1. Volume 24, Issue 5, Pages 676-686 (May 2016)
Structural Basis for the Magnesium-Dependent Activation and Hexamerization of the Lon AAA+ Protease 
Shih-Chieh Su, Chien-Chu Lin, Hui-Chung Tai, Mu-Yueh Chang, Meng-Ru Ho, C. Satheesan Babu, Jiahn-Haur Liao, Shih-Hsiung Wu, Yuan-Chih Chang, Carmay Lim, Chung-I Chang 
Structure 
Volume 24, Issue 5, Pages (May 2016)
DOI: /j.str
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2. Structure 2016 24, 676-686DOI: (10.1016/j.str.2016.03.003)
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3. Figure 1
Structural Models of the Degradation Chamber of LonA in Closed and Open Pore Conformations
(A) Domain arrangement in LonA. The boxed region corresponds to MtaLonA Core-E423Q, which lacks the flexible N-terminal domain (NTD).
(B) Top view of the structure of the enclosed chamber of Core-E423Q-ADP depicted as a semitransparent isosurface representation of the mass density, computed at 15 Å resolution from the crystal structure (PDB: 4YPL) shown in ribbons. The pore-loop Tyr residues are shown in red spheres.
(C) Two side views of the MtaLonA protomers in distinct ADP-bound (magenta) and nucleotide-free (cyan) conformations, superimposed on the protease domains. The AAA-α/β, -α, and protease domains are encircled by red, green, and black dashes, respectively. Arrowheads mark the nucleotide-binding sites.
(D) Top view of an open-chamber model of LonA constructed with six nucleotide-free protomers. See also the structural comparison of two Lon-like proteases in Figure S1.
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4. Figure 2 EM Analysis of LonA in Mg2+-Activated and Mg2+-ATP States
(A) Side (left) and top (right) views of the cryo-EM map of full-length MtaLonA in Mg2+-activated state shown in semitransparent isosurface representation. The map is docked with the nucleotide-free hexameric model of LonA as shown in Figure 1D. One protomer is colored in yellow.
(B) A representative reference-free class average image of negatively stained particles of MtaLonA Core-E423Q-Mg2+ without imposing symmetry.
(C) Views similar to those in (A) of the cryo-EM map of full-length MtaLonA in Mg2+-ATP state, docked with the crystal structure of the Core-E423Q-ADP complex, with one protomer colored in yellow. See also cryo-EM micrographs, 2D views, and 3D reconstructions in Figures S2–S4.
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5. Figure 3 Mg2+-Activated LonA Performs ATP-Independent Proteolysis
(A) Degradation of α-casein (open triangle) and Ig2 (black triangle) by MtaLonA in the presence of Mg2+ and ATP at 55°C.
(B) Fluorescence measurement for cleavage of FITC-casein by MtaLonA. “C” denotes control with no protease present. Apo denotes MtaLonA without Mg2+. Data are represented as mean ± SEM, n = 3.
(C) Degradation of the fluorogenic peptide suc-FLF-MNA.
(D) Degradation of Ig2 in various temperature-induced folding states in the presence of ATP and Mg2+ (top-left), of ADP and Mg2+ (top-right), and of Mg2+ alone (bottom-left). (D bottom-right) No degradation of Ig2 by apo-MtaLonA.
(E) Degradation of α-casein by MtaLonA in the presence of Mg2+ with or without ATP.
(F) Degradation of α-casein by the Y397G/I398G mutant of MtaLonA in the presence of Mg2+ with or without ATP. The reactions in (D–F) were analyzed by SDS-PAGE and stained with Coomassie Blue.
Asterisks denote product fragments. See also CD spectra of the substrates in Figure S5 and the temperature-dependent activity in Figure S6.
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6. Figure 4
Proposed Mg2+- and Nucleotide-Dependent Assembly Pathway of LonA
Cartoon illustrations of LonA in various assembly states. The Mg2+-free apo state exists in low oligomeric forms (Park et al., 2006; Rudyak et al., 2001). Summary of the structure and function relationship of the distinct assembled states of LonA is listed in the table shown below.
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7. Figure 5
Structure of MtaLonA Protease Domain Bound to Mg2+ and Bortezomib
(A) Hexameric protease ring viewed along the 6-fold symmetry axis with each monomer shown in different colors in ribbons. The bound bortezomib is shown in spheres. The AAA-α domains in the crystal structure are omitted for clarity.
(B) Bortezomib covalently bound to Ser678 with its σA-weighted Fo − Fc omit map contoured at 3σ.
(C) Close-up view of bortezomib bound to the substrate-binding groove. Interacting residues are labeled. Dashed lines denote hydrogen bonds. See also sequence alignment in Figure S7.
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8. Figure 6
Mg2+-Bound Protease Domain Forms an Extended Proteolytic Active-Site Structure Involved in Hexameric Interaction
(A) Stereo view of the Mg2+-binding site. The magnesium atom is shown in gray spheres and the water molecule involved in coordinating the dication is shown in red spheres. The displayed purple density around the magnesium atom is a σA-weighted Fo − Fc omit map contoured at 7.0σ. The carbon atom of the isobutyl group of bortezomib occupying a potential Mg2+-coordinating site is highlighted with a dotted sphere.
(B) Close-up view of the hexameric interface between two protease domains with one in orange ribbon and the other in yellow in a surface diagram. The ribbon model is superimposed with the inactive apo structure of the human LonA protease domain (gray; PDB: 2X36). Residues in the adjacent protomer in yellow interacting with the β4–α3 loop are highlighted. Bortezomib is displayed in spheres in only one of the protease domains for clarity.
(C and D) Plots of melting temperature change (ΔTm) of the purified AP induced by various concentrations of MgCl2 by CD and DSF. The data points are the ΔTm values of samples and the curves represent an exponential fit to the ΔTm values plotted against MgCl2 concentrations. The calculated Mg2+ association constants are also shown. See also structural, CD, and biochemical analyses in Figures S8 and S9.
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9. Figure 7
Open and Closed Structures of the Proteolytic Groove in an Mg2+-Activated Protease Domain
(A) Superposition of the Cα traces of the protease ring structures of AP-Mg2+ (magenta) and AP-Mg2+-bortezomib (green). The extended β4–α3 loops are marked by dashed circles and shaded in yellow. The protomer with a closed β1–β2 hairpin (blue) is indicated by the arrowhead.
(B and C) Superposition of the open (magenta in B) and closed (blue in C) structures of AP-Mg2+ with that of AP-Mg2+-bortezomib (gray).
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10. Figure 8
Roles of Mg2+ in the Deprotonation of the Catalytic Lys721 and in the Stability of the Proteolytic Active Site Evaluated by MD Simulations
(A) Rmsds of the protein backbone (BKB) atoms derived from simulations with Lys721 deprotonated in the presence (green) and absence (magenta) of Mg2+.
(B) Same as (A) but with Lys721 protonated (blue and red).
(C) Rmsds of the non-H atoms of protein residues within a radii of 10 Å from magnesium (Mg10) derived from the simulations in the presence of Mg2+ with Lys721 deprotonated (green) and protonated (blue). See also the simulation structure in Figure S10.
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