Mapping the MinE site involved in interaction with the MinD division site selection protein of Escherichia coli

Interactions between the MinD and MinE proteins are required for proper placement of the Escherichia coli division septum. The site within MinE that is required for interaction with MinD was mapped by studying the effects of site-directed minE mutations on MinD-MinE interactions in yeast two-hybrid and three-hybrid experiments. This confirmed that the MinE N-terminal domain is responsible for the interaction of MinE with MinD. Mutations that interfered with the interaction defined an extended surface on one face of the alpha-helical region of the MinE N-terminal domain, consistent with the idea that the MinE-MinD interaction involves formation of a coiled-coil structure by interaction with a complementary helical surface within MinD.     

Positioning of the MinE binding site on the MinD surface suggests a plausible mechanism for activation of the Escherichia coli MinD ATPase during division site selection

Division site selection in Escherichia coli requires that the MinD protein interact with itself and with MinC and MinE. MinD is a member of the NifH-ArsA-Par-MinD subgroup of ATPases. The MinE-MinD interaction results in activation of MinD ATPase activity in the presence of membrane vesicles. The sites within MinD responsible for its interaction with MinC and MinE were studied by site-directed mutagenesis and yeast two-hybrid analysis, guided by the known three-dimensional structure of MinD proteins. This provided evidence that MinC and MinE bind to overlapping sites on the MinD surface. The results also suggested that MinE and the invariant Lys11 residue in the ATPase P-loop of MinD compete for binding to a common site within the MinD structure, thereby providing a plausible structural basis for the ability of MinE to activate the ATPase activity of MinD.     

Membrane redistribution of the Escherichia coli MinD protein induced by MinE

Escherichia coli cells contain potential division sites at midcell and adjacent to the cell poles. Selection of the correct division site at midcell is controlled by three proteins: MinC, MinD, and MinE. It has previously been shown (D. Raskin and P. de Boer, Cell 91:685-694, 1997) that MinE-Gfp localizes to the midcell site in an MinD-dependent manner. We use here Gfp-MinD to show that MinD associates with the membrane around the entire periphery of the cell in the absence of the other Min proteins and that MinE is capable of altering the membrane distribution pattern of Gfp-MinD. Studies with the isolated N-terminal and C-terminal MinE domains indicated different roles for the two MinE domains in the redistribution of membrane-associated MinD.     

ATP-dependent interactions between Escherichia coli Min proteins and the phospholipid membrane in vitro

Proper placement of the division apparatus in Escherichia coli requires pole-to-pole oscillation of the MinC division inhibitor. MinC dynamics involves a membrane association-dissociation cycle that is driven by the activities of the MinD ATPase and the MinE topological specificity factor, which themselves undergo coupled oscillatory localization cycles. To understand the biochemical mechanisms underlying Min protein dynamics, we studied the interactions of purified Min proteins with phospholipid vesicles and the role of ATP in these interactions. We show that (i) the ATP-bound form of MinD (MinD.ATP) readily associates with phospholipid vesicles in the presence of Mg(2+), whereas the ADP-bound form (MinD.ADP) does not; (ii) MinD.ATP binds membrane in a self-enhancing fashion; (iii) both MinC and MinE can be recruited to MinD.ATP-decorated vesicles; (iv) MinE stimulates dissociation of MinD.ATP from the membrane in a process requiring hydrolysis of the nucleotide; and (v) MinE stimulates dissociation of MinC from MinD.ATP-membrane complexes, even when ATP hydrolysis is blocked. The results support and extend recent work by Z. Hu et al. (Z. Hu, E. P. Gogol, and J. Lutkenhaus, Proc. Natl. Acad. Sci. USA 99:6761-6766, 2002) and support models of protein oscillation wherein MinE induces Min protein dynamics by stimulating the conversion of the membrane-bound form of MinD (MinD.ATP) to the cytoplasmic form (MinD.ADP). The results also indicate that MinE-stimulated dissociation of MinC from the MinC-MinD.ATP-membrane complex can, and may, occur prior to hydrolysis of the nucleotide.     

