ScMreB5 is an active ATPase. Residues of ScMreB5 at the active site are compared with the monomeric CcMreB (PDB accession no. 4CZM), double-protofilament CcMreB (PDB accession no. 4CZJ), monomeric yeast actin (PDB accession no. 1YAG), and actin filament (PDB accession no. 5OOE) by superposing IIA domain of each structure onto IIA domain of ScMreB5–AMP-PNP structure (single protofilament conformation). The color of the residues are subdomain wise, for ScMreB5–AMP-PNP, IB (pink), IA (green), and IIA (sea green). For the other MreBs and actin structures, residues in subdomains are colored light blue. All distances marked by black dotted lines are <3.5 Å. (A) Superimposed active site residues holding the Mg2+ coordination sphere. Asp12, Glu134, and Asp156 residues of ScMreB5 are compared with the corresponding residues in monomeric CcMreB, monomeric actin, and actin filament. (B) Superimposed active site residues at the catalytic water interface. Glu134 and Thr161 residues of ScMreB5 are compared with the corresponding residues in monomeric CcMreB, monomeric actin, and actin filament. (C) Interacting interface for Asp70 of ScMreB5–AMP-PNP is shown with respect to corresponding residues present in double-protofilament CcMreB (PDB accession no. 4CZJ) and actin filament (PDB accession no. 5OOE). (D) ATPase activity characterization of ScMreB5. kobs (min−1) for the ScMreB5WT, active site mutants, and the polymerization mutant (ScMreB5WT [WT; N = 3; n = 15], ScMreB5E134A [E134A; N = 3; n = 8], ScMreB5D12A [D12A; N = 2; n = 7], ScMreB5D156A [D156A; N = 2; n = 3], ScMreB5D70A [D70A; N = 2; n = 7], ScMreB5T161A [T161A; N = 1; n = 8], and ScMreB5K57A [K57A; N = 2; n = 12]; N, number of independent protein purification batches; n, total number of repeats). The error bar denotes mean with SEM; unpaired t test, two-tailed; ***, P < 0.0001; **, P = 0.001–0.002. 10 µM protein, 1 mM ATP, and 1 mM MgCl2 were used in this assay. (E) Intra-protofilament polymerization interface of ScMreB5 (PDB accession no. 7BVY). Inset: Zoomed-in view of the interface showing the residue Lys57 of subdomain IB interacting with Asp276 and Pro149 of subdomain IIA. The distances in Å are labeled for the interactions.
Figure 2.

ScMreB5 is an active ATPase. Residues of ScMreB5 at the active site are compared with the monomeric CcMreB (PDB accession no. 4CZM), double-protofilament CcMreB (PDB accession no. 4CZJ), monomeric yeast actin (PDB accession no. 1YAG), and actin filament (PDB accession no. 5OOE) by superposing IIA domain of each structure onto IIA domain of ScMreB5–AMP-PNP structure (single protofilament conformation). The color of the residues are subdomain wise, for ScMreB5–AMP-PNP, IB (pink), IA (green), and IIA (sea green). For the other MreBs and actin structures, residues in subdomains are colored light blue. All distances marked by black dotted lines are <3.5 Å. (A) Superimposed active site residues holding the Mg2+ coordination sphere. Asp12, Glu134, and Asp156 residues of ScMreB5 are compared with the corresponding residues in monomeric CcMreB, monomeric actin, and actin filament. (B) Superimposed active site residues at the catalytic water interface. Glu134 and Thr161 residues of ScMreB5 are compared with the corresponding residues in monomeric CcMreB, monomeric actin, and actin filament. (C) Interacting interface for Asp70 of ScMreB5–AMP-PNP is shown with respect to corresponding residues present in double-protofilament CcMreB (PDB accession no. 4CZJ) and actin filament (PDB accession no. 5OOE). (D) ATPase activity characterization of ScMreB5. kobs (min−1) for the ScMreB5WT, active site mutants, and the polymerization mutant (ScMreB5WT [WT; N = 3; n = 15], ScMreB5E134A [E134A; N = 3; n = 8], ScMreB5D12A [D12A; N = 2; n = 7], ScMreB5D156A [D156A; N = 2; n = 3], ScMreB5D70A [D70A; N = 2; n = 7], ScMreB5T161A [T161A; N = 1; n = 8], and ScMreB5K57A [K57A; N = 2; n = 12]; N, number of independent protein purification batches; n, total number of repeats). The error bar denotes mean with SEM; unpaired t test, two-tailed; ***, P < 0.0001; **, P = 0.001–0.002. 10 µM protein, 1 mM ATP, and 1 mM MgCl2 were used in this assay. (E) Intra-protofilament polymerization interface of ScMreB5 (PDB accession no. 7BVY). Inset: Zoomed-in view of the interface showing the residue Lys57 of subdomain IB interacting with Asp276 and Pro149 of subdomain IIA. The distances in Å are labeled for the interactions.

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