A) and ¹³C-¹⁵N CO-N (B) correlation spectra of 100 µM ${\text{PRD}}_{703-800}^{\text{Strep}}$ in the absence (red) and presence (blue) of TSG101-UEV (molar ratio: 1:3). Some isolated cross-peaks that exhibit significant reduction in intensities on addition of TSG101-UEV are labeled. (C and D) The reduction in ¹H-¹⁵N (C) and ¹³C′-¹⁵N (D) cross-peak intensities of ${\text{PRD}}_{703-800}^{\text{Strep}}$ on addition of TSG101-UEV is indicative of intermediate exchange on the chemical-shift time scale. Color scheme is as follows: ${\text{PRD}}_{703-800}^{\text{Strep}}$ + TSG101-UEV molar ratio: green, 1:0.25; magenta, 1:1.5; blue, 1:3. Affected regions are highlighted with semitransparent gray rectangles; primary sequences of each interacting PRD site (, , and 3) are shown above the graphs, with recurring PTAP-like motifs underlined and labeled in dark purple. The position of the C-terminal strep tag (residues 801 to 808) is denoted by semitransparent orange rectangles.

Fig. 3.

NMR and structural analyses of interactions of TSG101-UEV with ${\text{PRD}}_{703-800}^{\text{Strep}}.$ (A) Overlay of expanded region of the ¹H-¹⁵N TROSY correlation spectra of ¹⁵N/²H-labeled 100 µM TSG101-UEV in the absence (red) and presence (blue) of ${\text{PRD}}_{703-800}^{\text{Strep}}$ (molar ratio: 1:4). Some isolated cross-peaks that exhibit significant reduction in intensities upon addition of ${\text{PRD}}_{703-800}^{\text{Strep}}$ are labeled. Cross-peaks that undergo chemical-shift changes on addition of ${\text{PRD}}_{703-800}^{\text{Strep}}$ are marked by circles. (B and C) The reduction in ¹H-¹⁵N cross-peak intensities (B) and the perturbations in ¹H_N/¹⁵N chemical shifts (C) of TSG101-UEV on addition of ${\text{PRD}}_{703-800}^{\text{Strep}}.$ Affected regions are highlighted in semitransparent red rectangles (secondary structure elements are indicated above the graphs). Semitransparent gray rectangles and dashed blue lines indicate the residues that could not be assigned unambiguously. (D) A ribbon diagram of model of TSG101-UEV + PRD_711–730 complex. TSG101-UEV and PRD_711–730 are colored in white and blue, respectively. Regions marked in red represent residues of TSG101-UEV that are most affected on addition of ${\text{PRD}}_{703-800}^{\text{Strep}}.$ Gray ribbons indicate residues around the binding site that could not be assigned unambiguously. For PRD_711–730, the side chains of individual residues are also shown; ⁷¹⁷PSAP⁷²⁰ motif (site 1) is marked with dark purple labels.

Fig. 4.

Quantitative analyses of interactions of ${\text{PRD}}_{703-800}^{\text{Strep}}$ with TSG101-UEV. (A) Representative backbone ¹⁵N-CPMG relaxation dispersion profiles observed for 100 µM ${\text{PRD}}_{703-800}^{\text{Strep}}$ on addition of TSG101-UEV (molar ratio: 1:5); dispersions were recorded at 600 MHz (red) and 800 MHz (blue). The experimental data are displayed as circles, and the solid lines represent the global best fits to a two-state exchange model. Control relaxation dispersions at 800 MHz obtained in the absence of TSG101-UEV are shown in black. For the control data, black lines are used to guide the eye. (B) Summary of kinetic parameters obtained upon globally best fitting all CPMG data to a two-site exchange mode. Each interacting site (1, 2, and 3) is fit individually. $k_{\text{on}}^{\text{app}}$ is an apparent pseudo–first-order association rate constant that pertains to 100 μM ${\text{PRD}}_{703-800}^{\text{Strep}}$ and 500 μM TSG101-UEV used in the CPMG experiments. k_off is the dissociation rate constant. The equilibrium dissociation constant, K_D, for each individual site is given by k_off/k_on, where $k_{\text{on}}=k_{\text{on}}^{\text{app}}/\left[\mathrm{L}\right]$ and [L] is the concentration of unbound TSG101-UEV. (C) Populations of 1:1 and 1:2 complexes formed between ${\text{PRD}}_{703-800}^{\text{Strep}}$ and TSG101-UEV, calculated using K_D values for each individual PRD site and mass-action law. Note that sites 2 and 3 are mutually exclusive as they cannot simultaneously interact with two TSG101-UEV molecules due to steric hindrance (cf., Fig. 2C). (D) Scheme depicting potential modes of interactions of ${\text{PRD}}_{703-800}^{\text{Strep}}$ with TSG101-UEV; populations of bound sites are labeled.

