Mechanism of the asymmetric activation of the MinD ATPase by MinE

Abstract

MinD is a component of the Min system involved in the spatial regulation of cell division. It is an ATPase in the MinD/ParA/Mrp deviant Walker A motif family which is within the P loop GTPase superfamily. Its ATPase activity is stimulated by MinE; however, the mechanism of this activation is unclear. MinD forms a symmetric dimer with two binding sites for MinE; however, a recent model suggested that MinE occupying one site was sufficient for ATP hydrolysis. By generating heterodimers with one binding site for MinE we show that one binding site is sufficient for stimulation of the MinD ATPase. Furthermore, comparison of structures of MinD and related proteins led us to examine the role of N45 in the switch I region. An asparagine at this position is conserved in four of the deviant Walker A motif subfamilies (MinD, chromosomal ParAs, Get3 and FleN) and we find that N45 in MinD is essential for MinE-stimulated ATPase activity and suggest that it is a key residue affected by MinE binding.

Fig. 1

The impact of MinE binding on the structure of MinD and comparison to other deviant Walker A motif members. The structures analyzed were MinD^D40AΔ10 (3Q9L), MinD^D40AΔ10 with bound MinE peptide (12–31) (3R9I) and Get3 in the transition state containing ADP and AlF₄⁻ (2WOJ). (A) Residues in MinD move to accommodate R21 of MinE. Depicted are MinE (orange), free MinD (cyan) and MinD (magenta) from the complex with the MinE peptide. The dotted lines indicate interactions between residues. (B) Comparison of the ATP binding region from the MinD-MinE peptide (12–31) complex to the free MinD dimer (cyan) reveals that the orientations of N45 and E146 are altered in the MinD-E peptide complex. In the free MinD dimer, E146 forms hydrogen bonds with an ATP molecule (green). However, in the MinD-MinE (12–31) peptide complex, E146 rotates away from the ADP molecule (dark blue) and forms a hydrogen bond with S149 (not shown) and a close contact of 3.8Å with S221 which in turn forms a hydrogen bond with R21 of MinE. N45 in the free MinD dimer hydrogen bonds a water molecule that interacts with a Mg²⁺ ion and moves away from the ADP molecule in the MinD-MinE (12–31) peptide complex to form a hydrogen bond with Asp 152 (not shown). In this position it would be near the γ-phosphate if it was present. (C) Location of N45/N61 and K11/K26 of MinD and Get3, respectively, with respect to ATP. The ATP binding regions of MinD-MinE (12–31) peptide complex (magenta), the free MinD dimer (cyan) and GET3 (gray, PDB: 2WOJ) were superimposed. The ATP molecule in the free MinD dimer (green), ADP in the MinD-MinE (12–31) peptide complex (blue) and ADP in GET3 (purple) are shown. The latter also contains AlF₄⁻ (red) and an Mg²⁺ ion (orange sphere). The signature lysine from MinD and Get3 (gray) overlap and the position of N61 of Get3 is between the positions of N45 observed in free MinD and the MinD-E peptide complex. (D) Alignment of residues in the Switch I region from MinD and several other deviant Walker A family members. Only a small sample of the sequences examined is shown. The positions of D40 and N45 in MinD are indicated. These residues are conserved in MinD, FleN, Get3 and chromosomal ParAs, however, Asn is not conserved in plasmid ParAs. It is also not conserved in ParC, which is closely related to ParA, but involved in location of chemotaxis proteins (Ringgaard et al., 2011).

Fig. 2

Analysis of the interaction of the MinD-N45A mutant with MinE. (A) The binding of MinD-N45A to the membrane, MinC and MinE was determined by phospholipid vesicle sedimentation assay in vitro. MinD-N45A (4 µµM) was incubated with phospholipid vesicles (400 µg/ml) in the presence of ADP or ATP (1 mM). MalE-MinC (4 µM) or MinE (4 µM) was added as indicated and the vesicles collected by centrifugation. Proteins bound to vesicles were assessed by SDS-PAGE. (B) MinD-N45A is deficient in MinE stimulated ATP hydrolysis. MinD-N45A, MinD or MinDD40A (4 µM) was incubated with phospholipid vesicles (400 µg/ml), ATP (1 mM) and MinE (4 µM) and the release of phosphate was assessed.

