Structural and functional studies of MinD ATPase: implications for the molecular recognition of the bacterial cell division apparatus

Abstract

Proper placement of the bacterial cell division site requires the site-specific inactivation of other potential division sites. In Escherichia coli, selection of the correct mid-cell site is mediated by the MinC, MinD and MinE proteins. To clarify the functional role of the bacterial cell division inhibitor MinD, which is a membrane-associated ATPase that works as an activator of MinC, we determined the crystal structure of a Pyrococcus furiosus MinD homologue complexed with a substrate analogue, AMPPCP, and with the product ADP at resolutions of 2.7 and 2.0 A, respectively. The structure reveals general similarities to the nitrogenase iron protein, the H-Ras p21 and the RecA-like ATPase domain. Alanine scanning mutational analyses of E.coli MinD were also performed in vivo. The results suggest that the residues around the ATP-binding site are required for the direct interaction with MinC, and that ATP binding and hydrolysis play a role as a molecular switch to control the mechanisms of MinCDE-dependent bacterial cell division.

Fig. 1. Structure of P.furiosus MinD. (A) Stereo diagram of MinD in complex with AMPPCP. The helices are shown as red cylinders, β-strands as green arrows. The bound nucleotide is shown with thickened blue bonds. (B) Ribbon diagram of NIP (a), H-Ras (b) and NSF (c), shown in the same orientation as in (A). Structural elements compared with MinD are coloured in blue (see text). The bound nucleotides, ADP-AlF₄^– (a), GTP (b) and ATP (c) are shown in red; the 4Fe:4S of NIP is in green. (C) A plot of the average main chain temperature factors for MinD–nucleotide complexes (red line for the ATP form and black for the ADP form). Secondary structural elements are indicated on the top.

Fig. 1. Structure of P.furiosus MinD. (A) Stereo diagram of MinD in complex with AMPPCP. The helices are shown as red cylinders, β-strands as green arrows. The bound nucleotide is shown with thickened blue bonds. (B) Ribbon diagram of NIP (a), H-Ras (b) and NSF (c), shown in the same orientation as in (A). Structural elements compared with MinD are coloured in blue (see text). The bound nucleotides, ADP-AlF₄^– (a), GTP (b) and ATP (c) are shown in red; the 4Fe:4S of NIP is in green. (C) A plot of the average main chain temperature factors for MinD–nucleotide complexes (red line for the ATP form and black for the ADP form). Secondary structural elements are indicated on the top.

Fig. 1. Structure of P.furiosus MinD. (A) Stereo diagram of MinD in complex with AMPPCP. The helices are shown as red cylinders, β-strands as green arrows. The bound nucleotide is shown with thickened blue bonds. (B) Ribbon diagram of NIP (a), H-Ras (b) and NSF (c), shown in the same orientation as in (A). Structural elements compared with MinD are coloured in blue (see text). The bound nucleotides, ADP-AlF₄^– (a), GTP (b) and ATP (c) are shown in red; the 4Fe:4S of NIP is in green. (C) A plot of the average main chain temperature factors for MinD–nucleotide complexes (red line for the ATP form and black for the ADP form). Secondary structural elements are indicated on the top.

Fig. 2. Nucleotide binding of P.furiosus MinD. (A and B) Scheme showing the interactions between P.furiosus MinD and the nucleotide [AMPPCP (A) and ADP (B)]. Dashed lines indicate hydrogen bonds (<3.5 Å). Water molecules are indicated by ‘w’; van der Waals contacts are indicated by arcs. The differences between AMPPCP- and ADP-bound MinD are highlighted in red. The attacking water molecule is shown in white inside the closed circle. (C) Walker motif sequence conservation in MinD family proteins. Bacterial MinD homologues (Eco, E.coli; Bsu, Bacillus subtilis; Nme, Neisseria meningitidis; Xfa, Xylella fastidiosa; Aae, Aquifex aeolicus), archaeal MinD homologues (Mja, Methanococcus jannaschii; Pfu, P.furiosus) and MinD-related proteins (Avi, Azotobacter vinelandii) were aligned with CLUSTAL_W (Thompson et al., 1994) and edited manually. Key motifs are highlighted in yellow. The Lys11 residue is boxed. Triangles indicate residues involved in the specific binding of phosphate (red), magnesium (yellow) and attacking water (blue).

Fig. 2. Nucleotide binding of P.furiosus MinD. (A and B) Scheme showing the interactions between P.furiosus MinD and the nucleotide [AMPPCP (A) and ADP (B)]. Dashed lines indicate hydrogen bonds (<3.5 Å). Water molecules are indicated by ‘w’; van der Waals contacts are indicated by arcs. The differences between AMPPCP- and ADP-bound MinD are highlighted in red. The attacking water molecule is shown in white inside the closed circle. (C) Walker motif sequence conservation in MinD family proteins. Bacterial MinD homologues (Eco, E.coli; Bsu, Bacillus subtilis; Nme, Neisseria meningitidis; Xfa, Xylella fastidiosa; Aae, Aquifex aeolicus), archaeal MinD homologues (Mja, Methanococcus jannaschii; Pfu, P.furiosus) and MinD-related proteins (Avi, Azotobacter vinelandii) were aligned with CLUSTAL_W (Thompson et al., 1994) and edited manually. Key motifs are highlighted in yellow. The Lys11 residue is boxed. Triangles indicate residues involved in the specific binding of phosphate (red), magnesium (yellow) and attacking water (blue).

Fig. 3. Sequence conservation for MinD homologous proteins mapped on the P.furiosus structure. The alignment analysis was done with the seven MinD homologous proteins described in Figure 2C. Variable regions are coloured in white, and increasing conservation is indicated with deepening red colour. Two views of the protein are shown, including the bound nucleotide represented by thickened blue bonds. The conserved residues indicated in the right panel are located mostly at the N-termini of the β-strands.

Fig. 4. Effects of the local conformation around the K11 residue. (A) Stereo diagram of the interface between the Walker A motif region (green) and α7 (red). K11, ADP and the coordinating side chains are shown as ball-and-stick, with ADP and oxygens in red, and nitrogens in blue. (B) Phenotypes of mutations at residues around E.coli K11 (ecK11). Fixed cells were stained with DAPI to observe the nucleoids and photographed. From left to right: BL21(DE3) [minCDE⁺] with control pET21a, pEMDwt [P_T7::minD⁺], pEMD11 [P_T7::minD^ecK11A], pEMD16 [P_T7::minD^ecK16A], pEMD146 [P_T7::minD^ecE146A] and pEMD152 [P_T7::minD^ecD152A]. The amino acids in P.furiosus (pfu) are also indicated in parentheses.