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. 2016 Nov;26(11):1197-1211.
doi: 10.1038/cr.2016.123. Epub 2016 Nov 4.

Complex Structure of the Fission Yeast SREBP-SCAP Binding Domains Reveals an Oligomeric Organization

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Free PMC article

Complex Structure of the Fission Yeast SREBP-SCAP Binding Domains Reveals an Oligomeric Organization

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

Abstract

Sterol regulatory element-binding protein (SREBP) transcription factors are master regulators of cellular lipid homeostasis in mammals and oxygen-responsive regulators of hypoxic adaptation in fungi. SREBP C-terminus binds to the WD40 domain of SREBP cleavage-activating protein (SCAP), which confers sterol regulation by controlling the ER-to-Golgi transport of the SREBP-SCAP complex and access to the activating proteases in the Golgi. Here, we biochemically and structurally show that the carboxyl terminal domains (CTD) of Sre1 and Scp1, the fission yeast SREBP and SCAP, form a functional 4:4 oligomer and Sre1-CTD forms a dimer of dimers. The crystal structure of Sre1-CTD at 3.5 Å and cryo-EM structure of the complex at 5.4 Å together with in vitro biochemical evidence elucidate three distinct regions in Sre1-CTD required for Scp1 binding, Sre1-CTD dimerization and tetrameric formation. Finally, these structurally identified domains are validated in a cellular context, demonstrating that the proper 4:4 oligomeric complex formation is required for Sre1 activation.

