Structure And Function Of Suppressor Of Fused And Gli/Ci Protein Complex In The Hedgehog Signaling Pathway

Hedgehog(Hh) signaling pathway controls a myriad of key development processes such as regulation of embryonic development and adult tissue homeostasis. Mutations of its pathway components in this pathway such as Patched, Smoothened, Suppressor of fused(Sufu) and Gli predispose to cancers and congenital anomalies. The Gli/Ci family of proteins mediates the Hh transcriptional response. Sufu is a major regulator of the Gli/Ci transcription factors in both vertebrates and invertebrates, and plays a more critical role in mammals. Both Sufu and Gli/Ci proteins are among the most highly conserved components of the Hh signaling pathway. Mutations in human Sufu(hSufu) are associated with various kinds of cancers including medulloblastoma(the most frequent malignant pediatric brain tumor) and Gorlin syndrome(also termed nevoid basal cell carcinoma syndrome or basal cell nevus syndrome).Sufu consists of an N-terminal domain(NTD) and a C-terminal domain(CTD). Although the structure of h Sufu-NTD has been reported, a full-length(FL) Sufu structure from any species is still lacking. Moreover, the molecular mechanism of how Sufu employs its two domains to recognize Gli/Ci is controversial. In order to understand the molecular mechanism of how Sufu recognizes and regulates Gli/Ci, we carried out a systematic and extensive study on the structure and function of Sufu and Sufu-Gli protein complex.By continuous attemps and efforts, we expressed and purified Sufu proteins from different species using prokaryotic and eukaryotic(insect cells) expression system. After the prediction by bioinformatics and restriction enzyme digestion, some deletion constucts with the flexible surface loop residues deleted were constructed, and crystallization was optimized using various methods. Finaly, crystals with good quality were obtained, and the diffraction resolution is beyond 3 ?. We solved the structure of full-length Sufu and the mutants with loop deleted on the surface of hSufu-CTD that influenc the growth and quality of crystals. According to the structure, the normal mode analysis shows that there is a breathing motion between the two domains of Sufu.After solving the structure of full-length Sufu, we focus our structural study of protein complex of Sufu and Gli/Ci. Molecular biology and biochemistry techniques were used to map the shortest sequence on Gli that can bind Sufu and be purified in vitro. We undertook an extensive effort of crystallizing various hSufu or dSufu constructs in complexes with different lengths of Gli1, Gli2, Gli3, or Ci peptides encompassing the Sufu-binding “SYGHLS” motif. We finally succeeded in determining the crystal structure of h SufuΔ60 in complex with human Gli1(hGli1)(residues 112-128), to the resolution of 1.70 ?. Our structural and FRET results suggest that the “open”-to-“closed” conformational transition of Sufu is correlated with its binding to Gli, and is under the regulation of Hh signaling.The importance of critical residues on both NTD and CTD of Sufu in recognizing and regulating Gli is further demonstrated by mutagenesis combined with pull-down, ITC, co-immunoprecipitation, subcellular fractionation, immunofluorescence, and luciferase assays and found that mutations of critical interface residues disrupt the Sufu-Gli protein complex, and prevent Sufu from repressing Gli-mediated transcription, tethering Gli in the cytoplasm, and protecting Gli from the 26 S proteasome-mediated degradation.In conclusion, we performed a continuous investigation on the structure and function of Sufu and Sufu-Gli/Ci protein complex. Our study provides mechanistic insight into the mutual recognition and regulation between Sufu and Gli/Ci from the perspective of structure.