The bacterial cell-division protein ZipA and its interaction with an FtsZ fragment revealed by X-ray crystallography

Abstract

In Escherichia coli, FtsZ, a homologue of eukaryotic tubulins, and ZipA, a membrane-anchored protein that binds to FtsZ, are two essential components of the septal ring structure that mediates cell division. Recent data indicate that ZipA is involved in the assembly of the ring by linking FtsZ to the cytoplasmic membrane and that the ZipA-FtsZ interaction is mediated by their C-terminal domains. We present the X-ray crystal structures of the C-terminal FtsZ-binding domain of ZipA and a complex between this domain and a C-terminal fragment of FtsZ. The ZipA domain is a six-stranded beta-sheet packed against three alpha-helices and contains the split beta-alpha-beta motif found in many RNA-binding proteins. The uncovered side of the sheet incorporates a shallow hydrophobic cavity exposed to solvent. In the complex, the 17-residue FtsZ fragment occupies this entire cavity of ZipA and binds as an extended beta-strand followed by alpha-helix. An alanine-scanning mutagenesis analysis of the FtsZ fragment was also performed, which shows that only a small cluster of the buried FtsZ side chains is critical in binding to ZipA.

Fig. 1. Structure and topology of ZipA/M185. (A) Ribbon diagram of the ZipA/M185 monomer. For clarity, β-strands have been labeled by number only. The β–α–β split motif is shown in yellow (1–5–α2–6). The insert in the split motif (α1–2–3–4) and helix α3, which immediately follows the motif, are colored purple. Prepared using the program RIBBONS (Carson, 1991). (B) Surface potential representation of ZipA/M185 (GRASP, Nicholls et al., 1991). Regions with electrostatic potential less than –11.5 kBT are red, while those greater than +10.5 kBT are blue (kB, Boltzmann constant, T, absolute temperature). The view is from the uncovered side of the β-sheet showing the cavity of neutral charge, which extends to ∼20 Å across the sheet and has space to accommodate a ligand. The orientation of the ZipA/M185 molecule is similar to that in (A). (C) Topological diagram showing the β–α–β fold of ZipA/M185. The diagram is arranged to coincide with the orientation in (A) and (B). β-strands are represented as arrows, while α-helices are cylinders. The color coding and secondary structural element numbering are the same as in (A). The assignment of the secondary structure of ZipA/M185 was done using the algorithm of Kabsch and Sander, as implemented in PROCHECK (Laskowski et al., 1993).

Fig. 2. Overview of the complex between ZipA/M185 and the FtsZ fragment. (A) Electrostatic potential surface of the FtsZ-binding cavity of ZipA/M185 (GRASP, Nicholls et al., 1991) as it is seen in complex with the FtsZ peptide (yellow ribbons). Contouring scheme for potentials is the same as in Figure 1B. FtsZ residues interacting with the cavity are shown. ZipA/M185 residues that contribute most of the total contacts are designated by name and number (black); those in the peptide, by number only (yellow). The peptide binds as an extended β-strand (residues 367–373) followed by α-helix (residues 374–383). Inserts 1–3, on the right, show peptide side chains (yellow) in hydrophobic pockets of ZipA/M185 (with side chains in white and van der Waals surface in green). Interaction between the peptide (yellow) and ZipA/M185 (white) backbones and internal hydrogen bonding within the peptide (see text) are shown in inserts 4 and 5, respectively. Hydrogen bonds are indicated by dashed white lines. Lys250 of ZipA/M185 does not make contacts with acidic residues Asp370 and Asp373 of the peptide; instead, both Asp370 and Lys250 are involved in hydrogen bonding with water molecules (not shown), and the side chain of Asp373 is hydrogen bonded to the NH group of Ala376. (B) Stereo diagram showing superposition of C_α traces of the ZipA domains as they are seen in the uncomplexed structure (blue lines) and in complex with the FtsZ peptide (purple lines). The most significant changes are found in the segment from the β4–β5 loop (residues 247–251) and in the β6–α3 loop (residues 303–306). From this orientation, the side-chain rearrangement of both Lys250 and Arg 305, upon peptide binding, is evident. Representative contact residues from ZipA/M185 (designated by number in black) and from the peptide (designated by number in purple) are shown, with the C_α trace of the peptide shown as magenta sticks. The orientation is similar to that in (A), and allows for direct comparison of the ZipA/M185–FtsZ interactions. (B) was prepared using the program RIBBONS (Carson, 1991).

