Research ArticleStructural Biology

The apo-structure of the leucine sensor Sestrin2 is still elusive

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Science Signaling  20 Sep 2016:
Vol. 9, Issue 446, pp. ra92
DOI: 10.1126/scisignal.aah4497

Figures

  • Fig. 1 Identification of ligand in the apo-Sestrin2 structure reported by Kim et al. (11).

    (A) Ribbon diagrams of the reported apo-Sestrin2 structure (5CUF; pink, left) and the leucine-bound structure (5DJ4; light blue, right). The largest peak in the 3.5 Å 5CUF Fobs-Fcalc difference map, contoured at 3σ, is shown as a green mesh in the 5CUF structure. The bound leucine built into the 5DJ4 structure is shown in orange. (B) Close-up view of the leucine-binding pocket of Sestrin2 in the 3.5 Å 5CUF (left) and the 2.7 Å 5DJ4 structures (right), refined without leucine built into the model. The Fobs-Fcalc (green mesh) and 2Fobs-Fcalc (blue mesh) electron density maps were contoured at 3σ and 1σ, respectively, and shown in the location of the bound leucine. (C) The 5CUF and 5DJ4 structures were refined with leucine built into the model and shown in the same view as in (B). The Fobs-Fcalc and 2Fobs-Fcalc electron density maps are depicted as in (B). Hydrogen bonds and electrostatic interactions are shown as black dashed lines. (D) The 5CUF structure was refined with malonate built into the model and shown in the same view as in (B). The Fobs-Fcalc and 2Fobs-Fcalc electron density maps are depicted as in (B). (E) The Rwork/Rfree(%) for the 5CUF structure refined with no ligand, leucine, or malonate modeled.

  • Fig. 2 Sestrin2 crystallizes in complex with leucine without the addition of exogenous leucine.

    (A) Close-up view of the leucine-binding pocket in Sestrin2 (5T0N; this study) refined without leucine built into the model. The Fobs-Fcalc (green mesh) and 2Fobs-Fcalc (blue mesh) electron density maps, contoured at 3σ and 1σ, respectively, are shown as in Fig. 1B. (B) Close-up view of the leucine-binding pocket in Sestrin2 refined with leucine built into the model. The Fobs-Fcalc and 2Fobs-Fcalc electron density maps are depicted as in (A). Hydrogen bonds and electrostatic interactions are shown as black dashed lines. (C) Close-up view of the leucine-binding pocket in Sestrin2 refined with malonate built into the model. The Fobs-Fcalc and 2Fobs-Fcalc electron density maps are depicted as in (A). Hydrogen bonds and electrostatic interactions are shown as black dashed lines. (D) The Rwork/Rfree(%) for the 5T0N structure (this study) refined with no ligand, leucine, or malonate modeled.

Tables

  • Table 1 Data collection and refinement statistics. RMS, root mean square.

    Values in parentheses are those for the highest resolution shell.

    Protein“Pseudo-apo”–Sestrin2
      OrganismHomo sapiens
      PDB ID5T0N
    Data collection
      Space groupI23
        a, b, and c (Å)292.26, 292.26, and 292.26
        α, β, and γ (°)90.0, 90.0, and 90.0
      Wavelength (Å)0.9792
      Resolution range (Å)200.0–3.0 (3.05–3.00)
      Completeness (%)100 (100)
      Redundancy40.7 (41.9)
      Rmeas (%)28.0 (>100)
      Rpim (%)4.4 (37.8)
      I19.5 (2.0)
    Refinement
      Resolution range (Å)103.3–3.0
      Rwork (%)18.9
      Rfree (%)22.5
      Number of reflections
        Total82,205
        Rfree reflections1,637
      Number of nonhydrogen atoms14,648
        Protein atoms14,648
      RMS deviations
        Bond lengths (Å)0.010
        Bond angles (°)1.037
      Average B-factor (Å2)
        Protein49.0
      Ramachandran (%)
        Favored96.4
        Allowed3.0
        Outlier0.7

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