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• W2984467720 abstract "Article Figures and data Abstract eLife digest Introduction Results and discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Stu2p/XMAP215 proteins are essential microtubule polymerases that use multiple αβ-tubulin-interacting TOG domains to bind microtubule plus ends and catalyze fast microtubule growth. We report here the structure of the TOG2 domain from Stu2p bound to yeast αβ-tubulin. Like TOG1, TOG2 binds selectively to a fully ‘curved’ conformation of αβ-tubulin, incompatible with a microtubule lattice. We also show that TOG1-TOG2 binds non-cooperatively to two αβ-tubulins. Preferential interactions between TOGs and fully curved αβ-tubulin that cannot exist elsewhere in the microtubule explain how these polymerases localize to the extreme microtubule end. We propose that these polymerases promote elongation because their linked TOG domains concentrate unpolymerized αβ-tubulin near curved subunits already bound at the microtubule end. This tethering model can explain catalyst-like behavior and also predicts that the polymerase action changes the configuration of the microtubule end. https://doi.org/10.7554/eLife.03069.001 eLife digest Dynamic filaments of proteins, called microtubules, have several important roles inside cells. Microtubules provide structural support for the cell; they help to pull chromosomes apart during cell division; and they guide the trafficking of proteins and molecules across the cell. The building blocks of microtubules are proteins called αβ-tubulin, which are continually added to and removed from the ends of a microtubule, causing it to grow and shrink. Other proteins that interact with the microtubules can help to speed up these construction and deconstruction processes. Ayaz et al. took a closer look at the structure of one particular family of proteins that make it easier for the microtubules to grow, using a technique called X-ray crystallography. The resulting images show two sites—called TOG1 and TOG2—on the enzymes that attach to the αβ-tubulin proteins. Ayaz et al. found that this binding can only occur when αβ-tubulin has a curved shape, which only happens when the tubulins are not included in, or are only bound weakly to the end of, a microtubule. Previous research suggested that the two binding sites might work together to provide ‘scaffolding’ that stabilizes the microtubule. However, genetic experiments by Ayaz et al. show that microtubules will grow even if one of the binding sites is missing. Both TOG1 and TOG2 bind to αβ-tubulin in the same way, and by using computer simulations Ayaz et al. found that this helps to speed up the growth of microtubules. This is because the enzyme's two sites concentrate the individual tubulin building blocks at the ends of the filament. For example, TOG2 could bind to the end of the microtubule, while TOG1 holds an αβ-tubulin protein nearby and ready to bind to the filament's end. This tethering allows the microtubules to be assembled more efficiently. https://doi.org/10.7554/eLife.03069.002 Introduction Microtubules are dynamic polymers of αβ-tubulin that have critical roles in chromosome segregation and intracellular organization (reviewed in Desai and Mitchison, 1997). The polymerization dynamics of microtubules are regulated by multiple cellular factors. Evolutionarily conserved proteins in the Stu2p/XMAP215 family (Gard and Kirschner, 1987; Ohkura et al., 1988; Wang and Huffaker, 1997) regulate microtubule dynamics by promoting fast microtubule elongation. These essential proteins use multiple αβ-tubulin binding tumor overexpressed gene (TOG) domains to selectively recognize the growing microtubule end and promote its elongation (Al-Bassam et al., 2006; Brouhard et al., 2008; Widlund et al., 2011; Al-Bassam et al., 2012). A significant advance in understanding occurred with a landmark study of XMAP215 in which in vitro reconstitution assays demonstrated that the polymerase affected the rate of microtubule elongation without affecting the apparent equilibrium (Brouhard et al., 2008). This and other observations (e.g., Shirasu-Hiza et al., 2003; van Breugel et al., 2003) formed the basis for describing XMAP215 as a catalyst for microtubule elongation. Catalytic action in turn led to the concept that TOG-containing polymerases might stabilize an otherwise rate-limiting intermediate along the polymerization pathway (Brouhard et al., 2008). However, an understanding of mechanism has been limited because the nature of this intermediate, and how TOG domains might