Key role of two terminal domains in the bidirectional polymerization of FtsA protein

Abstract

The effect of two different truncations involving either the 1C domain or the simultaneous absence of the S12-13 β-strands of the FtsA protein from Streptococcus pneumoniae, located at opposite terminal sides in the molecular structure, suggests that they are essential for ATP-dependent polymerization. These two truncated proteins are not able to polymerize themselves but can be incorporated to some extent into the FtsA(+) polymers during the assembling process. Consequently, they block the growth of the FtsA(+) polymers and slow down the polymerization rate. The combined action of the two truncated proteins produces an additive effect on the inhibition of FtsA(+) polymerization, indicating that each truncation affects a different interaction site within the FtsA molecule.

FIGURE 1.

Three-dimensional representations of structural models for FtsA dimer. A, the bioinformatics model as proposed in Carettoni et al. (11) and fitted to the sequence of the FtsA from S. pneumoniae. B, the crystallographic model of the FtsA dimer obtained from the crystal structure of T. maritima FtsA bound to a FtsZ protein peptide (not shown).⁴ The orientation of both models has been selected to show the best view of the interacting regions. Insets show graphics of the polymers. Monomers are differentiated using red or blue hues, and the interacting domains are abbreviated as S for the S12-S13 β strands or 1C for domain 1C. Surfaces available for interaction at the ends of the polymer are indicated by a pink border. Blue arrows indicate the potential to integrate one monomer.

FIGURE 2.

Nucleotide-dependent polymerization of FtsA⁺ and its variants. A, Coomassie Blue-stained 12% SDS-PAGE gel of 4 μg of the purified FtsA⁺ (lane A⁺) and its variants FtsAΔ1C (lane 1C) and FtsAΔS12–13 (lane S12–13). Molecular weight standards are shown in the left lane (MW). A faint lower band in lane A⁺ is a degradation product of FtsA⁺ that was already described in Lara et al. (4). B, analysis of the polymerization process followed by 90° light scattering. Purified proteins FtsA⁺ (○), FtsAΔ1C (□), and FtsAΔS12–13 (△) at the concentration of 5 μm were equilibrated separately for 5 min in a spectrofluorometer cuvette before the addition of 2 mm ATP. The polymerization reaction at 25 °C was followed during 20 min. The light-scattering intensity was measured as arbitrary units (A. U.). The insets show graphics of the polymer formed by FtsA⁺ as in Fig. 1, and the monomers of the two truncated forms. Surfaces available for monomer addition are outlined in pink, arrows indicate the possibility to add one monomer, whereas crosses indicate that the missing interaction prevents polymerization. C, sedimentation velocity analysis of the purified FtsA variants in the absence (upper panel) and in the presence of ATP (bottom panel). FtsAΔ1C (red line), FtsAΔS12–13 (blue line), and a mixture of equimolar concentrations of FtsAΔ1C and FtsAΔS12–13 (green line) are represented. For comparison, the sedimentation coefficient of FtsA⁺ derived from Lara et al. (4) is 3.2 S.

FIGURE 3.

Kinetics of inhibition of FtsA polymerization. A, light-scattering measurements upon the addition of 2 mm ATP to 5 μm FtsA⁺ alone (○) or in combination with an equimolar amount of one of the truncated proteins, FtsAΔ1C (■) or FtsAΔS12–13 (▴). The discontinuous line shows the best-fit trend line obtained from measurements of the ATP-dependent polymerization of 5 μm FtsA in the presence of 5 μm BSA used as a control. Insets show graphics of the polymers formed by each mixture. Monomers are differentiated using red or blue hues, and the interacting domains are abbreviated as S for the S12-S13 β strands or 1C for domain 1C. Surfaces available for interaction at the ends of the polymer are indicated by a pink border. Blue arrows indicate the potential to integrate one monomer. A. U., arbitrary units. B, light-scattering measurements upon the addition of 2 mm ATP to 5 μm FtsA⁺ alone, as in A (○), or to mixtures containing 5 μm FtsA⁺ plus 1.25 μm FtsAΔ1C (□), 1.25 μm FtsAΔS12–13 (△), or 1.25 μm FtsAΔ1C plus 1.25 μm FtsAΔS12–13 (●). Measurements were taken every 2 s; points are plotted for 30-s intervals. Plotted curves are tendency lines calculated according to the Hill function using the best-fit parameter values given in supplemental Table S1. The inset shows the graphic of one polymer blocked at both ends by the incorporation of one monomer of a truncated protein with the two different truncations occupying opposite ends. Graphic conventions are as for panel A. C, maximum polymerization levels of 5 μm wild type FtsA⁺ in the presence of 2 mm ATP and increasing concentrations, as indicated in the graph, of the truncated proteins FtsAΔ1C (□) or FtsAΔS12–13 (△). The data were calculated from the empirical light-scattering measurements according to the Hill function (see “Experimental Procedures”) and normalized to the results obtained in the absence of any competitor protein (100%). D, the time required to reach 50% of the maximum polymerization level (t₅₀) at increasing concentrations of the competitor proteins is represented for the data shown in panel C (continuous line for FtsAΔ1C and discontinuous line for FtsAΔS12–13). The error bars correspond to 2 ± S.D. for a 95% confidence limit as calculated from the Hill equation.

FIGURE 4.

Electron microscopy of polymers formed by FtsA. Polymerization of mixtures containing the proteins indicated in each frame was initiated by the addition of 2 mm ATP and left to proceed for 20 min. Samples from each mixture were applied to copper grids and negatively stained as described under “Experimental Procedures.” Numbers between brackets indicate the ratio of the protein components in the reaction mixtures, where 4 corresponds to 5 μm. The insets show graphics of the polymer formed by FtsA⁺ as in Fig. 1B and the polymer blocked at either end or at both ends by the incorporation of one monomer of a truncated protein or one of each truncated protein, respectively, with the two different truncations occupying opposite ends. Monomers are differentiated using red or blue hues, and the interacting domains are abbreviated as S for the S12-S13 β strands or 1C for domain 1C. Surfaces available for interaction at the ends of the polymer are indicated by a pink border. Blue arrows indicate the potential to integrate one monomer.

FIGURE 5.

Composition of FtsA polymers formed by FtsA⁺ with increasing concentrations of FtsAΔ1C or FtsAΔS12–13 as in Fig. 3. Polymerization was allowed to proceed for 20 min, and high molecular weight material was then pelleted at 13,000 rpm for 20 min in a tabletop microcentrifuge. The material contained in the pellets was resuspended in an equivalent volume of polymerization buffer (see “Experimental Procedures”). Equal volumes of each sample were run on an SDS-PAGE and stained with Coomassie Blue (supplemental Fig. S3). The densitometric values of the bands corresponding to the amount of FtsA⁺ (gray bars) and each variant (black bars) present in the pellets are shown. The total protein present in each supernatant was quantified and used to check that the recovery of the protein present in the two fractions added up to the total amount contained in each initial mixture.