Fig. 3. Sequence alignments. (A) Alignment of amino acid sequences of ZipA C-terminal regions from seven species: Escherichia coli (Ecoli); Haemophila influenzae (Haein); Salmonella typhimurium (Salty); Yersinia pestis (Yerpe); Shewanella putrefaciens (Shepu); Actinobacillus actinomycetemcomitans (Actac) and Pseudomonas aeruginosa (Pseae). The numbering is sequential for E.coli ZipA. The sequences share between 34 and 89% identity (X substitutes low-complexity sequences) and were identified by BLAST search (Altschul et al., 1997) using completely and partially sequenced genomes. Residues are highlighted as highly conserved (red) through to moderately well-conserved (blue and gray). Arrows and cylinders below the sequences indicate secondary structure elements observed in the crystal structure of E.coli ZipA, with coloring as in Figure 2. Lines represent areas of turns or loops. The asterisks mark residues involved in interaction with the FtsZ fragment. Residues with main-chain atoms hydrogen bonded to FtsZ are marked by H. (B) Sequence alignment of FtsZ C-terminal fragments from 11 species (BLAST search using complete genomes): E.coli (Ecoli); Bacillus subtilis (Bacsu); Pseudomonas aeruginosa (Pseae); Pseudomonas putida (Psepu); Azotobacter vinelandi (Azovi); Buchnera aphidicola (Bucap); Rickettsia prowazekii (Ricpr); Borrelia burgdorferi (Borbu); Caulobacter crescentus (Caucr); Rhizobium meliloti (Rhime) and Wolbachia sp. (Wolsp). Residues are highlighted using the same definition as in (A). The conformation of the bound FtsZ fragment is represented by an arrow and a cylinder below the sequences. The asterisks mark residues interacting with ZipA/M185 and marked by H are residues with the main-chain atoms hydrogen bonded to ZipA/M185.

Fig. 4. Biosensor assays of FtsZ peptides binding to ZipA/M185. (A) BIAcore sensorgrams of wild-type FtsZ peptide (residues 367–383) binding to immobilized ZipA/M185. Binding curves are expressed in resonance units (RU) as a function of time (s). One representative set of a triplicate experiment is shown, with concentrations of injected peptide indicated on the sensograms. (B) Plots of equilibrium binding responses (R_eq) versus the concentrations of FtsZ peptides. Each binding curve (colored lines) was derived from biosensor experiments performed in triplicate for each FtsZ variant (experimental sensorgrams obtained for mutants are not shown). In those experiments in which the binding responses closely approach or do not reach equilibrium, the maximal binding response (R_max) was estimated to be 110–115 RU at higher concentrations of injected peptides.

Fig. 5. A comparison of the FtsZ-binding domain of ZipA with the RNA-binding domain of the U1A spliceosomal protein. C_α stereo superposition of ZipA/M185 (green) and U1A (purple) is based on C_α atoms in the β strands of the β–α–β split motif (β1, β5, β6) and includes a ball-and-stick representation of bound RNA fragment and a C_α-model of FtsZ-peptide (sticks in cyan). The RNA-binding loop in U1A and the peptide-binding loop in ZipA/M185 (residues 247–251, designated by number in cyan) are highlighted. The N- and C-termini for the superimposed domains are indicated. Figure prepared using the program RIBBONS (Carson, 1991).

Fig. 6. Electron density maps. (A) Experimental map of ZipA/M185 at 2 Å resolution, calculated with SIRAS phases improved by density modification. The map, contoured at 1.2 σ, is superimposed on the refined coordinates of residues B263–B270. (B) Difference electron density in the region of the FtsZ peptide bound to ZipA/M185. Electron density is from an F_obs – F_calc map (25–1.95 Å, contoured at 1.7σ) calculated using model phases, with the peptide atoms omitted from all calculations. Figure prepared using the program RIBBONS (Carson, 1991).