selectively promote it, remained unclear. A recent study from our group revealed that the TOG1 domain from Stu2p binds preferentially to a ‘curved’ conformation of αβ-tubulin that cannot be incorporated into the body of the microtubule (Ayaz et al., 2012). Our study also showed that a TOG1-TOG2 construct could bind two αβ-tubulins (Ayaz et al., 2012). This latter observation suggested that two TOG domains might cooperate to stabilize an αβ-tubulin:αβ-tubulin interface, and consequently that cooperative binding to αβ-tubulin might contribute to polymerase activity. Our study did not determine how the polymerase recognizes the extreme microtubule end, but we speculated based on apparent biochemical differences between the TOG1 and TOG2 domains (Al-Bassam et al., 2006; Ayaz et al., 2012) that selective interactions between TOG2 and a different, end-specific conformation of αβ-tubulin might be important. In the present study we sought to gain insight into the mechanism of end recognition by determining the conformation of αβ-tubulin recognized by TOG2 and by testing whether cooperative TOG:αβ-tubulin interactions contributed to polymerase activity. We first determined the crystal structure of a TOG2:αβ-tubulin complex. This structure reveals that TOG2 binds to the same curved conformation of αβ-tubulin that TOG1 does. Our biochemical experiments underscore this structural similarity by demonstrating that the two TOG domains have comparable affinities for αβ-tubulin, with KD ∼100 nM. Next, we used analytical ultracentrifugation to show that in TOG1-TOG2 the two linked TOG domains bind non-cooperatively to two αβ-tubulins. Non-cooperative binding indicates that TOG1-TOG2 does not stabilize an αβ-tubulin:αβ-tubulin interface. Together with biochemical and genetic experiments, our results lead to a model that explains how the polymerase activity can emerge from the action of two tethered TOG domains that each bind independently to a conformation of αβ-tubulin that is incompatible with the microtubule lattice. We propose that the polymerase activity arises because linked TOG domains selectively increase the effective concentration of αβ-tubulin near weakly bound, curved αβ-tubulins already on the microtubule end. A computational realization of this model supports our proposal by recapitulating catalyst-like behavior. The model further suggests that the polymerase achieves its effect in part by transiently altering the configuration of the growing end. Results and discussion TOG2, like TOG1, binds curved αβ-tubulin We previously showed that the TOG1 domain from Stu2p binds preferentially to a curved conformation of αβ-tubulin (Ayaz et al., 2012). However, TOG1 is dispensable for the plus-end binding of Stu2p (Al-Bassam et al., 2006), and because of apparent differences in the biochemical behavior of TOG1 and TOG2 (Al-Bassam et al., 2006; Ayaz et al., 2012) we speculated that TOG2 might bind to a different, lattice-induced conformation of αβ-tubulin (Ayaz et al., 2012). We solved the crystal structure of a TOG2:αβ-tubulin complex (Figure 1) to resolve this ambiguity. The structure was determined by molecular replacement from crystals that diffracted anisotropically to a minimum Bragg spacing of 2.8 Å (Table 1). The final model has good geometry (Table 1; Molprobity [Chen et al., 2010] clash score 1.79; 95.6% favored residues in Ramachandran plot) and has been refined to an Rfree of 0.259 (Rwork = 0.217). Figure 1 Download asset Open asset TOG2 binds to curved αβ-tubulin analogously to TOG1. (A) Structure of the TOG2:αβ-tubulin complex (TOG2: slate, α-tubulin: pink, β-tubulin: green), with the important binding residues W341 and R519 represented as spheres. The semi-transparent gray cartoon shows the previously observed binding mode of TOG1, with its binding residues W23 and R200 depicted as spheres. (B) Close-up of the TOG2:αβ-tubulin interface, colored as in A, and showing in spheres important interacting residues based on an earlier study. (C) Structural superposition of αβ-tubulin-bound (slate) and unbound (gray) TOG2 (PDB 2QK1). The two structures of TOG2 show only small local deviations, arguing against any significant conformational change associated with αβ-tubulin binding. (D) Structural superposition of the αβ-tubulin conformations in the TOG2 (colored) and TOG1 (gray) complexes. In both complexes αβ-tubulin adopts very similar curved conformations. (E) Structural superposition of TOG2-bound αβ-tubulin (colored) and the straight conformation of αβ-tubulin (PDB 1JFF, gray), showing the ∼13° rotation of β-tubulin relative to α-tubulin