ATP-dependent polymerization dynamics of bacterial actin proteins involved in Spiroplasma swimming

Abstract

MreB is a bacterial protein belonging to the actin superfamily. This protein polymerizes into an antiparallel double-stranded filament that determines cell shape by maintaining cell wall synthesis. Spiroplasma eriocheiris, a helical wall-less bacterium, has five MreB homologous (SpeMreB1-5) that probably contribute to swimming motility. Here, we investigated the structure, ATPase activity and polymerization dynamics of SpeMreB3 and SpeMreB5. SpeMreB3 polymerized into a double-stranded filament with possible antiparallel polarity, while SpeMreB5 formed sheets which contained the antiparallel filament, upon nucleotide binding. SpeMreB3 showed slow P_i release owing to the lack of an amino acid motif conserved in the catalytic centre of MreB family proteins. Our SpeMreB3 crystal structures and analyses of SpeMreB3 and SpeMreB5 variants showed that the amino acid motif probably plays a role in eliminating a nucleophilic water proton during ATP hydrolysis. Sedimentation assays suggest that SpeMreB3 has a lower polymerization activity than SpeMreB5, though their polymerization dynamics are qualitatively similar to those of other actin superfamily proteins, in which pre-ATP hydrolysis and post-P_i release states are unfavourable for them to remain as filaments.

Keywords: Mollicutes; MreB; X-ray crystallography; bacterial cytoskeleton; cell motility; electron microscopy.

Figure 1.

Structures of SpeMreB3 and SpeMreB5 filaments observed by EM. (a,b) Negative-staining EM image of (a) 10 µM SpeMreB3 and (b) 5 µM SpeMreB5. The samples were diluted to 3 µM prior to placement onto an EM grid. (c,d) Two-dimensional averaged image of (d) SpeMreB3 and (d) SpeMreB5 filaments averaged from 2874 to 652 particles, respectively. The estimated subunit repeats are 5.1 ± 0.1 and 5.2 ± 0.2 nm for SpeMreB3 and SpeMreB5, respectively. A weak electron density connecting the protofilaments is indicated by a triangle. (e) A two-dimensional averaged image of the five-stranded SpeMreB5 sheet structure averaged from 1575 particles. The estimated subunit repeat is 5.2 ± 0.2 nm. The protofilaments in the juxtaposed filament and the other protofilaments are indicated by solid and open triangles, respectively.

Figure 2.

Crystal structures of SpeMreB3. (a) Protofilament structures in crystals of Nf-SpeMreB3 and SpeMreB3 AMPPNP complexes. Two different conformations (Mol-A and B) in the asymmetric unit of the SpeMreB3 AMPPNP complex crystal are shown in the centre and right panels, respectively. Two subunits in the protofilaments are labelled as i and i-1. The subunit repeat is indicated at the right of each i-1 subunit. The four subdomains (IA, IB, IIA and IIB) and N- and C-termini are labelled on the i subunit. The boxed regions on Nf and AMPPNP complex Mol-A protofilaments are magnified in b and c, respectively, to represent the intra-protofilament interactions. (b,c) Close up view of the subunit interface in protofilaments in the crystal of (b) Nf-SpeMreB3 and (c) the SpeMreB3 AMPPNP complex Mol-A. Subdomains IA, IB, IIA and IIB are indicated by ribbon representations coloured with light blue, orange, magenta and green, respectively. Hydrogen bonds and electrostatic interactions are indicated by broken lines. The residues involved in the hydrogen bonding or electrostatic interaction network are indicated by stick models or blue (backbone nitrogen atom) or red (backbone oxygen atom) spheres with labels. Water molecules involved in the interactions are shown as small red spheres. (d) Structural comparison of Mol-A and B in the SpeMreB3 AMPPNP complex and Nf-SpeMreB3. The structures are superimposed onto the subdomains IIA and IIB of Mol-A. The movement of the subdomain IA and IB relative to Nf-SpeMreB3 is indicated by red arrows. (e) A ribbon representation of the filament structure of SpeMreB3 AMPPNP Mol-A fit to the EM image (figure 1c; electronic supplementary material, figure S6E).

Figure 3.

