Conformation of the Cell Division Regulator MinE:  Evidence for Interactions between the Topological Specificity and Anti-MinCD Domains

Abstract

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.

Appropriation of the MinD protein-interaction motif by the dimeric interface of the bacterial cell division regulator MinE

“…Using standard heteronuclear solution NMR techniques, an ensemble of structures was determined for a full-length Ng-MinE sample. This was done using the E46A mutant, because it exhibited more favorable solubility characteristics than the wild-type protein, and it retained structural characteristics that were highly similar to that of WTas determined by circular dichroism and backbone NMR secondary chemical shifts (17,18). As shown in Fig.…”

“…The ability to use peptides to test stimulation of MinD ATPase activity allowed us to address the specific role of Leu 22 as well as additional regions of the anti-MinCD domain in the MinD interaction. Although the L22D mutant of the full-length protein was not active, this may have been due to the significant structural perturbation that results from the introduction of a negative charge in the hydrophobic core (17). However, if Leu 22 plays a direct role in MinD interactions, then an L22D version of Ng-MinE 1-22 would also be expected to show reduced activity relative to the WT peptide, because the activity of the peptide does not require the globular core.…”

“…According to this model, the anti-MinCD domain is unfolded and solventexposed, allowing free interactions with membrane-bound MinD. However, we have shown that in the full-length MinE protein from Neisseria gonorrhoeae (Ng) the anti-MinCD domain is stably folded, with interactions involving the topological specificity domain (17).…”

The tail of the ParG DNA segregation protein remodels ParF polymers and enhances ATP hydrolysis via an arginine finger-like motif

“…We propose that the N-terminal domain of MinE might activate MinD ATP hydrolysis by an arginine finger-like mechanism, supplying one or both conserved arginines in trans into the ATP-binding pocket in MinD. Moreover, this charge-balancing strategy of ATPase activation might be a universally conserved feature of regulatory partners of ParA-like proteins, including Spo0J implicated in chromosome (21,22,36). The E. coli MinE N-terminal domain (residues 1-35) is predicted to consist of an extended or nascent helix (22).…”

Modeling Three-Dimensional Spatial Regulation of Bacterial Cell Division (Dissertation)

“…The domains at the two terminals of MinE appear to have separate roles in the polar zone oscillations although they have been found to interact with each other (Ramos et al, 2006).…”