Limits...
Structural basis of SUFU-GLI interaction in human Hedgehog signalling regulation.

Cherry AL, Finta C, Karlström M, Jin Q, Schwend T, Astorga-Wells J, Zubarev RA, Del Campo M, Criswell AR, de Sanctis D, Jovine L, Toftgård R - Acta Crystallogr. D Biol. Crystallogr. (2013)

Bottom Line: Despite its central importance, little is known about SUFU regulation and the nature of SUFU-GLI interaction.It is demonstrated that GLI binding is associated with major conformational changes in SUFU, including an intrinsically disordered loop that is also crucial for pathway activation.These findings reveal the structure of the SUFU-GLI interface and suggest a mechanism for an essential regulatory step in Hedgehog signalling, offering possibilities for the development of novel pathway modulators and therapeutics.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biosciences and Nutrition and Center for Biosciences, Karolinska Institutet, Novum, Hälsovägen 7, SE-141 83 Huddinge, Sweden.

ABSTRACT
Hedgehog signalling plays a fundamental role in the control of metazoan development, cell proliferation and differentiation, as highlighted by the fact that its deregulation is associated with the development of many human tumours. SUFU is an essential intracellular negative regulator of mammalian Hedgehog signalling and acts by binding and modulating the activity of GLI transcription factors. Despite its central importance, little is known about SUFU regulation and the nature of SUFU-GLI interaction. Here, the crystal and small-angle X-ray scattering structures of full-length human SUFU and its complex with the key SYGHL motif conserved in all GLIs are reported. It is demonstrated that GLI binding is associated with major conformational changes in SUFU, including an intrinsically disordered loop that is also crucial for pathway activation. These findings reveal the structure of the SUFU-GLI interface and suggest a mechanism for an essential regulatory step in Hedgehog signalling, offering possibilities for the development of novel pathway modulators and therapeutics.

Show MeSH

Related in: MedlinePlus

Mechanism of GLI binding. (a, b, c) Interactions between MBPA216H_K220H-SUFU-ΔW61D_L62S_G63F_P453A_Δ454–456_K457A and GLI3p. (a) The peptide (blue) is clamped between the N-terminal (beige) and C-terminal (green) domains. (b) GLI3p (blue residue labels) binds in a channel with His336 and Lys337 protruding into deep pockets. (c) SUFU–GLI3p interactions, with side-chain hydrogen bonds highlighted in yellow, and comparison of GLI1p and GLI3p. Residues that are conserved in GLI1, GLI2 and GLI3 are shown in red. Blue boxes indicate residues with visible electron density. (d, e) Mutations around the GLI3p binding site have varying effects on the ability of SUFU-FL to repress GLI1-induced reporter gene activity in HEK 293 cells (d) and to suppress constitutive pathway activity in Sufu−/− cells (e).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3852661&req=5

fig6: Mechanism of GLI binding. (a, b, c) Interactions between MBPA216H_K220H-SUFU-ΔW61D_L62S_G63F_P453A_Δ454–456_K457A and GLI3p. (a) The peptide (blue) is clamped between the N-terminal (beige) and C-terminal (green) domains. (b) GLI3p (blue residue labels) binds in a channel with His336 and Lys337 protruding into deep pockets. (c) SUFU–GLI3p interactions, with side-chain hydrogen bonds highlighted in yellow, and comparison of GLI1p and GLI3p. Residues that are conserved in GLI1, GLI2 and GLI3 are shown in red. Blue boxes indicate residues with visible electron density. (d, e) Mutations around the GLI3p binding site have varying effects on the ability of SUFU-FL to repress GLI1-induced reporter gene activity in HEK 293 cells (d) and to suppress constitutive pathway activity in Sufu−/− cells (e).