Determination of the structure of the MinD-ATP complex reveals the orientation of MinD on the membrane and the relative location of the binding sites for MinE and MinC

The three Min proteins spatially regulate Z ring positioning in Escherichia coli and are dynamically associated with the membrane. MinD binds to vesicles in the presence of ATP and can recruit MinC or MinE. Biochemical and genetic evidence indicate the binding sites for these two proteins on MinD overlap. Here we solved the structure of a hydrolytic-deficient mutant of MinD truncated for the C-terminal amphipathic helix involved in binding to the membrane. The structure solved in the presence of ATP is a dimer and reveals the face of MinD abutting the membrane. Using a combination of random and extensive site-directed mutagenesis additional residues important for MinE and MinC binding were identified. The location of these residues on the MinD structure confirms that the binding sites overlap and reveals that the binding sites are at the dimer interface and exposed to the cytosol. The location of the binding sites at the dimer interface offers a simple explanation for the ATP dependence of MinC and MinE binding to MinD.     

Structural basis for the topological specificity function of MinE

Correct positioning of the division septum in Escherichia coli depends on the coordinated action of the MinC, MinD and MinE proteins. Topological specificity is conferred on the MinCD division inhibitor by MinE, which counters MinCD activity only in the vicinity of the preferred midcell division site. Here we report the structure of the homodimeric topological specificity domain of Escherichia coli MinE and show that it forms a novel alphabeta sandwich. Structure-directed mutagenesis of conserved surface residues has enabled us to identify a spatially restricted site on the surface of the protein that is critical for the topological specificity function of MinE.     

The MinD protein is a membrane ATPase required for the correct placement of the Escherichia coli division site.

The proper placement of the cell division site in Escherichia coli requires the site-specific inactivation of potential division sites at the cell poles in a process that is mediated by the MinC, MinD and MinE proteins. During the normal division cycle MinD plays two roles. It activates the MinC-dependent mechanism that is responsible for the inactivation of potential division sites and it also renders the division inhibition system sensitive to the topological specificity factor MinE. MinE suppresses the division block at the normal division site at mid-cell but not all cell poles, thereby ensuring the normal division pattern. In this study the MinD protein was purified to homogeneity and shown to bind ATP and to have ATPase activity. When the putative ATP binding domain of MinD was altered by site-directed mutagenesis, the mutant protein was no longer able to activate the MinC-dependent division inhibition system. Immunoelectron microscopy showed that MinD was located in the inner membrane region of the cell envelope. These results show that MinD is a membrane ATPase and suggest that the ATPase activity plays an essential role in the functions of the MinD protein during the normal division process.     

Expression of Escherichia coli Min system in Bacillus subtilis and its effect on cell division

In both rod-shaped Bacillus subtilis and Escherichia coli cells, Min proteins are involved in the regulation of division septa formation. In E. coli , dynamic oscillation of MinCD inhibitory complex and MinE, a topological specificity protein, prevents improper polar septation. However, in B. subtilis no MinE is present and no oscillation of Min proteins can be observed. The function of MinE is substituted by that of an unrelated DivIVA protein, which targets MinCD to division sites and retains them at the cell poles. We inspected cell division when the E. coli Min system was introduced into B. subtilis cells. Expression of these heterologous Min proteins resulted in cell elongation. We demonstrate here that E. coli MinD can partially substitute for the function of its B. subtilis protein counterpart. Moreover, E. coli MinD was observed to have similar helical localization as B. subtilis MinD.     

Division site placement in E.coli: mutations that prevent formation of the MinE ring lead to loss of the normal midcell arrest of growth of polar MinD membrane domains

The MinE protein functions as a topological specificity factor in determining the site of septal placement in Escherichia coli. MinE assembles into a membrane-associated ring structure near midcell and directs the localization of MinD and MinC into a membrane- associated polar zone that undergoes a characteristic pole-to-pole oscillation cycle. Single (green fluorescent protein) and double label (yellow fluorescent protein/cyan fluorescent protein) fluorescence labeling experiments showed that mutational alteration of a site on the alpha-face of MinE led to a failure to assemble the MinE ring, associated with loss of the ability to support a normal pattern of division site placement. The absence of the MinE ring did not prevent the assembly and disassembly of the MinD polar zone. Mutant cells lacking the MinE ring were characterized by the growth of MinD polar zones past their normal arrest point near midcell. The results suggested that the MinE ring acts as a stop-growth mechanism to prevent the MinCD polar zone from extending beyond the midcell division site.     

