Thermal adaptation of mesophilic and thermophilic FtsZ assembly by modulation of the critical concentration

Cytokinesis is the last stage in the cell cycle. In prokaryotes, the protein FtsZ guides cell constriction by assembling into a contractile ring-shaped structure termed the Z-ring. Constriction of the Z-ring is driven by the GTPase activity of FtsZ that overcomes the energetic barrier between two protein conformations having different propensities to assemble into polymers. FtsZ is found in psychrophilic, mesophilic and thermophilic organisms thereby functioning at temperatures ranging from subzero to >100 degrees C. To gain insight into the functional adaptations enabling assembly of FtsZ in distinct environmental conditions, we analyzed the energetics of FtsZ function from mesophilic Escherichia coli in comparison with FtsZ from thermophilic Methanocaldococcus jannaschii. Presumably, the assembly may be similarly modulated by temperature for both FtsZ orthologs. The temperature dependence of the first-order rates of nucleotide hydrolysis and of polymer disassembly, indicated an entropy-driven destabilization of the FtsZ-GTP intermediate. This destabilization was true for both mesophilic and thermophilic FtsZ, reflecting a conserved mechanism of disassembly. From the temperature dependence of the critical concentrations for polymerization, we detected a change of opposite sign in the heat capacity, that was partially explained by the specific changes in the solvent-accessible surface area between the free and polymerized states of FtsZ. At the physiological temperature, the assembly of both FtsZ orthologs was found to be driven by a small positive entropy. In contrast, the assembly occurred with a negative enthalpy for mesophilic FtsZ and with a positive enthalpy for thermophilic FtsZ. Notably, the assembly of both FtsZ orthologs is characterized by a critical concentration of similar value (1-2 mu M) at the environmental temperatures of their host organisms. These findings suggest a simple but robust mechanism of adaptation of FtsZ, previously shown for eukaryotic tubulin, by adjustment of the critical concentration for polymerization.Becas Chile and Programa de Mejoramiento de la Calidad y Equidad de la Educacion Comision Nacional de Investigacion Cientifica y Tecnologica 24090139 Fondo Nacional de Desarrollo Cientifico y Tecnologico 113071

Measured parameters for the GTPase activity and polymerization of FtsZ from <i>Escherichia coli</i>.

<p>Measured parameters for the GTPase activity and polymerization of FtsZ from <i>Escherichia coli</i>.</p

Global analysis of the critical concentration using the integrated van′t Hoff equation.

<p>The combined data obtained with the GTP hydrolysis and polymerization assays were fit to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185707#pone.0185707.e008" target="_blank">Eq 6</a> using nonlinear regression (solid lines), for mesophilic EcFtsZ (A) and thermophilic MjFtsZ (B). The fitting coefficients for EcFtsZ are: <i>a</i> = -787.59, <i>b</i> = 38,745 and <i>c</i> = 117.86, and the coefficients obtained for MjFtsZ: <i>a</i> = 776.24, <i>b</i> = -40,253 and <i>c</i> = -110.53. The temperature-dependent parameters Δ<i>H</i>⁰ and Δ<i>S</i>⁰ were calculated using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185707#pone.0185707.e010" target="_blank">Eq 7</a> (Panels C and D), as well as the temperature-independent heat capacity change Δ<i>C</i>_<i>p</i> (see text).</p

Measured parameters for the GTPase activity and polymerization of FtsZ from <i>Methanocaldococcus jannaschii</i>.

<p>Measured parameters for the GTPase activity and polymerization of FtsZ from <i>Methanocaldococcus jannaschii</i>.</p

Eyring plots of the GTPase activity and polymerization of FtsZ.

<p>Top row, mesophilic EcFtsZ. Bottom row, thermophilic MjFtsZ. The kinetic rates of GTP hydrolysis, <i>k_cat</i> (A and C), and filament depolymerization, <i>k_depol</i> (B and D), are plotted as a function of temperature. The apparent enthalpies (Δ<i>H</i>^0‡) and entropies (Δ<i>S</i>^0‡) of the transition state were calculated by nonlinear regression using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185707#pone.0185707.e006" target="_blank">Eq 4</a> (solid lines), and the best-fit values are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185707#pone.0185707.t003" target="_blank">Table 3</a>.</p

The morphology of FtsZ polymers was characterized by negative-stain transmission electron microscopy.

<p>Top row, 10 μM EcFtsZ was polymerized at 10, 20 and 30°C (A-C). Bottom row, 10 μM MjFtsZ was polymerized at 40, 60 and 80°C (D–F). The arrowheads in D point to examples of short and curved polymers. The scale bars represent 100 and 500 nm for the top and bottom rows, respectively (black bars).</p

Temperature dependence of the GTP hydrolysis rates of FtsZ.

<p>The effect of temperature and protein concentration on the GTPase activity of FtsZ was examined for mesophilic EcFtsZ (A) and for thermophilic MjFtsZ (B). The catalytic constant <i>k_cat</i> was determined from the slopes of the linear regressions indicated by the solid lines (the substrate was used at saturating concentrations). The critical concentrations C_C-GTPase were obtained from the intersection of the linear regressions with the protein concentration axis. The symbols and error bars are the averages and the standard deviations from triplicate samples. The calculated values of C_C-GTPase and <i>k_cat</i> are presented in Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185707#pone.0185707.t001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185707#pone.0185707.t002" target="_blank">2</a>.</p

Transition state enthalpy, entropy and free energy of FtsZ assembly.

<p>Transition state enthalpy, entropy and free energy of FtsZ assembly.</p

Single tryptophan mutants of FtsZ: Nucleotide binding/exchange and conformational transitions

Artículo de publicación ISICell division protein FtsZ cooperatively self-assembles into straight filaments when bound to GTP. A set of conformational changes that are linked to FtsZ GTPase activity are involved in the transition from straight to curved filaments that eventually disassemble. In thiswork, we characterized the fluorescence of single Trp mutants as a reporter of the predicted conformational changes between the GDP- and GTP-states of Escherichia coli FtsZ. Steady-state fluorescence characterization showed the Trp senses different environments and displays low solvent accessibility. Time-resolved fluorescence data indicated that the main conformational changes in FtsZ occur at the interaction surface between the N and C domains, but also minor rearrangements were detected in the bulk of the N domain. Surprisingly, despite its location near the bottomprotofilament interface at the C domain, the Trp 275 fluorescence lifetime did not report changes between the GDP and GTP states. The equilibrium unfolding of FtsZ features an intermediate that is stabilized by the nucleotide bound in the N-domain aswell as by quaternary protein–protein interactions. In this context,we characterized the unfolding of the Trpmutants using time-resolved fluorescence and phasor plot analysis. A novel picture of the structural transition from the native state in the absence of denaturant, to the solvent-exposed unfolded state is presented. Taken together our results show that conformational changes between the GDP and GTP states of FtsZ, such as those observed in FtsZ unfolding, are restricted to the interaction surface between the N and C domains.This work was supported by FP7 EC DIVINOCELL grant 223431 and Fondo Nacional de Desarrollo Científico y Tecnológico grant 1130711 (to O.M.). F.M.-F. received fellowships from Becas Chile and Programa de Mejoramiento de la Calidad y Equidad de la Educación, and was supported by Comisión Nacional de Investigación Científica y Tecnológica grant 24090139