Crystal structure of the bacterial cell division inhibitor MinC

Abstract

Bacterial cell division requires accurate selection of the middle of the cell, where the bacterial tubulin homologue FtsZ polymerizes into a ring structure. In Escherichia coli, site selection is dependent on MinC, MinD and MINE: MinC acts, with MinD, to inhibit division at sites other than the midcell by directly interacting with FTSZ: Here we report the crystal structure to 2.2 A of MinC from Thermotoga maritima. MinC consists of two domains separated by a short linker. The C-terminal domain is a right-handed beta-helix and is involved in dimer formation. The crystals contain two different MinC dimers, demonstrating flexibility in the linker region. The two-domain architecture and dimerization of MinC can be rationalized with a model of cell division inhibition. MinC does not act like SulA, which affects the GTPase activity of FtsZ, and the model can explain how MinC would select for the FtsZ polymer rather than the monomer.

Fig. 1. Stereo drawings of the final 2F_o – F_c map, looking into the hydrophobic core of the β-helix of the C-terminal domain. Turn 2 with strands S2A, S2B and S2C is shown; turn 3 is visible underneath. Side ‘A’ of the triangular domain forms the dimer interface. Made with MAIN (Turk, 1992).

Fig. 2. Ribbon drawings of MinC. (A) An asymmetric unit contains two different MinC dimers, highlighting the flexibility of the linker region (linker, grey; N-terminal domain, yellow; C-terminal domain, blue). Face ‘A’ of the triangular C-terminal domain forms the dimer interface alone in dimer AB (top). (B) Stereo drawing of the N-terminal domain (residues 1–95) with the flexible linker (residues 96–102). (C) Top and side view of the C-terminal domain. The domain folds into a small triangular, right-handed β-helix with a hydrophobic core. The length of the sides is: A, four; B, three; and C, five residues in β-conformation. The strands in the domain have been numbered to reflect their position with respect to the turn number and the side of the β-helix. Made with MOLSCRIPT and RASTER3D (Kraulis, 1991; Merritt and Bacon, 1997).

Fig. 3. Sequence alignment of T.maritima and E.coli MinC proteins. The secondary structure of the T.maritima crystal structure is indicated above. Identical residues are boxed. Made with ALSCRIPT (Barton, 1993).

Fig. 4. Sequence repeats in the C-terminal domain of T.maritima MinC giving rise to the symmetrical β-helical fold with exclusively small, hydrophobic sidechains in the core of the β-helix. Sidechain orientation can be ‘I’ with sidechains pointing into the hydrophobic core or ‘O’, pointing outwards.

Fig. 5. Structural alignment of the N-terminal domain of MinC from T.maritima, SpoIIAA from B.subtilis (PDB 1AUZ; Kovacs et al., 1998) and FtsA from T.maritima (PDB 1E4F; van den Ent and Löwe, 2000). FtsA shows the highest DALI score of 3.7, r.m.s.d. 3.2 Å over 74 residues. SpoIIAA has a DALI score against the N-terminal domain of MinC of 3.5, r.m.s.d. 3.6 Å over 72 almost consecutive residues. Aligned stretches are coloured, all other residues are shown in grey. Made with MOLSCRIPT and RASTER3D (Kraulis, 1991; Merritt and Bacon, 1997).

Fig. 6. Schematic overview of the model of MinC function. FtsZ forms linear head-to-tail polymers (protofilaments) with a repeat of 40 Å (Löwe and Amos, 1999). MinC forms tight head-to-head dimers via the C-terminal domain (blue). To enable binding of MinC to FtsZ protofilaments, the N–terminal domains of MinC (orange) have to be rotated, which is facilitated by a flexible linker region. By forming a dimer, MinC will bind much more tightly to FtsZ protofilaments than FtsZ monomers. Although the binding site of MinD on MinC is not known, we speculate that it may pre-orient the N-terminal domains of MinC, thereby greatly enhancing binding affinity to FtsZ. MinD’s ATPase activity could be required for changing conformation in MinC to switch between different affinities or FtsZ polymers. MinC’s binding site on FtsZ is thought to overlap with FtsA’s binding site (Justice et al., 2000), which includes the conserved C-terminal peptide in FtsZ. ZipA can bind to FtsZ in the presence of MinCD, whereas FtsA cannot (Justice et al., 2000). SulA, an SOS response factor, interferes with FtsZ polymerization directly (Mukherjee et al., 1998). By inhibiting FtsA binding, MinC can block septum formation, since the recruitment of all other septum proteins is dependent on localization of FtsZ and FtsA.