The dimerization and topological specificity functions of MinE reside in a structurally autonomous C-terminal domain

Correct placement of the division septum in Escherichia coli requires the co-ordinated action of three proteins, MinC, MinD and MinE. MinC and MinD interact to form a non-specific division inhibitor that blocks septation at all potential division sites. MinE is able to antagonize MinCD in a topologically sensitive manner, as it restricts MinCD activity to the unwanted division sites at the cell poles. Here, we show that the topological specificity function of MinE residues in a structurally autonomous, trypsin-resistant domain comprising residues 31-88. Nuclear magnetic resonance (NMR) and circular dichroic spectroscopy indicate that this domain includes both alpha and beta secondary structure, while analytical ultracentrifugation reveals that it also contains a region responsible for MinE homodimerization. While trypsin digestion indicates that the anti-MinCD domain of MinE (residues 1-22) does not form a tightly folded structural domain, NMR analysis of a peptide corresponding to MinE1-22 indicates that this region forms a nascent helix in which the peptide rapidly interconverts between disordered (random coil) and alpha-helical conformations. This suggests that the N-terminal region of MinE may be poised to adopt an alpha-helical conformation when it interacts with the target of its anti-MinCD activity, presumably MinD.     

Dynamic localization cycle of the cell division regulator MinE in Escherichia coli

The MinC protein directs placement of the division septum to the middle of Escherichia coli cells by blocking assembly of the division apparatus at other sites. MinD and MinE regulate MinC activity by modulating its cellular location in a unique fashion. MinD recruits MinC to the membrane, and MinE induces MinC/MinD to oscillate rapidly between the membrane of opposite cell halves. Using fixed cells, we previously found that a MinE-green fluorescent protein fusion accumulated in an annular structure at or near the midcell, as well as along the membrane on only one side of the ring. Here we show that in living cells, MinE undergoes a rapid localization cycle that appears coupled to MinD oscillation. The results show that MinE is not a fixed marker for septal ring assembly. Rather, they support a model in which MinE stimulates the removal of MinD from the membrane in a wave-like fashion. These waves run from a midcell position towards the poles in an alternating sequence such that the time-averaged concentration of division inhibitor is lowest at midcell.     

The N terminus of MinD contains determinants which affect its dynamic localization and enzymatic activity

MinD is involved in regulating the proper placement of the cytokinetic machinery in some bacteria, including Neisseria gonorrhoeae and Escherichia coli. Stimulation of the ATPase activity of MinD by MinE has been proposed to induce dynamic, pole-to-pole oscillations of MinD in E. coli. Here, we investigated the effects of deleting or mutating conserved residues within the N terminus of N. gonorrhoeae MinD (MinD(Ng)) on protein dynamism, localization, and interactions with MinD(Ng) and with MinE(Ng). Deletions or mutations were generated in the first five residues of MinD(Ng), and mutant proteins were evaluated by several functional assays. Truncation or mutation of N-terminal residues disrupted MinD(Ng) interactions with itself and with MinE. Although the majority of green fluorescent protein (GFP)-MinD(Ng) mutants could still oscillate from pole to pole in E. coli, the GFP-MinD(Ng) oscillation cycles were significantly faster and were accompanied by increased cytoplasmic localization. Interestingly, in vitro ATPase assays indicated that MinD(Ng) proteins lacking the first three residues or with an I5E substitution possessed higher MinE(Ng)-independent ATPase activities than the wild-type protein. These results indicate that determinants found within the extreme N terminus of MinD(Ng) are implicated in regulating the enzymatic activity and dynamic localization of the protein.     