Fig. 5.

Amyloid fibrils and gel formed by GB1–PRD_800–868. (A and B) Absorbance spectra of CR (A) and emission spectra of ThT (B) for aggregates formed by GB1–PRD_800–868 (red). GB1–${\text{PRD}}_{703-800}^{\text{Strep}}$ samples (blue) were used as controls. Raw data of three replicates (n = 3) are plotted against wavelength (nm). (C and D) Negatively stained EM images of GB1–PRD_800–868 amyloid fibrils. (Scale bars, 200 nm.) (E) Formation of a gel by GB1–PRD_800–868. To initiate the gel formation, a 0.1 mM GB1–PRD_800–868 sample was kept at room temperature under quiescent conditions for ∼3 d. (F) EM images of GB1–PRD_800–868 gel.

Fig. 6.

Formation and dissolution of GB1–PRD_800–868 amyloid fibrils. (A–D) ThT fluorescence was monitored to determine the effects of pH (A), ionic strength (B), concentration (C), and temperature (D) on the aggregation kinetics of GB1–PRD_800–868 (raw data of three replicates [n = 3] are plotted against time). Control curves (dark red) (A) are collected using GB1–${\text{PRD}}_{703-800}^{\text{Strep}}.$ Control experiments were also carried out on GB1 fusion tag and showed no ThT signal. For A–C, the measurements were carried out at 30 °C. (E) Dissolution of GB1–PRD_800–868 amyloid fibrils at 4 °C. ThT emission spectra of GB1–PRD_800–868 (n = 3). Samples were incubated at 40 °C for ∼2 h (0 and 2-h time points are shown in light and dark red, respectively). Temperature was dropped to 4 °C, and fluorescence was measured until the reading was stabilized (3 to 7 h; light-to-dark blue gradient). The blue and red gradient color bars denote incubation time at 4 and 40 °C, respectively. The gel formed by GB1–PRD_800–868 at 40 °C became fragile at 4 °C (E, Lower). ThT assays were also carried out on PRD_800–868. Poor solubility of PRD_800–868 resulted in large variations in kinetic curves (n = 2) (F). a.u., arbitrary units.

Fig. 7.

Dissolution of GB1–PRD_800–868 amyloids upon Src-mediated tyrosine phosphorylation. (A–D) Characterization of in vitro phosphorylation of PRD constructs using Phos-tag SDS-PAGE (A), Western blotting (B), and MS (C and D). For Phos-tag gel, the following constructs, namely ${\text{PRD}}_{703-800}^{\text{Strep}},$ GB1–PRD_800–868, PRD_800–868, and the GB1 tag, were incubated with Src (substrate to kinase molar ratio: 1:0.01). Phosphorylated products were visualized by silver staining and are marked by pink asterisks. (B) In vitro phosphorylation of ${\text{PRD}}_{703-800}^{\text{Strep}}$ and PRD_800–868 by Western blotting. (C and D) LC-ESI-TOFMS and LC-MS/MS analyses of in vitro phosphorylation reactions revealed hyperphosphorylated states of GB1–PRD_800–868 (C) and PRD_800–868 (D). A schematic representation of GB1–PRD_800–868 along with phosphorylated tyrosines (dashed rectangle) are shown above the graph in C. The numbers in red represent the number of phosphorylated tyrosines, labeled as pY (peaks marked with blue asterisks represent sodium/iron adducts; SI Appendix, Table S2). (E–G) The impact of tyrosine phosphorylation on aggregation kinetics of GB1–PRD_800–868 was assessed using ThT assays (E and F) and NMR spectroscopy (G). For ThT assays (n = 3) (E), 150 µM GB1–PRD_800–868 samples were incubated at 30 °C with 2 mM ATP and varying concentrations of Src (molar ratios: 1:0.1 [gray], 1:0.02 [red], and 1:0.01 [green]). a.u., arbitrary units. Control experiments were carried out on a GB1–PRD_800–868 + Src mixture in the absence of ATP (black; molar ratio: 1:0.1). (F) Samples of 150 µM GB1–PRD_800–868 were incubated at 30 °C without Src (n = 5) for ∼11 h. Src + ATP were then added to three samples (red), whereas the remaining two (black) received only Src. G shows the overlay of expanded regions of the ¹H-¹⁵N TROSY-HSQC spectra of phosphorylated GB1–PRD_800–868 (red) and the phosphorylated GB1 tag (blue). Both were incubated with Src (molar ratio: 1:0.1) in the presence of 2 mM ATP for 5 h at 30 °C. (G, Inset) Corresponding one-dimensional profiles of ¹⁵N-labeled GB1–PRD_800–868 recorded at 0 and 5 h (black and red, respectively) after addition of unlabeled Src and ATP.