Fig. 3

Assessment of MinD mutants for interaction with MinE and self interaction. (A) The interaction between MinE and MinD was assessed with the bacterial 2-hybrid system. Note the last column assesses the interaction between MinE^R21A and MinD. For each sample three colonies were picked from the plasmid transformation into media and 100 µl of each spotted on indicator plates. (B) The interaction of various MinD mutants with MinD^WT was assessed. Note the last column assesses the interaction between two MinD mutants: MinD^D40A and the double mutant (DM), MinD^{E53A N222A}.

Fig. 4

MinE-R21A fails to stimulate the ATPase activity of MinD. MinE-R21A was incubated with phospholipid vesicles (400 µg/ml), ATP (1 mM) and MinD (4 µM) and the release of phosphate was assessed. The MinE-R21A concentration ranged as indicated.

Fig. 5

ATPase assay of MinD heterodimers. (A) Scheme depicting heterodimer formation and a sequential model for MinE activation of MinD ATPase. The red cross symbol indicates the double mutations (E53A/N222A). As MinE binds to the heterodimer, R21 contacts the MinD^WT subunit resulting in the hydrolysis of ATP bound to that subunit, which in turn activates the MinD^E53A/N222A subunit to hydrolyze ATP bound to it. If the WT subunit is replaced with the D40A mutant the ATP bound to MinD^E53A/N222A is still hydrolyzed. (B) Heterodimer ATPase assay. The ATPase activity of the MinD heterodimer composed of MinD^WT and MinD^E53A/N222A (designated DM) is not affected by an excess of MinD^E53A/N222A mutant protein. The ATPase activity was determined after mixing the various proteins, phospholipid vesicles (400 µg/ml) and ATP (1 mM).

Fig. 6

MinE stimulation of MinD heterodimers. (A) The MinD^D40A-MinD^E53A/N222A heterodimer is stimulated by MinE. WT, D40A and DM refer to wild type (MinD^WT), catalytic-deficient mutant (MinD^D40A) and double mutant (MinD^E53A/N222A) protein, respectively. (B) Stimulation of the MinD^WT -MinD^{D40A/E53A/N222A} heterodimer by MinE. MinD^WT and MinD^{D40A/E53A/N222A}, which is catalytic deficient and unable to bind MinE, were incubated together. The observation that hydrolysis occurs indicates the WT subunit within the heterodimer is able to hydrolyze ATP in response to MinE. TM refers to the triple mutant (MinD^{D40A/E53A/N222A}) protein.

Fig. 7

Effect of ATP and MinE on MinD residues involved in ATP hydrolysis. In the presence of ADP MinD is a monomer an K11 interacts with S148 and D152. Upon the addition of ATP this interaction is broken and K11 is free to interact with ATP bound to another subunit to promote dimerization, while S148 interacts with N45 across the dimer interface. Upon MinE binding the orientation of N45 changes and it interacts with the γ-phosphate of ATP in the same subunit to participate in ATP hydrolysis.

Fig. 8

Comparison of the γ-phosphate binding regions of MinD, the MinD-MinE peptide complex, Get3-AlF₄⁻ and Ras-GAP-AlF₃ (PDB:1WQ1). Shown are MinD and Get3 residues that are known or proposed to stabilize the transition state. The AlF₄ is from Ras-GAP complex and the AlF₃ is from the Get3 structure. Free MinD residues are cyan, the MinD-E complex residues are magenta, Get3 residues are gray and residues from the Ras-GAP structure are green. The lysine on the lower right is the conserved lysine common to all Walker A motifs. On the upper left are the signature lysines from Get3 and MinD, which overlap the arginine finger from Ras-GAP. On the lower left is the catalytic glutamine from Ras. On the upper right are the asparagines from MinD and Get3. The glutamine in Ras has two roles, aligning the attacking water molecule and interacting with AlF₃. In MinD and Get3 an aspartic acid residue (not shown) aligns the attacking water molecule while in Get3 Asn 61 binds to the γ-phosphate in the transition state. We propose that N45 of MinD would do this in the transition state of MinD.