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. 2015 Apr;25(4):401-11.
doi: 10.1038/cr.2015.32. Epub 2015 Mar 13.

Structure of the WD40 Domain of SCAP From Fission Yeast Reveals the Molecular Basis for SREBP Recognition

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Free PMC article

Structure of the WD40 Domain of SCAP From Fission Yeast Reveals the Molecular Basis for SREBP Recognition

Xin Gong et al. Cell Res. .
Free PMC article

Abstract

The sterol regulatory element-binding protein (SREBP) and SREBP cleavage-activating protein (SCAP) are central players in the SREBP pathway, which control the cellular lipid homeostasis. SCAP binds to SREBP through their carboxyl (C) domains and escorts SREBP from the endoplasmic reticulum to the Golgi upon sterol depletion. A conserved pathway, with the homologues of SREBP and SCAP being Sre1 and Scp1, was identified in fission yeast Schizosaccharomyces pombe. Here we report the in vitro reconstitution of the complex between the C domains of Sre1 and Scp1 as well as the crystal structure of the WD40 domain of Scp1 at 2.1 Å resolution. The structure reveals an eight-bladed β-propeller that exhibits several distinctive features from a canonical WD40 repeat domain. Structural and biochemical characterization led to the identification of two Scp1 elements that are involved in Sre1 recognition, an Arg/Lys-enriched surface patch on the top face of the WD40 propeller and a 30-residue C-terminal tail. The structural and biochemical findings were corroborated by in vivo examinations. These studies serve as a framework for the mechanistic understanding and further functional characterization of the SREBP and SCAP proteins in fission yeast and higher organisms.

Figures

Figure 1
Figure 1
In vitro reconstitution of the Sre1-Scp1 cytosolic complex. (A) Schematic illustration of the domain organizations of Scp1 and Sre1. Transmembrane helices are colored red and the carboxyl terminal tail of Scp1 is colored green. “SSD” stands for sterol sensing domain. The eight WD40 repeats of Scp1 are numbered 1-8. (B) Purified recombinant proteins of the C-terminal domains of Scp1 and Sre1 form complex in vitro. The MBP-fused regulatory domain of Sre1 (residues 628-900, and named MBP-Sre1) was immobilized on amylose resin to pull down Scp1-WD40 (residues 567-1 085). MBP was tested as negative control. Details of the experiments can be found in Materials and Methods.
Figure 2
Figure 2
Crystal structure of the Scp1 WD40 domain. (A) Protein purification of the Scp1-WD40 protein (residues 567-961 and 986-1 054, C618S/C671S/C680S/C756S/C873S/C901S/C920S/C941S/C1010S) used for crystallization studies. Two fragments (residues 567-961 and 986-1 054) were co-expressed in E.coli. A representative chromatogram of size exclusion chromatography of the recombinant Scp1-WD40 is shown. Peak fractions were analyzed by SDS-PAGE and visualized by Coomassie blue staining. (B) Overall structure of the Scp1 WD40 domain. Two perpendicular views are shown. The structure is shown in rainbow color, with the N- and C-termini colored blue and red, respectively. The eight blades are numbered 1 to 8 and the four strands in each blade are labeled A to D from the center to the outer ring. All structure figures were prepared with PyMol.
Figure 3
Figure 3
Structural comparison of the eight blades in the Scp1-WD40 β-propeller. (A) Structural superimposition of the blades 1/2/4/5/8. These five blades share similar repeat structures. (B) Blades 3 and 7 have elongated B/C/D strands. Shown here are structural superimpositions of blade 1 with blades 3 (left) and 7 (right). (C) Blade 6 has elongated AB and CD loops extending out of the bottom face. Left: structural comparison between blades 1 and 6. The 6AB and 6CD loops are highlighted in cyan and purple, respectively. Right: The 2Fo-Fc omit electron density map of 6CD loop (residues 870-908), contoured at 1σ, is shown as blue mesh.
Figure 4
Figure 4
Identification of Sre1-binding surface based on the Scp1-WD40 structure. (A) The top surface of the Scp1-WD40 propeller. The left and right panels depict the electrostatic surface potential and the corresponding cartoon representations, respectively. Six basic residues from blades 1 and 2 constitute a positively charged surface patch, the “RK patch”. (B) A close-up view on the residues involved in Sre1 binding. Six basic residues, one acidic residue and two Ser residues are colored blue, red and yellow, respectively. (C) Interaction between Scp1-WD40 variants and Sre1 regulatory domain assessed by MBP-mediated pull-down assay. The WT (residues 567-1 085) and Scp1-Δ24 were included as control. Scp1-M1/M2/M3 almost completely lost association with Sre1 (Lanes 10-12, boxed in red). Scp1-M4/M5/M6 showed decreased binding with Sre1 (Lanes 13-15, boxed in green). The experiments were performed with the same protocol as that for Figure 1B.
Figure 5
Figure 5
Functional analysis of Scp1 mutants in vivo. (A) Western blot of membrane fractions from WT and the indicated scp1 mutant S. pombe strains probed with anti-Sre1 serum, anti-Myc IgG or anti-Dsc5 serum. (B) Western blot of whole-cell lysates from WT and the indicated scp1 mutant S. pombe strains in the presence or absence of hrd1+ probed with anti-Sre1 serum or anti-Dsc5 serum. P denotes Sre1 precursor form. Asterisks indicate cross-reacting protein. (C) WT and mutant yeast (5 000, 1 000, 200, 40, and 8 cells) were grown on rich medium (3 days) or rich medium containing cobalt chloride (1.6 mM; 6 days). (D) Western blot of whole-cell lysates from WT and the indicated scp1 mutant S. pombe strains grown in the presence or absence of oxygen for 3 h probed with anti-Sre1 serum or anti-Dsc5 serum. P and N denote Sre1 precursor and nuclear forms, respectively. Asterisks indicate cross-reacting proteins. (E) Two potential models for Sre1-Scp1 complex formation.

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References

    1. Horton JD, Goldstein JL, Brown MS. SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver. J Clin Invest. 2002;109:1125–1131. - PMC - PubMed
    1. Brown MS, Goldstein JL. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell. 1997;89:331–340. - PubMed
    1. Shao W, Espenshade PJ. Expanding roles for SREBP in metabolism. Cell Metab. 2012;16:414–419. - PMC - PubMed
    1. Espenshade PJ, Hughes AL. Regulation of sterol synthesis in eukaryotes. Annu Rev Genet. 2007;41:401–427. - PubMed
    1. Goldstein JL, DeBose-Boyd RA, Brown MS. Protein sensors for membrane sterols. Cell. 2006;124:35–46. - PubMed

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