Analysis of MinD mutations reveals residues required for MinE stimulation of the MinD ATPase and residues required for MinC interaction

The MinD ATPase is critical to the oscillation of the Min proteins, which limits formation of the Z ring to midcell. In the presence of ATP, MinD binds to the membrane and recruits MinC, forming a complex that can destabilize the cytokinetic Z ring. MinE, which is also recruited to the membrane by MinD, displaces MinC and stimulates the MinD ATPase, resulting in the oscillation of the Min proteins. In this study we have investigated the role of lysine 11, present in the deviant Walker A motif of MinD, and the three residues in helix 7 (E146, S148, and D152) that interact electrostatically with lysine 11. Lysine 11 is required for interaction of MinD with the membrane, MinC, MinE, and itself. In contrast, the three residues in helix 7 that interact with lysine 11 are not required for binding to the membrane or activation of MinC. They are also not required for MinE binding; however, they are required for MinE to stimulate the MinD ATPase. Interestingly, the D152A mutant self-interacts, binds to the membrane, and recruits MinC and MinE in the presence of ADP as well as ATP. This mutant provides evidence that dimerization of MinD is sufficient for MinD to bind the membrane and recruit its partners.     

Conformation of the cell division regulator MinE: evidence for interactions between the topological specificity and anti-MinCD domains

Symmetric division of Gram-negative bacteria depends on the combined action of three proteins that ensure correct positioning of the cell division septum, namely, MinC, MinD, and MinE. To achieve this function, MinC and MinD form a membrane-bound complex that blocks cell division at all potential sites. Opposing this inhibition is MinE, which interacts with MinD via its N-terminal anti-MinCD domain to site-specifically counter the action of the MinCD complex. The anti-MinCD domain has been proposed to bind MinD in a helical conformation; however, little is actually known about the structure of this functionally critical region. To understand how MinE can perform its anti-MinCD function, we have therefore investigated the conformation of the full-length MinE from Neisseria gonorrhoeae by solution NMR. Although solubility considerations required the use of sample conditions that limit the observation of amide resonances to regions that are protected from solvent exchange, backbone chemical shifts from both N- and C-terminal domains could be assigned. In contrast to previous models, secondary chemical shift analysis of these solvent-protected regions shows that parts of the N-terminal anti-MinCD domain are stably folded with many functionally important residues localizing to a beta-structure. In addition, this N-terminal domain may be interacting with the C-terminal topological specificity domain, since mutations made in one domain led to NMR spectral changes in both domains. The nonfunctional MinE mutant L22D showed even larger evidence of structural perturbations in both domains, with significant destabilization of the entire MinE structure. Overall, these results suggest that there is an intimate structural association between the anti-MinCD and topological specificity domains, allowing the functional properties of the two domains to be modulated through this interaction.     

MinD and MinE Interact with Anionic Phospholipids and Regulate Division Plane Formation in Escherichia coli

The Min proteins (MinC, MinD, and MinE) form a pole-to-pole oscillator that controls the spatial assembly of the division machinery in Escherichia coli cells. Previous studies identified that interactions of MinD with phospholipids positioned the Min machinery at the membrane. We extend these studies by measuring the affinity, kinetics, and ATPase activity of E. coli MinD, MinE, and MinDE binding to supported lipid bilayers containing varying compositions of anionic phospholipids. Using quartz crystal microbalance measurements, we found that the binding affinity (K_d) for the interaction of recombinant E. coli MinD and MinE with lipid bilayers increased with increasing concentration of the anionic phospholipids phosphatidylglycerol and cardiolipin. The K_d for MinD (1.8 μm) in the presence of ATP was smaller than for MinE (12.1 μm) binding to membranes consisting of 95:5 phosphatidylcholine/cardiolipin. The simultaneous binding of MinD and MinE to membranes revealed that increasing the concentration of anionic phospholipid stimulates the initial rate of adsorption (k_on). The ATPase activity of MinD decreased in the presence of anionic phospholipids. These results indicate that anionic lipids, which are concentrated at the poles, increase the retention of MinD and MinE and explain its dwell time at this region of bacterial cells. These studies provide insight into interactions between MinD and MinE and between these proteins and membranes that are relevant to understanding the process of bacterial cell division, in which the interaction of proteins and membranes is essential.     

Roles of MinC and MinD in the site-specific septation block mediated by the MinCDE system of Escherichia coli.