that is characteristic of the curved conformation. https://doi.org/10.7554/eLife.03069.003 Table 1 Data collection and refinement statistics https://doi.org/10.7554/eLife.03069.004 Data collection Space groupC2 Cell dimensions a, b, c (Å)111.91, 89.57, 135.51 β (°)112.31 Resolution (Å)50.0–2.81 (2.92–2.81)* Rsym0.143 (0.924) <I>/<σI>9.6 (1.1) Wilson B-value (Å)48.9 Anisotropy (Å) relative to best direction (001) ΔB in (100) direction, ΔB in (010) direction+29.95, +8.38 CC1/2 in high resolution shell0.542 Completeness (%)98.2 (91.3) Redundancy4.1 (3.2)Refinement Resolution (Å)2.81 No. reflections26,235 Completeness(%)86.5† (35.3) Rwork/ Rfree (%)21.8/25.9 (33.0/41.3) Maximum likelihood estimated coordinate error (Å)0.42 No. atoms8524 Protein (non-hydrogen)8437 Ligand/ion66 Water21 B-factors Protein44.7 Ligand/ion52.0 Water27.3 Rms deviations Bond lengths (Å)0.003 Bond angles (°)0.66 Ramanchandran plot Favored (%)95.0 Allowed (%)4.25 Disallowed (%)0.75 Rotamer outliers (%)3.2 Molprobity clash score1.5 * Highest resolution shell is shown in parenthesis. † The data were corrected for anisotropy in HKL2000. This treatment eliminated weak reflections and reduced the completeness of the data used for refinement compared to the completeness reported for data collection. The structure of the TOG2:αβ-tubulin complex is remarkably similar to that of the TOG1:αβ-tubulin complex (Figure 1; Ayaz et al., 2012). Conserved residues like W341 and R519 on the tubulin-binding surface of TOG2 make very similar contacts with αβ-tubulin as their equivalents in TOG1 (W23 and R200, respectively) (Figure 1A,B). The structure of TOG2 bound to αβ-tubulin superimposes with the structure of ‘free’ TOG2 (Slep and Vale, 2007) with 0.7 Å rmsd over 237 Cα atoms (Figure 1C). Because the TOG2 domains pack differently in the ‘bound’ and ‘free’ crystals, the similarity in structure suggests that these TOG domains are rigid modules that do not change conformation when they bind αβ-tubulin. Even though the structure reported here was obtained from a new crystal form, the conformation of αβ-tubulin in complex with TOG2 is nearly identical to that seen for TOG1-bound αβ-tubulin (Figure 1D) (12.3° of curvature in the TOG2 complex vs 13.1° in the TOG1 complex), and is characteristically distinct from the straight conformation of αβ-tubulin (1° of curvature by our measure) (Löwe et al., 2001; Figure 1E). Underscoring this similarity, individual tubulin chains between the TOG1 and TOG2 complexes superimpose on each other with 0.4 Å rmsd over Cα atoms. Thus, both TOG1 and TOG2 bind preferentially to the same, curved conformation of αβ-tubulin. By extension, other TOGs in this family probably also bind to curved αβ-tubulin. The shared preference of TOG1 and TOG2 for curved αβ-tubulin has implications for the mechanism of end recognition. Because TOG:αβ-tubulin interactions are required for plus-end localization of Stu2p/XMAP215 polymerases (Al-Bassam et al., 2006), preferential binding of TOGs to curved αβ-tubulin suggests that curved αβ-tubulin itself is the distinctive end-specific feature the polymerase recognizes. In contrast, other plus-end tracking proteins like Eb1 recognize lattice-specific features and consequently show ‘comet-like’ localization that extends into the microtubule body (Nakamura et al., 2012; Maurer et al., 2014). By invoking binding to an epitope that cannot exist in the body of the microtubule, our model explains how Stu2p/XMAP215 polymerases localize to the extreme microtubule end (Nakamura et al., 2012; Maurer et al., 2014). The shared preference for fully curved αβ-tubulin also poses an apparent paradox, because it indicates that the polymerase is constructed from ‘parts’ that bind most strongly to a conformation of αβ-tubulin that cannot exist in the microtubule lattice. We explore and propose a resolution for this apparent contradiction in later sections. TOG1 and TOG2 each bind αβ-tubulin with comparable affinity To determine if the structural similarity between the TOG1 and TOG2 complexes with αβ-tubulin extends to a biochemical similarity, we measured the affinity of TOG:αβ-tubulin interactions. Previously we had used polymerization-blocked αβ-tubulin mutants (Johnson et al., 2011) for TOG binding assays. To eliminate the possibility that blocking mutations and/or fluorescent labeling might mask or alter αβ-tubulin:αβ-tubulin interactions, we developed a label-free assay in which polymerization-competent αβ-tubulin could be used. Analytical ultracentrifugation monitored by absorbance at 230 nm (A230) allowed us to work at concentrations