Figures

Figure 1
Figure 1
Sre1-CTD and Scp1-WD4 can form a 4:4 complex. (A) The Sre1-CTD and Scp1-WD40 form a high-order oligomer on the size exclusion chromatography (SEC). For better solution behavior, the Scp1-WD40 protein was obtained by co-expressing segments containing residues 567-961 and residues 986-1 085 as reported previously. (B) The negative staining EM analysis of the unprocessed (WT) and GraFixed complex of Sre1-CTD and Scp1-WD40. The WT proteins display considerable degree of sample heterogeneity, indicating complex dissociation. In contrast, the GraFixed samples were more homogeneous, sharing similar structural feature with a subgroup of WT particles. (C) GraFixed Sre1-CTD and Scp1-WD40 complex gave rise to well-behaved proteins. The experimental details of GraFix cross-linking can be found in Materials and Methods. The GraFixed samples were eluted from SEC at a slightly advanced volume, suggesting that the WT complex may undergo dissociation at the tested concentration. (D) Reconstruction of the negative staining EM structure of the GraFixed Sre1-CTD and Scp1-WD40 complex. The overall structure resembles a flattened dumbbell with a seemingly four-fold symmetry related by two perpendicular axes. Each of the four corners of the EM map exhibits a distinct donut shape that can be docked with the WD40 domain of Scp1. All structure figures and the EM maps were prepared with PyMol and Chimera, respectively. See Supplementary information, Figures S1 and S2 for additional characterizations.
Figure 2
Figure 2
Sre1-CTD exists as a dimer of dimer. (A) The Sre1-CTD may exist as an oligomer. The Sre1-CTD (residues 628-896) was eluted from SEC at a position corresponding to the molecular weight of a tetramer. The cross-linked Sre1-CTD, which showed a molecular weight of ∼120 kDa on SDS-PAGE, was eluted at a similar position to the WT protein on SEC. (B) Static light scattering (SLS) analysis of the Sre1-CTD. SLS analysis of the WT and cross-linked samples suggested that the Sre1-CTD may form a less stable tetramer as the cross-linked protein was measured with a molecular weight of a tetramer, whereas the WT protein exhibited a slightly reduced molecular weight. (C) Analytical ultracentrifugation analysis (AUC) of the Sre1-CTD. Consistent with the SLS analysis, the sedimentation velocity AUC (AUC-SV) analysis of the WT and cross-linked Sre1-CTD supported the formation of an unstable tetramer of Sre1-CTD. (D) Sedimentation equilibrium AUC analysis of the Sre1-CTD. The AUC-SE analysis yielded a dissociation coefficient (Kd) of 1 μM between tetramer and dimer, suggesting a dimer of dimer organization of the Sre1-CTD. See also Supplementary information, Figure S3.
Figure 3
Figure 3
Crystal structure of Sre1-CTD dimer at 3.5 Å resolution. (A) The Sre1-CTD forms a dimer in the crystal structure. The two protomers within each asymmetric unit exhibits a two-fold pseudo-symmetry relative by an axis that traverses the interface constituted by the anti-parallel β-strands and the α4-α6 helices of the two protomers. (B) The overall structure of a Sre1-CTD protomer. Shown here is Mol A in each asymmetric unit. There are three short helices on the amino termini of Mol A that are invisible in Mol B. The shared structure core of the two protomers comprises 8 α helices and a short β strand that connects α4 and α5. Right panel: The topological structure of Sre1-CTD. (C) Biochemical validation of the structurally revealed interface. PPP mutant that was designed to disrupt the anti-parallel β strands led to disruption of the oligomer. Mutations of four additional amino acids (K766E/V809D/R812E/F818D) on helices α4 and α6 completely disrupted of the oligomeric interface (designated PPP+4 mutant). The C terminus-deleted (designated ΔC-tail) protein remained as an oligomer. See also Supplementary information, Figures S4 and S5.
Figure 4
Figure 4
Cryo-EM structure of the Sre1-CTD/Scp1-WD40 complex. (A) Cryo-EM imaging of the GraFixed Sre1-CTD/Scp1-WD40 complex. Shown on the left and right are representative micrographs as well as 2D classifications. (B) The overall EM map of the Sre1-CTD and Scp1-WD40 complex. (C) Docking of the crystal structures of Sre1-CTD dimer and Scp1-WD40 into the EM map. The crystal structures of the individual components can be easily docked into the EM map in Chimera. The two protomers in each Sre1-CTD dimer are colored green and blue for visual clarity. The modest resolution does not allow discrimination of any conformational differences between the two protomers within each dimer. See also Supplementary information, Figure S6.
Figure 5
Figure 5
Interface between Sre1-CTD and Scp1-WD40. (A) The interface of the Sre1-CTD and Scp1-WD40 involves helix α8 and the succeeding C-tail helix, which is missing in the crystal structure, and the RK patch of Scp1, which was biochemically identified previously. (B) Biochemical verification of the interface between Sre1-CTD and Scp1-WD40. Deletion of the C-tail or mutation of the conserved residues on helix α8 resulted in compromised complex formation. (C) WT, sre1Δ and the indicated sre1 mutants (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, sre1Δ and ΔC-tail and ERG sre1 mutants grown in the presence or absence of oxygen for 3 h with anti-Sre1 serum. P and N denote Sre1 precursor and nuclear forms, respectively. Asterisks indicate cross-reacting proteins. (E) Western blot of membrane fractions from WT and sre1 mutant strains (ΔC-tail and ERG) with or without hrd1+ probed with anti-Sre1 serum or anti-Dsc5 serum.
Figure 6
Figure 6
Tetrameric formation of Sre1-CTD. (A) The N-terminus of the α1 helix contains two aromatic residues Trp702/Tyr703 that are highly conserved in related species and are shown as sticks. (B) AUC-SV analysis of the Sre1-CTD oligomeric mutants. The AUC-SV analysis of the WT, PPP+4 mutant and WY mutant of Sre1-CTD supported that the conserved WY motif contributed to the tetrameric formation. (C) SLS analysis of the Sre1-CTD oligomeric mutants. SLS characterization of WY mutant produced two peaks corresponding to 57.5 kDa and 30 kDa, respectively, suggesting less stable dimer formation. (D) WT, sre1Δ and sre1 WY mutant cells (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). (E) Western blot of whole-cell lysates from WT, sre1Δ and sre1 WY cells grown in the presence or absence of oxygen for 3 h with anti-Sre1 serum. P and N denote Sre1 precursor and nuclear forms, respectively. Asterisks indicate cross-reacting proteins. (F) Western blot of membrane fractions from WT and WY mutant cells with or without hrd1+ probed with anti-Sre1 serum or anti-Dsc5 serum.
Figure 7
Figure 7
Three distinct regions in the Sre1-CTD required for Scp1 binding, Sre1-CTD dimerization and tetrameric formation. The simplified cartoon highlights the three distinct surface areas of Sre1-CTD for homo- and heterotypic interactions. (I) the dimer interface, (II) the interface between dimers, (III) the interface with Scp1-WD40. The green and blue colors of the two protomers in each Sre1-CTD are for visual clarity only.

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