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Crystal structure of a mycobacterial Insig homolog provides insight into how these sensors monitor sterol levels

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Science  10 Jul 2015:
Vol. 349, Issue 6244, pp. 187-191
DOI: 10.1126/science.aab1091
  • Fig. 1 The crystal structure of MvINS, a mycobacterial homolog of mammalian Insig proteins.

    (A) The overall structure of an MvINS monomer. Two perpendicular views are shown. It reveals a novel fold that may be shared by all Insig proteins. (B) TM1/TM2 can be superimposed to TM5/TM6. (C) Three adjacent MvINS molecules form a homotrimer in the crystal. The side and periplasmic views of the overall trimer are shown. (D) The trimeric interface is mediated exclusively by hydrophobic residues on TM3 and TM4. Arg77 on TM3 was mutated to Cys for disulfide-bond formation with Cys117 on TM4 from the adjacent protomer. Both residues are labeled red. (E) MvINS is a trimer in solution. Wild-type and cross-linked MvINS-R77C were subjected to size exclusion chromatography. The peak fractions were applied to SDS–polyacrylamide gel electrophoresis followed by Coomassie blue staining. All structure figures were prepared with PyMol (37).

  • Fig. 2 Each MvINS protomer accommodates one DAG molecule.

    (A) The MvINS protomer encloses a V-shaped cavity. Shown here is a cut-through view of the surface electrostatic potential (calculated in PyMol). The 2Fo-Fc electron density map, contoured at 1σ, is shown as cyan mesh in the inset. (B) Electron density for the bound DAG molecule. The 2Fo-Fc electron density for the bromine-derived DAG molecule, shown in magenta mesh, is contoured at 0.8σ. The anomalous signal for Br, shown as green mesh, is contoured at 3.5σ. (C) Overall structure of MvINS bound to DAG. The bromine (colored dark red)–derived DAG is shown in gray spheres. (D) Coordination of DAG by polar residues in MvINS. The residues that are hydrogen-bonded to DAG are shown in sticks. Hydrogen bonds are represented by red dashed lines.

  • Fig. 3 Structure-guided identification of functional residues in human Insig-2.

    (A) The recombinantly expressed human Insig-2 interacts with the transmembrane domain of mouse Scap (mScap-TMD) in a 25HC-dependent manner. The recombinant proteins of Insig-2 and mScap-TMD were overexpressed in baculovirus-infected Sf-9 cells. (B) Examination of the previously identified functional residues using the insect cell assay system. Consistent with the previous report (24), single point mutations F115A, Q132Q, W145A, and D149A led to loss of Scap binding even in the presence of 25HC. (C) Identification of additional Insig-2 residues that are involved in the 25HC-dependent Scap binding. The three residues Ala113/Gly117/His120 are outward-facing residues on TM3. Single point mutations of these residues led to diminished Scap binding even in the presence of 25HC. In contrast, substitution of Val114 and Val116 with Phe retained complex formation with Scap. Shown here are representative results of at least three repeating experiments.

  • Fig. 4 Identification of residues involved in 25HC binding based on the homologous model of Insig-2.

    (A) Identification of Insig-2 pocket residues that may contribute to 25HC binding. The pocket residues that are in the vicinity of Phe115 were analyzed. Single point mutations G39F, C77D, or G200F resulted in compromised mScap-TMD binding in the presence of 25HC. These three residues and Phe115 are positioned at a similar height within the central pocket of the structural model of Insig-2. (B) The measurement of direct binding between [3H]25HC and indicated Insig-2 variants. The experiments were performed following the identical protocol as reported previously (24). The data points represent the average of duplicate assays.

Supplementary Materials

  • Crystal structure of a mycobacterial Insig homolog provides insight into how these sensors monitor sterol levels

    Ruobing Ren, Xinhui Zhou, Yuan He, Meng Ke, Jianping Wu, Xiaohui Liu, Chuangye Yan, Yixuan Wu, Xin Gong, Xiaoguang Lei, S. Frank Yan, Arun Radhakrishnan, Nieng Yan

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Supplementary Text
    • Figs. S1 to S8
    • Tables S1 and S2

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