Mentions: In order to determine how SUFU interacts with GLI transcription factors, we attempted to co-crystallize MBP-SUFU-FL and MBP-SUFU-Δ with GLI1p as well as corresponding peptides from human GLI2 (GLI2p; residues 267–283) and GLI3 (GLI3p; residues 328–344) (Supplementary Table S1b). Despite extensive screening, these attempts were unsuccessful. Therefore, the residues WLG61–63 of SUFU were mutated to DSF in MBP-SUFU-Δ to disrupt crystal contacts between a loop within the SUFU N-terminal domain and residues in the C-terminal domain (Supplementary Fig. S2b) previously implicated in GLI binding (Merchant et al., 2004 ▶). Furthermore, another flexible loop in SUFU was shortened and residues 216 and 220 in the MBP moiety were mutated to histidine in order to promote metal ion-mediated crystallization of the fusion protein (Laganowsky et al., 2011 ▶). The resulting construct, MBPA216H_K220H-SUFU-ΔW61D_L62S_G63F_P453A_Δ454–456_K457A, produced crystals with GLI1p and GLI3p that diffracted to 2.8 Å resolution (R = 19.7%, Rfree = 23.4% and R = 20.1%, Rfree = 23.4%, respectively). Crystals with both peptides belonged to space group P21 and contained four molecules in the asymmetric unit which all exhibited a rotation, via a flexible linker, of 58° relative to the apo crystal structures (Fig. 5 ▶a and Supplementary Video S1). Each molecule had clear density for the peptide between domains (Fig. 5 ▶b). Peptide modelled into this density forms a β-strand clamped between the two domains, creating one continuous 13-strand β-sheet spanning both domains. Interactions between SUFU His164 and Glu376 secure the closed conformation (Fig. 6 ▶a). HDX protection analysis and SAXS experiments confirmed that this protein/peptide conformation occurs in solution and is not a crystallization artifact (Fig. 7 ▶, Supplementary Table S2 and Supplementary Figs. S6, S8 and S9). In both structures the GLI peptide fits snugly into a narrow channel with the histidine from the SYGH motif (Dunaeva et al., 2003 ▶; His123 in GLI1 and His336 in GLI3) protruding into a pocket where it forms hydrogen-bonding interactions with Tyr147 and Asp159 (Figs. 6 ▶b and 6 ▶c and Supplementary Table S3). The mutation of Tyr147, Asp159 or Glu376 in MBP-SUFU-FL abolished detectable binding to GLI1p in the microscale thermophoresis assay (data not shown). To determine whether these binding differences were translated into functional differences in the cell, we examined the transcriptional activity of GLI1 in HEK 293 cells transiently transfected with mutated SUFU constructs (Fig. 6 ▶d). The mutation of Asp159 and Tyr147 had a significant effect on the ability of SUFU to repress GLI, whereas the mutation of Glu376 and His164 had a smaller effect. A similar pattern was observed in experiments measuring constitutive Hh pathway activity in Sufu−/− MEFs (Fig. 6 ▶e). Notably, the leucine immediately following the GLI SYGH motif, which is also completely conserved, packs tightly into a hydrophobic pocket formed by SUFU residues Val269, Ala271 and Leu380 (Fig. 6 ▶b). The following serine (conserved except in Xenopus and Ciona) is hydrogen bonded to Glu376. In agreement with these observations, a GLI3 peptide that terminates at His336 (GLI3p-SHC; residues 328–336; Supplementary Table S1b) is unable to protect MBP-SUFU-FL from deuteration in HDX experiments (data not shown). Hence, the critical binding motif extends beyond that previously described (Dunaeva et al., 2003 ▶).


Structural basis of SUFU-GLI interaction in human Hedgehog signalling regulation.

Cherry AL, Finta C, Karlström M, Jin Q, Schwend T, Astorga-Wells J, Zubarev RA, Del Campo M, Criswell AR, de Sanctis D, Jovine L, Toftgård R - Acta Crystallogr. D Biol. Crystallogr. (2013)

Mechanism of GLI binding. (a, b, c) Interactions between MBPA216H_K220H-SUFU-ΔW61D_L62S_G63F_P453A_Δ454–456_K457A and GLI3p. (a) The peptide (blue) is clamped between the N-terminal (beige) and C-terminal (green) domains. (b) GLI3p (blue residue labels) binds in a channel with His336 and Lys337 protruding into deep pockets. (c) SUFU–GLI3p interactions, with side-chain hydrogen bonds highlighted in yellow, and comparison of GLI1p and GLI3p. Residues that are conserved in GLI1, GLI2 and GLI3 are shown in red. Blue boxes indicate residues with visible electron density. (d, e) Mutations around the GLI3p binding site have varying effects on the ability of SUFU-FL to repress GLI1-induced reporter gene activity in HEK 293 cells (d) and to suppress constitutive pathway activity in Sufu−/− cells (e).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3852661&req=5