of αβ-tubulin at which higher-order oligomers of αβ-tubulin were nearly undetectable (Figure 2A). Indeed, at αβ-tubulin concentrations ranging from 0.08 to 1 μM there is a single dominant peak at 5.8 S with only 1.7 and 2.8% of the material sedimenting faster at the lowest and highest concentration tested. This uniform sedimentation behavior made it possible for us to use analytical ultracentrifugation as a quantitative binding assay. Figure 2 Download asset Open asset TOG1 and TOG2 bind αβ-tubulin with comparable affinity. (A) Sedimentation velocity analytical ultracentrifugation of polymerization competent yeast αβ-tubulin does not show signs of self-association between 80 nM and 1 μM concentration. The main plot shows c(s) distributions for a range of αβ-tubulin concentration. The inset shows that the s20,w is not increasing with αβ-tubulin concentration. Data points are color coded to match the c(s) distribution for that concentration. c(s), signal population as a function of s; s20,w, sedimentation coefficient standardized to pure water and 20°C. (B) Analysis of TOG1:αβ-tubulin interactions by sedimentation velocity. The main plot shows c(s) distributions (color coded by concentration) for seven concentrations of TOG1 (44 nM–4.5 μM) titrated into 0.35 μM αβ-tubulin. The inset shows the fit (gray line) of a 1:1 binding isotherm to the signal average sfast (dots colored to match the c(s) distribution for that concentration of TOG1) resulting in a dissociation constant of 70 nM. (C) Analysis of TOG2:αβ-tubulin interactions by sedimentation velocity. Plots are as described in (B). The fitted dissociation constant is 160 nM. https://doi.org/10.7554/eLife.03069.005 We measured the affinity of TOG1 and TOG2 for αβ-tubulin by separately titrating variable amounts of each TOG domain into a constant amount of αβ-tubulin and analyzing the resulting sedimentation behavior (Figure 2B,C). We fit the concentration-dependent sedimentation profiles using single-site binding isotherms (Dam and Schuck, 2005; Figure 2B,C). The fits indicate that both TOG domains form relatively tight, 1:1 complexes with αβ-tubulin: TOG1 binds with KD = 70 nM and TOG2 binds with KD = 160 nM. The KD for the TOG2:αβ-tubulin complex reported here is consistent with our prior measurement using fluorescence anisotropy and polymerization blocked αβ-tubulin (Ayaz et al., 2012). Thus, in addition to a shared preference for the same curved conformation of αβ-tubulin, TOG1 and TOG2 also bind αβ-tubulin with comparable affinities. From both a biochemical and a structural perspective, the TOG1 and TOG2 domains are remarkably similar to each other. TOG1-TOG2 binds noncooperatively to two αβ-tubulins When present together in a TOG1-TOG2 construct, each of the TOG domains can engage its own αβ-tubulin (Ayaz et al., 2012). We speculated that the two TOG-bound αβ-tubulins might also interact with each other, and that this cooperativity might make important contributions to polymerase function (Figure 3A). Figure 3 Download asset Open asset In TOG1-TOG2, the two TOG domains bind two αβ-tubulins without positive cooperativity. (A) Cartoons illustrating three different possible arrangements of a TOG1-TOG2:(αβ)2 complex: independent (left) denotes that an αβ-tubulin:αβ-tubulin interface does not provide additional stability to the complex, cooperative (right) denotes that an αβ-tubulin:αβ-tubulin interface (either longitudinal or lateral) provides additional stability to the complex. Predicted sedimentation coefficients (calculated using HYDROPRO [García De La Torre et al., 2000]) are indicated. αβ-tubulin is represented in pink and green, TOG1-TOG2 in shades of blue. (B) Sedimentation behavior of a TOG1-TOG2:(αβ)1 complex by sedimentation velocity AUC using two different mutations (W314A and R519A) that impair TOG2:αβ-tubulin interactions. The ‘one tubulin’ complex sediments at 7.1 S. (C) Placing limits on the sedimentation behavior of a TOG1-TOG2:(αβ)2 complex by sedimentation velocity AUC. At ∼5 molar equivalents of αβ-tubulin to TOG1-TOG2, the resulting complex sediments at 9.1 S. The inset shows the predicted fraction of TOG1-TOG2 engaged in ‘two αβ-tubulin complex’ under different assumptions about cooperativity. (D) Concentration dependence of TOG1-TOG2:αβ-tubulin interactions. Seven concentrations of TOG1-TOG2 were mixed with 0.3 μM αβ-tubulin and analyzed by sedimentation velocity AUC. Red dots indicate the signal-weighted sw values for the seven runs. The blue and pink swaths show the predicted behavior for TOG1 and TOG2 binding αβ-tubulin with 10-fold positive or