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

Abstract

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.

FIG. 1.

Sequence alignment of the N termini of MinD proteins. Abbreviations: Mj, Methanococcus jannaschii; Af, Archaeoglobus fulgidus; Pf, Pyrococcus furiosus; Ph, Pyrococcus horikoshii; Ng, Neisseria gonorrhoeae; Nm, Neisseria meningitidis; Ec, Escherichia coli; St, Salmonella enterica serovar Typhimurium;Yp, Yersinia pestis; Vc, Vibrio cholerae; Pa, Pseudomonas aeruginosa; Bm, Brucella melitensis; Bsui, Bacillus suis; At, Agrobacterium tumefaciens; Hp, Helicobacter pylori; Aa, Aquifex aeolicus; Tm, Thermotoga maritima; Bs, Bacillus subtilis; Lm, Listeria monocytogenes; Cp, Clostridium perfringens; Sy, Synechocystis sp.; Gt, Guillardia theta; Dr, Deinococcus radiodurans; Ct, Chlamydia trachomatis. Residues of the Walker A ATP-binding motif are aligned below the solid bar.

FIG. 2.

Localization of wild-type and N-terminal deletion derivatives of MinD_Ng in E. coli PB114. Oscillation cycles (from one pole to the other and back) of GFP-MinD_Ng fusions were measured by using E. coli rods that were 2.0 to 2.5 μm long. (A) Distinct pole-to-pole movement of wild-type GFP-MinD_Ng in E. coli. In the cell shown, one cycle of fusion protein movement required 30 s. Note the U-shaped fluorescent signal of the fusion protein alternately lining each cell polar region. The left panel shows a differential interference contrast image, and the remaining panels show corresponding fluorescence images. Bar = 5 μm (the magnifications for all other images are similar). (B) GFP-MinD_Ng localizes in longer E. coli cells as regularly spaced bands. Each band contains the fusion protein arranged within adjacent polymeric arrays (arrows) that are suggestive of a coil-like structure. (C) GFP-MinD_Ng-2aaNT exhibits pole-to-pole oscillation (arrows). The time required for the fusion protein to complete one oscillatory cycle in this cell was 30 s. (D) GFP-MinD_Ng-3aaNT can also exhibit pole-to-pole oscillation (arrows), and one cycle required only 15 s in the cell shown. However, much of the fusion protein signal is distributed throughout the cytosol, making visualization of GFP-MinD_Ng-3aaNT oscillation more difficult. (E) Raw image of E. coli PB114 expressing GFP-MinD_Ng-3aaNT. Note the nearly uniform cytosolic localization of the protein. (E′) Image enhancement of panel E, revealing the presence of GFP-MinD_Ng-3aaNT localizing within polymeric bands. (F) In the absence of MinE_Ng, GFP-MinD_Ng localizes along the entire inner cell periphery with no evidence of oscillation or polymeric arrays. (G) Western blot obtained by using anti-MinD_Ng antisera to detect GFP-MinD_Ng proteins in E. coli PB114. Lane 1, cell extract from untransformed E. coli PB114; lane 2, wild-type GFP-MinD_Ng (pSR15); lane 3, GFP-MinD_Ng-2aaNT (pSIA16); lane 4, GFP-MinD_Ng-3aaNT (pSIA17). (H) Western blot obtained by using anti-MinE_Ng antisera to detect MinE_Ng in E. coli PB114 transformed with no plasmid (lane 1), pSR15 (lane 2), pSIA16 (lane 3), or pSIA17 (lane 4).

FIG. 3.

Localization of GFP-MinD_Ng-I4Q in E. coli PB114. (A) Raw images of E. coli PB114 expressing GFP-MinD_Ng-I4Q. Bar = 5 μm (the magnifications for all other images are similar). Note the increased cytosolic localization of GFP-MinD_Ng-I4Q relative to wild-type GFP-MinD_Ng (Fig. 2A). GFP-MinD_Ng-I4Q required only 15 s to complete one cycle of oscillation. (B) Localization of GFP-MinD_Ng-I4Q in a longer E. coli cell. Note the increased cyosolic distribution of fluorescent signal relative to that of wild-type GFP-MinD_Ng (Fig. 2B). (B′) Image enhancement of panel B, showing that GFP-MinD_Ng-I4Q can still localize within bands, which is suggestive of a polymeric array (arrows). (C) GFP-MinD_Ng-I5A is mostly localized in the cytosol. (C′) Contrast enhancement of the image in panel C, showing that GFP-MinD_Ng-I5A retains the ability to localize as bands (arrows). (D) GFP-MinD_Ng-I5E is uniformly distributed throughout the cytoplasm. (E) Western blot obtained by using anti-MinD_Ng antisera to detect GFP-MinD_Ng in E. coli PB114 transformed with wild-type GFP-MinD_Ng (pSR15) (lane 1), GFP-MinD_Ng-K3E (pSIA18) (lane 2), MinD_Ng-K3I (pJS30) (lane 3), GFP-MinD_Ng-I4Q (pJS18) (lane 4), GFP-MinD_Ng-I5E (pJS29) (lane 5), GFP-MinD_Ng-I5A (pJS19) (lane 6), or no plasmid (lane 7). (F) Western blotting to detect MinE_Ng in E. coli transformed with no plasmid (lane 1), pSR15 (lane 2), pSIA18 (lane 3), pJS30 (lane 4), pJS18 (lane 5), pJS29 (lane 6), or pJS19 (lane 7).

FIG. 4.

MinD_Ng ATPase stimulation assays. Equal amounts of purified MinD_Ng, MinD_Ng-3aaNT, MinD_Ng-I5E, and MinD_Ng-K16Q were incubated with PG vesicles and 1 mM ATP. The ATPase activities of each mixture were tested in the presence and absence of MinE_Ng over a 90-min period. Inorganic phosphate released due to ATP hydrolysis was monitored by using a malachite green-based method. Buffer A was MinD_Ng storage buffer, and buffer B was MinE_Ng storage buffer. WT, wild type.

FIG. 5.

Structure of A. fulgidus MinD (PDB accession no. 1HYQ) (2), highlighting the N-terminal residues. Yellow indicates the extreme N-terminal amino acids at positions 1 to 5. The red residues (aa 6 to 9), in conjunction with the yellow residues (aa 1 to 5), form a β-strand that connects to the P-loop (Walker A ATP-binding motif [green]). The general position of the ATP-binding face of MinD is indicated. The ribbon diagram was generated with the RasMol molecular graphics visualization tool (version 2.7.2.1).