Structural basis of βTrCP1-associated GLI3 processing

Abstract

Controlled ubiquitin-mediated protein degradation is essential for various cellular processes. GLI family regulates the transcriptional events of the sonic hedgehog pathway genes that are implicated in almost one fourth of human tumors. GLI3 phosphorylation by Ser/Thr kinases is a primary factor for their transcriptional activity that incurs the formation of both GLI3 repressor and activator forms. GLI3 processing is triggered in an ubiquitin-dependent manner via SCF^βTrCP1 complex; however, structural characterization, mode of action based on sequence of phosphorylation signatures and induced conformational readjustments remain elusive. Here, through structural analysis and molecular dynamics simulation assays, we explored comparative binding pattern of GLI3 phosphopeptides against βTrCP1. A comprehensive and thorough analysis demarcated GLI3 presence in the binding cleft shared by inter-bladed binding grooves of β-propeller. Our results revealed the involvement of all seven WD40 repeats of βTrCP1 in GLI3 interaction. Conversely, GLI3 phosphorylation pattern at primary protein kinase A (PKA) sites and secondary casein kinase 1 (CK1) or glycogen synthase kinase 3 (GSK3) sites was carefully evaluated. Our results indicated that GLI3 processing depends on the 19 phosphorylation sites (849, 852, 855, 856, 860, 861, 864, 865, 868, 872, 873, 876, 877, 880, 899, 903, 906, 907 and 910 positions) by a cascade of PKA, GSK3β and CSKI kinases. The presence of a sequential phosphorylation in the binding induction of GLI3 and βTrCP1 may be a hallmark to authenticate GLI3 processing. We speculate that mechanistic information of the individual residual contributions through structure-guided approaches may be pivotal for the rational design of specific and more potent inhibitors against activated GLI3 with a special emphasis on the anticancer activity.

Figure 1

Schematic illustration of GLI3 translocation, processing, and degradation via SCF (SKP1, Cullin, F-box protein complex). In the absence of hedgehog (right panel), Ptch1 constitutively inhibits Smo, preventing its ciliary localization. In this state, GLI proteins are retained in a complex with Sufu, where PKA, CK1ε, and GSK3 phosphorylate them. Phosphorylated GLI3 binds the SCF complex that is partially ubiquitinated and processed by the proteasome into GLI3-R, which translocates to the nucleus and represses transcription of Hh target genes, including Akt, Gli1, Ptch1 etc., prior to degradation by an unknown E3 ligase complex. In the presence of Hh (Left panel), PKA phosphorylation is inhibited. The activated form of GLI3 not only prevents it from processing but also permits its subsequent transport to the nucleus to allow activation of transcription of Hh target genes. Following this, GLI3-A is ubiquitinated by the SPOP/CUL3 complex and degraded by the proteasome. P, phosphate group; Ptch1, Patched 1; SUFU, Suppressor of Fused; SMO, Smoothened; PKA, Protein kinase A, GSK3, Glycogen synthase kinase 3; CSKI, Casein kinase 1; CUL1, Cullin 1; CUL3, Cullin 3; RBX1, RING-box protein 1; SKP1, S-Phase Kinase-Associated Protein 1; SPOP, Speckle Type BTB/POZ Protein; GLI3-R, GLI3 repressor; GLI3-A, GLI3 activator.

Figure 2

Binding orientation of β-propeller due to phosphopeptide binding. 7 WD40 repeats of βTrCP1, comprising 25 beta sheets are organized to form a circular structure (β-propeller). Optimal docked complexes of βTrCP1 bound (A) GLI3^PKA (B) GLI3^GSK3β, (C) GLI3^CSKIϵ (D) GLI3-β1–4. βTrCP1 is shown in white colored ribbon, while GLI3 phosphopeptide is shown in red colored ribbon.

Figure 3

Binding mode and molecular interaction analysis of motif peptides. Optimal docked complexes of βTrCP1-bound (A) GLI3-β1 (B) GLI3-β2, (C) GLI3-β3 (D) GLI3-β4 and (E) GLI3-β1–4 peptides. βTrCP1 and GLI3 are shown in white and khaki colored ribbons with interacting residues in green and goldenrod colored ball and sticks, respectively.

Figure 4

Time-dependent analysis of 40 ns MD simulations of apo- versus GLI3 peptide-bound βTrCP1. (A) RMSD plotted as a time function computed through least square fitting of backbone Cα-atoms. (B) Rg plots of individual simulated complexes along the course of 40 ns of MD simulation. (C) RMSF per residue plot for each trajectory file. (D) Comparison of the most fluctuating residues is indicated by bar chart. Apo and bound forms of βTrCP1 with GLI3-β1, GLI3-β2, GLI3-β3, GLI3-β4 and GLI3-β1–4 are represented in blue, green, gold, orange, cyan and purple colors, respectively.

Figure 5

Binding energy and hydrogen bond versus time plots for 40 ns MD simulation. (A) LJ-SR binding energy profile. (B) Intermolecular hydrogen bonding pattern of βTrCP1-GLI3 complexes. GLI3-β1, GLI3-β2, GLI3-β3, GLI3-β4 and GLI3-β1–4 are represented in green, gold, orange, cyan and purple colors, respectively.

Figure 6

Structural details of βTrCP1 and GLI3 phosphopeptide binding. βTrCP1 is represented by light gray ribbon, while pale yellow ribbons represent phosphopeptide GLI3-β1–4 with interacting residues indicated by coral ball and stick mode. Illustration of four sequence motifs (β1 to β4) related to the βTrCP1 binding site are underlined that are phosphorylated by a putative cascade of PKA, GSK3β and CK1. PKA phosphorylated serines (phosphoserine) in the sequence motifs are colored in red. GSK3β phosphorylates serines (green) four residues N-terminal to a phosphoserine, while CK1 phosphorylates serines (blue) three residues C-terminal to a phosphoserine; both can chronologically multiphosphorylate GLI3 after priming. Middle panel shows the conservation pattern of βTrCP1 binding residues upon phosphopeptide binding. X-axis indicates the binding residues of βTrCP1 and Y-axis indicates the GLI3 phosphopeptides (GLI3-β1, GLI3-β2, GLI3-β3, GLI3-β4 and GLI3-β1–4). Dot represents the contribution of respective residue in binding to phosphopeptide.

Figure 7

Conformational switches of the GLI3 phosphopeptide structure upon binding to βTrCP1. Phosphopeptides of (A) GLI3-β1, (B) GLI3-β2, (C) GLI3-β3, (D) GLI3-β4 and (E) GLI3-β1–4 are represented in green, gold, orange, cyan and purple colors, respectively. Phosphorylated residues via PKA, GSK3β and CSKI are shown by red, light green and blue colors, respectively in ball and stick mode. Secondary structures are illustrated above the corresponding plots. Coils delineate α-helices, while line specifies loop. (F) Comparative RMSF versus time plot of significant phosphorylated residues.

Figure 8

The binding free decomposition on per residue basis calculated from 40 ns MD trajectories by MM/PBSA method. Binding free energy decomposition at residue basis for βTrCP1 upon binding to (A) GLI3-β4 (B) GLI3-β1–4 peptides. Binding free energy decomposition on a per-residue basis for (C) GLI3-β4 (D) GLI3-β1–4.