P_i release measurement and determination of active site structures of SpeMreB3 and SpeMreB5. (a) Time course plots of P_i release from 3 µM SpeMreB3 wild-type (cyan), SpeMreB3 D147E (light green), SpeMreB3 K174T (navy blue), SpeMreB3 K174T/S176D (purple), SpeMreB5 wild-type (red) and SpeMreB5 D162S (pink) in the presence of 2 mM ATP. The measurements were performed three times, and a representative curve is plotted in the graph. (b) P_i release rates from SpeMreBs estimated from (a) and electronic supplementary material, figure S8H and I. Error bars indicate the standard deviation (s.d.) from three repeated measurements. Symbols indicate p-value supported by Student's t-test (** p < 0.01, n.s. p > 0.05). (c) Concentration dependence of P_i release from SpeMreB3 (cyan) and SpeMreB5 (red). Error bars indicate the s.d. from three repeated measurements. (d–f) Close up view of the active sites of (d) the CcMreB AMPPNP complex (PDB: 4CZJ), (e) the SciMreB5 AMPPNP complex (PDB: 7BVY), and (f) the SpeMreB3 AMPPNP complex (Mol-A). Mg²⁺ and water molecules are indicated as green and red spheres, respectively. (g) Weblogos of amino acid sequences around the ATP hydrolysis region from: (left upper) 4832 MreB family proteins from non-Spiroplasma bacteria used in our previous study [9], (left lower) 29 Spiroplasma MreB3, and (right) 171 Spiroplasma MreBs (except for MreB3). The corresponding amino acid for the core amino acids motif for ATP hydrolysis (E140, T167 and E169 in CcMreB) are indicated by triangles. (h) A working model for ATP hydrolysis in MreB family proteins. Residues corresponding to T167 and E169 in CcMreB, the nucleophilic water molecule, and the γ-P_i of ATP are shown in the model. The unshared electron pairs of each atom on the residues and the water are indicated by two neighbouring dots. A putative electron transfer pathway is indicated by arrows. (i) Close up view of the active sites of AMPPNP-bound F-actin (PDB: 6DJM). Mg²⁺ and water molecules are indicated as green and red spheres, respectively.

Figure 4.

SpeMreB sedimentation assays. Each SpeMreB was incubated with buffer S (20 mM Tris–HCl pH 8.0, 1 M NaCl, 200 mM Arginine-HCl pH 8.0, 5 mM DTT, 2 mM MgCl₂ and 2 mM ATP) for 1 h after initiating polymerization and were ultracentrifuged at 436 000×g for 120 min at 23°C. Precipitates were resuspended with water equivalent to the sample amount. For SpeMreB3 and its variants, each fraction was diluted three times before the preparation of the sample for SDS-PAGE. Each fraction was loaded onto a 12.5% Laemmli gel and stained with Coomassie brilliant blue to quantify the protein concentration. Fractions derived from the same sample were loaded onto adjacent lanes, and the total concentration of the sample is indicated on the lanes. (a,b) Sedimentation assay of (a) 8 µM SpeMreB3 and (b) 3 µM SpeMreB5 in the presence (left half lanes of each panel, (+) ATP) or absence (right half lanes of each panel, (−) ATP) of Mg-ATP. Protein size standards are visualized in Lane M, with the molecular masses of each band on the left side. (c) Quantified precipitation amounts of sedimented SpeMreBs. The resulting concentrations of the precipitated fractions were plotted over the total SpeMreB concentrations with linear fitting. Error bars indicate the s.d. from five repeated measurements for SpeMreB5 wild-type polymerized with ATP and ATP-analogues and three repeated measurements for the others. Critical concentrations were estimated as the x-intercept of each linear fit and are summarized in table 1.

Figure 5.

Working model of SpeMreB polymerization. ATP and ADP are denoted as ‘T' and ‘D', respectively. The characters ‘G' and ‘F' indicate SpeMreBs in the monomeric and polymerized states, respectively, named analogous to actin states. Bound nucleotides on SpeMreBs are indicated as subscripts. In the filamentous states, the pre-hydrolysis and post-P_i release states, which showed high critical concentrations are indicated with asterisks. The schematic structures of monomeric and polymerized SpeMreB3 (cyan) and SpeMreB5 (red) are indicated beside the corresponding positions on the polymerization cycles with the filament characters.