Structural studies of Hedgehog pathway-related proteins

Valérie Biou (CNRS Researcher),
Annick Paquelin (CNRS technician)

The evolutionarily conserved Hedgehog (HH) pathway is crucial during multiple steps of animal embryo development as it controls a number of cell functions including division, survival, migration and differentiation. For example, HH controls the patterning of the wings and the appendages in flies, and the development of the limbs, nervous tube, brain, muscles and vessels in humans. In humans, HH is implicated in numerous pathologies such as developmental defects, cardiovascular diseases and cancers.

To understand the mechanism underlying the function of HH, the Drosophila model has been essential. The HH protein binds to its co-receptors at the plasma membrane. One of them, the transmembrane receptor Patched (PTC), catalytically inhibits the "G-Protein Coupled Receptor -like" Smoothened (SMO) protein in the absence of HH. Once activated, SMO acts on an intracellular multi-protein complex, the Hedgehog Transducing Complex (HTC), whose composition varies according to the HH activation state, but which contains and controls the zinc-finger transcription factor Cubitus interruptus (CI), which belongs to the Gli family. When the HH ligand is not bound to PTC, the HTC associates with microtubules. This triggers CI phosphorylation, ubiquitination and targeting to the proteasome. As a result, CI is partially proteolysed into a shorter form that acts as a repressor towards HH target genes. On the other hand, when HH binds to PTC, its inhibitory effect on SMO is suppressed and SMO accumulates as a hyperphosphorylated form at the cell surface. Consequently, the HTC bound to the SMO C-terminal tail leaves the microtubules and is also recruited at the plasma membrane. CI cleavage is thus prevented, and full-length CI triggers the transcription of target genes in the nucleus .

In mammals, a key component of HH signalling is the pioneer protein Suppressor of Fused (SUFU). This protein was initially discovered in flies for its ability to suppress the reduction in HH signalling resulting from mutations in a protein kinase called Fused (FU), pointing to SUFU’s inhibitory function. It was later shown to directly interact with FU and CI, and to be associated with the HTC in the cytoplasm. It acts as an ultimate gatekeeper that limits the activity of CI in the absence of HH both via limiting the nuclear entry of uncleaved CI, and probably as a cofactor that modulates CI transcriptional activity. In all organisms, SUFU is seen both in the cytoplasm and in the nucleus, is associated with CI/Gli, and seems to enhance Gli affinity to DNA.

Our work

We use a combination of biochemistry, biophysics, (small angle X-ray scattering, SEC-MALS, micro-calorimetry) spectroscopy and crystallography to understand the structure and function of Hedgehog-related proteins.

Our recent results showed the binding of zinc to SUFU [1] (www.nature.com/articles/s41598-017-11203-2). Guided by a sequence analysis that revealed a conserved potential metal binding site, we discovered that SUFU binds zinc. This binding was found to occur with a nanomolar affinity to SUFU from all three species. Mutation of one histidine from the conserved motif induces a moderate decrease in affinity for zinc, while circular dichroism indicates that the mutant remains structured. Our results reveal new metal binding affinity characteristics about SUFU that could be of importance for its regulatory function in HH. We used colorimetric methods and atomic absorption spectroscopy to measure the affinity and stoichiometry of zinc binding to SUFU. We showed that the affinity is pH-dependent and that replacing two histidine residues changes the affinity of SUFU for zinc.

Figure1

Figure 1 (up): Zinc binding measurements.
Dissociation constant of zinc for hSUFU (orange), zSUFU (blue), dSUFU wild type (red) and dSUFU His73A mutant (purple).

Figure 2 (right) CD spectra of dSUFU with zinc added and mutants and calculated pKa of Histidine residues.
A, (right up) CD spectra of dSUFU wild type (red solid line), His71A mutant (green dotted line), His73A mutant (purple hashed line) and dSUFU with zinc added (brown mixed line);
B, (right down)Secondary structure estimation using Bestsel analysis of CD spectra of dSUFU wild type, His71A and His73A mutants (same color code as A).

Figure2AFigure 2B

In parallel, we use small angle X-ray scattering (SAXS) to decipher its tertiary and quaternary structure in solution. Our studies revealed differences between the solution structures of SUFU from drosophila and vertebrates. We are investigating these differences further.

Bibliography (in blue from the lab):
  1. Jabrani, Makamte, Moreau,Gharbi, Plessis, Sanial & Biou (2017) Biophysical characterisation of the novel zinc binding property in Suppressor of Fused. Scientific Reports 7, Article number: 11139 https://doi.org/10.1038/s41598-017-11203-2

Last update: 17/09/06

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