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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.

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The SUFU IDR has distinct structural properties. (a) Thermal stability assays of MBP-SUFU constructs performed in triplicate, either alone (blue) or with GLI1p (red) or GLI1p-SH (green). All constructs bind to GLI1p; however, MBP-SUFU-FL has different physical properties upon initial GLI1p binding, as shown by the marked increase in fluorescence. (b) Microscale thermophoresis experiments with FAM-GLI1p and titrated MBP-SUFU constructs, showing an average of three separate experiments. All proteins have similar affinity, but the thermophoretic properties of FAM-GLI1p are modified differently between the MBP-SUFU-FL construct and the MBP-SUFU-Δ and MBP-SUFU-SH constructs, reflecting a difference in shape.
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fig4: The SUFU IDR has distinct structural properties. (a) Thermal stability assays of MBP-SUFU constructs performed in triplicate, either alone (blue) or with GLI1p (red) or GLI1p-SH (green). All constructs bind to GLI1p; however, MBP-SUFU-FL has different physical properties upon initial GLI1p binding, as shown by the marked increase in fluorescence. (b) Microscale thermophoresis experiments with FAM-GLI1p and titrated MBP-SUFU constructs, showing an average of three separate experiments. All proteins have similar affinity, but the thermophoretic properties of FAM-GLI1p are modified differently between the MBP-SUFU-FL construct and the MBP-SUFU-Δ and MBP-SUFU-SH constructs, reflecting a difference in shape.

Mentions: To determine whether the IDR affects the GLI-binding properties of SUFU, we used a human GLI1-derived peptide (GLI1p; residues 115–131; Supplementary Table S1b) containing the highly conserved SYGH motif important for interaction with SUFU (Dunaeva et al., 2003 ▶) in a thermal stability assay with MBP-SUFU-FL and MBP-SUFU-Δ (Fig. 4 ▶a). The addition of GLI1p stabilized both proteins: Tm for MBP-SUFU-FL was shifted by 4°C and that for MBP-SUFU-Δ was shifted by 3.6°C. No stabilization was provided by a control peptide comprising the same residues randomly shuffled (GLI1p-SH; Supplementary Table S1b). Interestingly, MBP-SUFU-FL exhibited high initial fluorescence values upon addition of GLI1p, but no such effect was observed with either MBP-SUFU-Δ or a third construct which was identical to MBP-SUFU-FL except that the 82 residues of the IDR were shuffled (MBP-SUFU-SH; Supplementary Table S1a). The GLI1 dose dependency of this effect was confirmed in a separate experiment with increasing GLI1p concentrations (Supplementary Fig. S7a). Furthermore, MBP-SUFU-FL was more stable (Tm = 50.1°C) than both the MBP-SUFU-Δ (Tm = 46.6°C) and MBP-SUFU-SH (Tm = 47.7°C) constructs. Taken together, these data show that despite being apparently disordered the IDR has properties which are different from those of a random loop and alter upon GLI1p peptide binding.


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)

The SUFU IDR has distinct structural properties. (a) Thermal stability assays of MBP-SUFU constructs performed in triplicate, either alone (blue) or with GLI1p (red) or GLI1p-SH (green). All constructs bind to GLI1p; however, MBP-SUFU-FL has different physical properties upon initial GLI1p binding, as shown by the marked increase in fluorescence. (b) Microscale thermophoresis experiments with FAM-GLI1p and titrated MBP-SUFU constructs, showing an average of three separate experiments. All proteins have similar affinity, but the thermophoretic properties of FAM-GLI1p are modified differently between the MBP-SUFU-FL construct and the MBP-SUFU-Δ and MBP-SUFU-SH constructs, reflecting a difference in shape.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: The SUFU IDR has distinct structural properties. (a) Thermal stability assays of MBP-SUFU constructs performed in triplicate, either alone (blue) or with GLI1p (red) or GLI1p-SH (green). All constructs bind to GLI1p; however, MBP-SUFU-FL has different physical properties upon initial GLI1p binding, as shown by the marked increase in fluorescence. (b) Microscale thermophoresis experiments with FAM-GLI1p and titrated MBP-SUFU constructs, showing an average of three separate experiments. All proteins have similar affinity, but the thermophoretic properties of FAM-GLI1p are modified differently between the MBP-SUFU-FL construct and the MBP-SUFU-Δ and MBP-SUFU-SH constructs, reflecting a difference in shape.
Mentions: To determine whether the IDR affects the GLI-binding properties of SUFU, we used a human GLI1-derived peptide (GLI1p; residues 115–131; Supplementary Table S1b) containing the highly conserved SYGH motif important for interaction with SUFU (Dunaeva et al., 2003 ▶) in a thermal stability assay with MBP-SUFU-FL and MBP-SUFU-Δ (Fig. 4 ▶a). The addition of GLI1p stabilized both proteins: Tm for MBP-SUFU-FL was shifted by 4°C and that for MBP-SUFU-Δ was shifted by 3.6°C. No stabilization was provided by a control peptide comprising the same residues randomly shuffled (GLI1p-SH; Supplementary Table S1b). Interestingly, MBP-SUFU-FL exhibited high initial fluorescence values upon addition of GLI1p, but no such effect was observed with either MBP-SUFU-Δ or a third construct which was identical to MBP-SUFU-FL except that the 82 residues of the IDR were shuffled (MBP-SUFU-SH; Supplementary Table S1a). The GLI1 dose dependency of this effect was confirmed in a separate experiment with increasing GLI1p concentrations (Supplementary Fig. S7a). Furthermore, MBP-SUFU-FL was more stable (Tm = 50.1°C) than both the MBP-SUFU-Δ (Tm = 46.6°C) and MBP-SUFU-SH (Tm = 47.7°C) constructs. Taken together, these data show that despite being apparently disordered the IDR has properties which are different from those of a random loop and alter upon GLI1p peptide binding.

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