The proper placement of the cell division site in Escherichia coli requires the site-specific inactivation of potential division sites at the cell poles in a process that requires the coordinate action of the MinC, MinD, and MinE proteins. In the absence of MinE, the coordinate expression of MinC and MinD leads to a general inhibition of cell division. MinE gives topological specificity to the division inhibition process, so that the septation block is restricted to the cell poles. At normal levels of expression, both MinC and MinD are required for the division block. We show here that, when expressed at high levels, MinC acts as a division inhibitor even in the absence of MinD. The division inhibition that results from MinC overexpression in the absence of MinD is insensitive to the MinE topological specificity factor. The results suggest that MinC is the proximate cause of the septation block and that MinD plays two roles in the MinCDE system--it activates the MinC-dependent division inhibition mechanism and is also required for the sensitivity of the division inhibition system to the MinE topological specificity factor.     

The Min oscillator uses MinD-dependent conformational changes in MinE to spatially regulate cytokinesis

In E. coli, MinD recruits MinE to the membrane, leading to a coupled oscillation required for spatial regulation of the cytokinetic Z ring. How these proteins interact, however, is not clear because the MinD-binding regions of MinE are sequestered within a six-stranded β sheet and masked by N-terminal helices. minE mutations that restore interaction between some MinD and MinE mutants were isolated. These mutations alter the MinE structure leading to release of the MinD-binding regions and the N-terminal helices that bind the membrane. Crystallization of MinD-MinE complexes revealed a four-stranded β sheet MinE dimer with the released β strands (MinD-binding regions) converted to α helices bound to MinD dimers. These results identify the MinD-dependent conformational changes in MinE that convert it from a latent to an active form and lead to a model of how MinE persists at the MinD-membrane surface.     

Recruitment of MinC,an inhibitor of Z-ring formation,to the membrane in Escherichia coli: role of MinD and MinE

In Escherichia coli, the min system prevents division away from midcell through topological regulation of MinC, an inhibitor of Z-ring formation. The topological regulation involves oscillation of MinC between the poles of the cell under the direction of the MinDE oscillator. Since the mechanism of MinC involvement in the oscillation is unknown, we investigated the interaction of MinC with the other Min proteins. We observed that MinD dimerized in the presence of ATP and interacted with MinC. In the presence of a phospholipid bilayer, MinD bound to the bilayer and recruited MinC in an ATP-dependent manner. Addition of MinE to the MinCD-bilayer complex resulted in release of both MinC and MinD. The release of MinC did not require ATP hydrolysis, indicating that MinE could displace MinC from the MinD-bilayer complex. In contrast, MinC was unable to displace MinE bound to the MinD-bilayer complex. These results suggest that MinE induces a conformational change in MinD bound to the bilayer that results in the release of MinC. Also, it is argued that binding of MinD to the membrane activates MinC.     

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

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.     

The three-dimensional structure of septum site-determining protein MinD from Pyrococcus horikoshii OT3 in complex with Mg-ADP

BACKGROUND: In Escherichia coli, the cell division site is determined by the cooperative activity of min operon products MinC, MinD, and MinE. MinC is a nonspecific inhibitor of the septum protein FtsZ, and MinE is the supressor of MinC. MinD plays a multifunctional role. It is a membrane-associated ATPase and is a septum site-determining factor through the activation and regulation of MinC and MinE. MinD is also known to undergo a rapid pole-to-pole oscillation movement in vivo as observed by fluorescent microscopy. RESULTS: The three-dimensional structure of the MinD-2 from Pyrococcus horikoshii OT3 (PH0612) has been determined at 2.3 A resolution by X-ray crystallography using the Se-Met MAD method. The molecule consists of a beta sheet with 7 parallel and 1 antiparallel strands and 11 peripheral alpha helices. It contains the classical mononucleotide binding loop with bound ADP and magnesium ion, which is consistent with the suggested ATPase activity. CONCLUSIONS: Structure analysis shows that MinD is most similar to nitrogenase iron protein, which is a member of the P loop-containing nucleotide triphosphate hydrolase superfamily of proteins. Unlike nitrogenase or other member proteins that normally work as a dimer, MinD was present as a monomer in the crystal. Both the 31P NMR and Malachite Green method exhibited relatively low levels of ATPase activity. These facts suggest that MinD may work as a molecular switch in the multiprotein complex in bacterial cell division.