fig6: Mechanism of GLI binding. (a, b, c) Interactions between MBPA216H_K220H-SUFU-ΔW61D_L62S_G63F_P453A_Δ454–456_K457A and GLI3p. (a) The peptide (blue) is clamped between the N-terminal (beige) and C-terminal (green) domains. (b) GLI3p (blue residue labels) binds in a channel with His336 and Lys337 protruding into deep pockets. (c) SUFU–GLI3p interactions, with side-chain hydrogen bonds highlighted in yellow, and comparison of GLI1p and GLI3p. Residues that are conserved in GLI1, GLI2 and GLI3 are shown in red. Blue boxes indicate residues with visible electron density. (d, e) Mutations around the GLI3p binding site have varying effects on the ability of SUFU-FL to repress GLI1-induced reporter gene activity in HEK 293 cells (d) and to suppress constitutive pathway activity in Sufu−/− cells (e).
Mentions: In order to determine how SUFU interacts with GLI transcription factors, we attempted to co-crystallize MBP-SUFU-FL and MBP-SUFU-Δ with GLI1p as well as corresponding peptides from human GLI2 (GLI2p; residues 267–283) and GLI3 (GLI3p; residues 328–344) (Supplementary Table S1b). Despite extensive screening, these attempts were unsuccessful. Therefore, the residues WLG61–63 of SUFU were mutated to DSF in MBP-SUFU-Δ to disrupt crystal contacts between a loop within the SUFU N-terminal domain and residues in the C-terminal domain (Supplementary Fig. S2b) previously implicated in GLI binding (Merchant et al., 2004 ▶). Furthermore, another flexible loop in SUFU was shortened and residues 216 and 220 in the MBP moiety were mutated to histidine in order to promote metal ion-mediated crystallization of the fusion protein (Laganowsky et al., 2011 ▶). The resulting construct, MBPA216H_K220H-SUFU-ΔW61D_L62S_G63F_P453A_Δ454–456_K457A, produced crystals with GLI1p and GLI3p that diffracted to 2.8 Å resolution (R = 19.7%, Rfree = 23.4% and R = 20.1%, Rfree = 23.4%, respectively). Crystals with both peptides belonged to space group P21 and contained four molecules in the asymmetric unit which all exhibited a rotation, via a flexible linker, of 58° relative to the apo crystal structures (Fig. 5 ▶a and Supplementary Video S1). Each molecule had clear density for the peptide between domains (Fig. 5 ▶b). Peptide modelled into this density forms a β-strand clamped between the two domains, creating one continuous 13-strand β-sheet spanning both domains. Interactions between SUFU His164 and Glu376 secure the closed conformation (Fig. 6 ▶a). HDX protection analysis and SAXS experiments confirmed that this protein/peptide conformation occurs in solution and is not a crystallization artifact (Fig. 7 ▶, Supplementary Table S2 and Supplementary Figs. S6, S8 and S9). In both structures the GLI peptide fits snugly into a narrow channel with the histidine from the SYGH motif (Dunaeva et al., 2003 ▶; His123 in GLI1 and His336 in GLI3) protruding into a pocket where it forms hydrogen-bonding interactions with Tyr147 and Asp159 (Figs. 6 ▶b and 6 ▶c and Supplementary Table S3). The mutation of Tyr147, Asp159 or Glu376 in MBP-SUFU-FL abolished detectable binding to GLI1p in the microscale thermophoresis assay (data not shown). To determine whether these binding differences were translated into functional differences in the cell, we examined the transcriptional activity of GLI1 in HEK 293 cells transiently transfected with mutated SUFU constructs (Fig. 6 ▶d). The mutation of Asp159 and Tyr147 had a significant effect on the ability of SUFU to repress GLI, whereas the mutation of Glu376 and His164 had a smaller effect. A similar pattern was observed in experiments measuring constitutive Hh pathway activity in Sufu−/− MEFs (Fig. 6 ▶e). Notably, the leucine immediately following the GLI SYGH motif, which is also completely conserved, packs tightly into a hydrophobic pocket formed by SUFU residues Val269, Ala271 and Leu380 (Fig. 6 ▶b). The following serine (conserved except in Xenopus and Ciona) is hydrogen bonded to Glu376. In agreement with these observations, a GLI3 peptide that terminates at His336 (GLI3p-SHC; residues 328–336; Supplementary Table S1b) is unable to protect MBP-SUFU-FL from deuteration in HDX experiments (data not shown). Hence, the critical binding motif extends beyond that previously described (Dunaeva et al., 2003 ▶).

Bottom Line: Despite its central importance, little is known about SUFU regulation and the nature of SUFU-GLI interaction.It is demonstrated that GLI binding is associated with major conformational changes in SUFU, including an intrinsically disordered loop that is also crucial for pathway activation.These findings reveal the structure of the SUFU-GLI interface and suggest a mechanism for an essential regulatory step in Hedgehog signalling, offering possibilities for the development of novel pathway modulators and therapeutics.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biosciences and Nutrition and Center for Biosciences, Karolinska Institutet, Novum, Hälsovägen 7, SE-141 83 Huddinge, Sweden.

ABSTRACT
Hedgehog signalling plays a fundamental role in the control of metazoan development, cell proliferation and differentiation, as highlighted by the fact that its deregulation is associated with the development of many human tumours. SUFU is an essential intracellular negative regulator of mammalian Hedgehog signalling and acts by binding and modulating the activity of GLI transcription factors. Despite its central importance, little is known about SUFU regulation and the nature of SUFU-GLI interaction. Here, the crystal and small-angle X-ray scattering structures of full-length human SUFU and its complex with the key SYGHL motif conserved in all GLIs are reported. It is demonstrated that GLI binding is associated with major conformational changes in SUFU, including an intrinsically disordered loop that is also crucial for pathway activation. These findings reveal the structure of the SUFU-GLI interface and suggest a mechanism for an essential regulatory step in Hedgehog signalling, offering possibilities for the development of novel pathway modulators and therapeutics.

Show MeSH
Related in: MedlinePlus