A biochemical mechanism for Stu2/XMAP215-family microtubule polymerases
- Department of Biophysics, University of Texas Southwestern Medical Center, United States
- Department of Biochemistry, University of Texas Southwestern Medical Center, United States
Figures
Figure 1
Similarities between microtubule and actin polymerases suggest a shared mechanism.
(A) Tumor overexpressed gene (TOG)-based microtubule polymerases catalyze faster microtubule growth. (B) Schematic cartoons showing how the number of TOG domains and oligomerization state of TOG-based polymerases can vary in different organisms. Box: A biochemical understanding to explain how polymerase activity results from the number of TOG domains and the rate constants governing their interactions with tubulin has not been established. (C) The microtubule polymerase Stu2 and the actin polymerase Ena/VASP may use a common mechanism: both polymerases are oligomers that contain subunit (tubulin or actin) binding domains connected by flexible linkers. (D) Cartoon illustrating an enzyme-like mechanism (Breitsprecher et al., 2011) that quantitatively explains Ena/VASP activity in terms of the number of subunit binding domains (N), the rate constants governing their interactions with actin (kon, koff, KD), and a transfer rate constant kt that describes how fast the bound subunit is added to the polymer end. Equation: relationship between the Ena/VASP-mediated growth rate (V) and the number and biochemical properties of the actin-binding domains (Breitsprecher et al., 2011), annotated to indicate its similarity to the Michaelis-Menten equation.
Figure 2
Stu2 polymerase activity at different tubulin concentrations follows Michaelis-Menten kinetics, consistent with the biochemical model.
(A) Domain organization of Stu2 constructs used in this work. (B) Representative kymographs from in vitro measurements of microtubule dynamics using the indicated constructs. TOG1* indicates a non-binding mutant (R200A mutation) that was used to vary the number of tubulin-binding tumor overexpressed gene (TOG) domains in the polymerase. (C) Average microtubule (MT) growth rates from assays containing 0.8 µM yeast tubulin, without (No Stu2) or with 100 nM of the indicated Stu2 variant. Error bars represent SEM, n=25 for all. The dotted horizontal gray line shows the growth rate in control; vertical arrows indicate the Stu2-mediated growth rate, obtained by subtracting the measured ‘No Stu2’ growth rate. (D) Stu2-mediated growth rates measured at multiple tubulin concentrations (filled circles), fit to the Michaelis-Menten equation. Error bars represent SEM, n=25 for all. The resulting Vmax and KM are shown in the inset table.
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Figure 2—source data 1
The file contains the numerical values corresponding to Figure 2C.
- https://cdn.elifesciences.org/articles/107818/elife-107818-fig2-data1-v1.xlsx
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Figure 2—source data 2
The file contains numerical values corresponding to the points in Figure 2D.
- https://cdn.elifesciences.org/articles/107818/elife-107818-fig2-data2-v1.xlsx
Figure 3
The amount of Stu2 at the microtubule end is independent of microtubule growth rate.
(A) Representative kymographs for measurements from assays containing 100 nM Stu2(TOG2-TOG2) at the indicated tubulin concentrations (0.6 and 1.4 µM), monitored by total internal reflection fluorescence (TIRF) microscopy. A minimum of two to three ‘comet’ intensities were measured using intensity line scans as indicated for each growth episode. (B) The fluorescence intensity at the microtubule end does not differ significantly (p=0.83; unpaired t-test with Welch’s correction) at low (0.6 μM, growth rate 21.8 subunits/s; n=30) and high (1.4 μM, growth rate 40.7 subunits/s; n=42) tubulin concentration. Similar fluorescent intensity values at both low and high tubulin concentrations indicate that the amount of Stu2 on the growing microtubule end does not vary with tubulin concentration. Bars indicate SD.
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Figure 3—source data 1
The file contains numerical values for the data plotted in Figure 3B.
- https://cdn.elifesciences.org/articles/107818/elife-107818-fig3-data1-v1.xlsx
Figure 4
Rate constants governing tumor overexpressed gene (TOG):tubulin interactions under similar conditions to where polymerase activity was measured.
(A) Schematic of site-specific, sortase-mediated biotinylation of tubulin (see Materials and methods). Yeast tubulin containing a sortase recognition sequence (LPETGG) at the C-terminus of β-tubulin was expressed and purified before reacting it with Sortase A and a separately prepared GGGC-Biotin peptide. (B) Sensograms from bio-layer interferometry measurements of different concentrations of TOG2 interacting with biotinylated tubulin immobilized on the sensor. Inset table: on- and off-rate constants obtained from globally fitting a 1:1 model to the data. (C) Steady-state response for TOG2:tubulin binding plotted vs TOG2 concentration (filled circles) and fit to a 1:1 binding model (line). No response was observed for TOG2*, a mutant that does not bind tubulin. The dissociation constant obtained from the steady-state analysis (KD = 9 nM) is consistent with the one calculated from kinetic measurements (KD = koff/kon = 0.0031/3.2=10 nM) and indicates a higher TOG:tubulin affinity than expected from prior measurements in different buffer conditions.
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Figure 4—source data 1
The file contains numerical values for the data plotted in Figure 4C.
- https://cdn.elifesciences.org/articles/107818/elife-107818-fig4-data1-v1.xlsx
Figure 5
Biochemical insights into the polymerase mechanism.
The cartoon summarizes the main insights obtained from the biochemical measurements and model. Only one polymerase is shown for clarity, and the plus-end specificity of the polymerase results from the polarity of tumor overexpressed gene (TOG):tubulin engagement and how they are placed relative to a basic region (not shown) that contributes lattice-binding affinity (Ayaz et al., 2012; Geyer et al., 2018). (A) Overview of the polymerase mechanism, illustrating the individual reactions: association and dissociation of TOG:tubulin complexes (horizontal arrows), ‘transfer’ of a TOG-bound tubulin to the microtubule end (solid vertical arrows), and uncatalyzed (polymerase-independent) association of unpolymerized tubulin with the microtubule end (dashed vertical arrow). Polymerase engagement with the microtubule end results from a combination of TOG binding to a microtubule-end-resident tubulin and the basic region. Only one polymerase is shown for simplicity. Arrow lengths reflect the relative rates of different steps but are not to scale. Subsequent panels are vignetted to emphasize different aspects of the mechanism. (B) The polymerase operates with high efficiency because the rate at which TOG-bound tubulins are transferred to the microtubule end (vertical downward arrow) greatly exceeds the rate at which TOG:tubulin complexes dissociate (horizontal leftward arrow). The polymerase is kinetically limited because the slowest step in the polymerase mechanism is tubulin binding to an ‘empty’ TOG (horizontal rightward arrow). (C) The polymerase can achieve substantial rate enhancement because each TOG-bound tubulin is transferred to the microtubule end at a rate that is comparable to the uncatalyzed one (vertical downward arrows), and there can be multiple TOGs from multiple polymerases participating.
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A biochemical mechanism for Stu2/XMAP215-family microtubule polymerases
eLife 14:RP107818.
https://doi.org/10.7554/eLife.107818.3
