Crystal structure of Helicobacter pylori MinE, a cell division topological specificity factor

Abstract

In Gram-negative bacteria, proper placement of the FtsZ ring, mediated by nucleoid occlusion and the activities of the dynamic oscillating Min proteins MinC, MinD and MinE, is required for correct positioning of the cell division septum. MinE is a topological specificity factor that counters the activity of MinCD division inhibitor at the mid-cell division site. Its structure consists of an anti-MinCD domain and a topology specificity domain (TSD). Previous NMR analysis of truncated Escherichia coli MinE showed that the TSD domain contains a long alpha-helix and two anti-parallel beta-strands, which mediate formation of a homodimeric alpha/beta structure. Here we report the crystal structure of full-length Helicobacter pylori MinE and redefine its TSD based on that structure. The N-terminal region of the TSD (residues 19-26), previously defined as part of the anti-MinCD domain, forms a beta-strand (betaA) and participates in TSD folding. In addition, H. pylori MinE forms a dimer through the interaction of anti-parallel betaA-strands. Moreover, we observed serial dimer-dimer interactions within the crystal packing, resulting in the formation of a multimeric structure. We therefore redefine the functional domain of MinE and propose that a multimeric filamentous structure is formed through anti-parallel beta-strand interactions.

Fig. 1

Crystal structure and sequence alignment of full-length HpMinE. A. Ribbon view showing the overall dimeric structure of HpMinE. Subunits MolA and MolB are coloured blue and orange respectively. The homodimeric interface of HpMinE is comprised of the α-face (the anti-parallel α-helices) and the β-face (six anti-parallel β-strands). These figures were made using PyMOL (DeLano, 2002). B. Ribbon view of the HpMinE monomer. The monomeric structure consists of an α-helix (αA) and three anti-parallel β-strands (βA, βB and βC). Helix αA and two β-strands (βB and βC) are coloured blue and orange respectively. The β-strand located at the dimer interface (βA) is coloured green. C. Comparison of the structures of the full-length HpMinE and truncated EcMinE (pdb id 1EV0) dimers. Full-length HpMinE is shown in blue, except the N-terminal strand (βA) is shown in green. Truncated EcMinE is shown in orange. D. Sequence alignment of HpMinE with homologous sequences from other Gram-negative bacteria. The amino acid sequences of MinE from H. pylori, E. coli, N. gonorrhoeae, Shigella flexneri, Salmonella typhi, Salmonella typhimurium, Yersinia pestis, Vibrio cholerae, Vibrio vulnificus, Pseudomonas aeruginosa, Pseudomonas putida, Xylella fastidiosa, Agrobacterium tumefaciens, Brucella melitensis and Deinococcus radiodurans were aligned using the Clustal X programme (Thompson et al., 1997). The locations of the anti-MinCD domain (orange) and TSD (blue) in the HpMinE are indicated. Residues that contribute to the hydrophobic core between the α-face and β-face of the HpMinE dimer are indicated by open circles above sequences.

Fig. 2

The dimer interface within the HpMinE structure. A. Close-up view of the interacting residues in the α-face (Y34, M38, E41, I42, V45 and Y49). Protomers MolA and MolB are coloured blue and orange respectively. The four-amino acid cluster comprised of E41 and V45 from each subunit is located at the centre of the α-face. In particular, the E41 residues (MolA/MolB) make hydrogen bonds with the respective Y49 residues. Hydrogen bonds are shown as dotted lines. B. Close-up view of the interacting residues in the β-face (L20, K21, L22, I23, L24 and A25). Hydrogen bonds are shown as dotted lines. C. The dimeric structure of HpMinE coloured according to a relative conservation index (1 to 100) based on homologous MinE sequences from other Gram-negative bacteria, including E. coli, N. gonorrhoeae, S. flexneri, S. typhi, S. typhimurium, Y. pestis, V. cholerae, V. vulnificus, P. aeruginosa, P. putida, X. fastidiosa, A. tumefaciens, B. melitensis and D. radiodurans. The relative conservation index was calculated using the Clustal X programme. The residues that form the hydrophobic core between the α-face and β-face (L20, L22, L24, I42, I43 and I46) are highly conserved.

Fig. 3

MinE residues important for topological specificity function and E-ring formation. A. Representation of HpMinE residues that correspond to mutated EcMinE or NgMinE residues used to determine which residues are important for topological specificity function and E-ring formation. Residues important for topological specificity and E-ring formation are coloured orange; less important residues are coloured blue. B. Summary of the effects of MinE mutants on the cell division phenotype and E-ring formation. E-ring formation indicates the percentage of cells with E-rings. Phenotype is classified into three distinct types based on the cell morphology. WT, wild-type phenotype; Rod, filamentous phenotype; Minicelling, minicelling phenotype containing a mixed population of minicells, wild-type cells and short filaments.

Fig. 4

The multimeric structure of HpMinE. A. Ribbon view of the multimeric structure of HpMinE generated by the sixfold symmetry in the crystal. The width and length of a single turn of the multimeric structure are approximately 5.5 and 12.5 nm respectively. The N-terminus of each HpMinE is shown as a red sphere. B. Close-up view of the interacting residues in the interface between two dimers (blue and green). Residues I55, H56 and F57 are involved in hydrophobic interaction, and H56 makes a hydrogen bond with D54.