negative cooperativity, respectively, and assuming the sedimentation coefficient of TOG1-TOG2:(αβ)2 falls in the range 9.1–10.9 S (see text). The gold swath shows the predicted behavior for noncooperatively binding TOGs using the same range of sedimentation coefficient for TOG1-TOG2:(αβ)2. The data are not consistent with cooperative binding of TOG1-TOG2 to two αβ-tubulins. Instead, they are much better described by independently binding TOG domains. https://doi.org/10.7554/eLife.03069.006 We used analytical ultracentrifugation to determine if cooperative interactions stabilized the TOG1-TOG2:(αβ-tubulin)2 complex. First, using mutations on TOG1-TOG2 that disrupt the slightly weaker TOG2:αβ-tubulin interactions, we determined that TOG1-TOG2 bound to a single αβ-tubulin sediments at 7.1 S (Figure 3B). Next, using a mixture in which αβ-tubulin was superstoichiometric with respect to TOG1-TOG2 (Figure 3C), we observed a larger species sedimenting at 9.1 S. This value places a lower limit on the sedimentation coefficient for the TOG1-TOG2:(αβ-tubulin)2 complex (Figure 3C, inset) that is consistent with hydrodynamic calculations (García De La Torre et al., 2000) using extended models in which the two αβ-tubulins do not contact each other and the TOG domains are separated by varying distances. Hydrodynamic calculations predict that compact models containing longitudinally or laterally of αβ-tubulins should sediment around 10.5–10.9 S (Figure 3A,C). Knowledge about the sedimentation behavior of the one and two tubulin complexes allowed us to analyze a titration of TOG1-TOG2 into αβ-tubulin under different assumptions about cooperativity (Figure 3D). The resulting signal weighted average sedimentation coefficients are not consistent with models that assume even modest 10-fold positive or negative cooperativity (Figure 3D). Instead, the data are much better described by a model in which each TOG domain in TOG1-TOG2 interacts independently with its αβ-tubulin (the best fit was obtained from a model invoking less than twofold negative cooperativity, not shown). Noncooperative binding contradicts our initial expectation and indicates that αβ-tubulin:αβ-tubulin contacts do not provide additional stability to the TOG1-TOG2:(αβ-tubulin)2 complex. The lack of positive cooperativity stabilizing the TOG1-TOG2:(αβ-tubulin)2 complex is striking because in this complex the two bound αβ-tubulins are physically constrained to occupy a relatively small volume and therefore are effectively at quite high concentration relative to each other. Indeed, allowing 55 Å for the length of each TOG domain and assuming that the ∼75 amino acid linker is flexible and can maximally span 220 Å we estimate that the two TOG1-TOG2-bound αβ-tubulins are minimally at an effective concentration of roughly 200 μM, likely higher because the linker will rarely be fully extended. This effective concentration of TOG1-TOG2-bound αβ-tubulin is in the range of current estimates for the longitudinal KD for αβ-tubulins (e.g., Gardner et al., 2011). Observing non-cooperative binding therefore suggests that some property of the TOG1-TOG2 linker antagonizes or counterbalances longitudinal interactions between αβ-tubulins (see below for experiments concerning the linker sequence). Our data do not rule out that linked TOG domains might stabilize lateral interactions between αβ-tubulins because these lateral interactions are thought to be much weaker (only molar affinity [Gardner et al., 2011]): even at effective αβ-tubulin concentrations approaching 1 mM a weak interface like this would not be populated and therefore would contribute little additional stability to the TOG1-TOG2:(αβ-tubulin)2 complex. Whatever the underlying mechanism, the unexpected observation that the two linked TOG domains behave as independent αβ-tubulin binding modules places significant constraints on biochemical models for the mechanism of these polymerases. Two TOG domains are required for Stu2p function, but they do not have to be different Stu2/XMAP215 family polymerases contain at least two different TOG domains. This property might indicate that the two domains have different functional specialization, but the structural and biochemical similarity of TOG1 and TOG2 (described above) does not seem consistent with separation of function. To investigate if different TOG domains are required for polymerase function we constructed a ‘TOG-swapped’ and other variants of Stu2p and assayed their ability to rescue the conditional depletion of endogenous Stu2p using a previously described assay (Kosco et al., 2001; Al-Bassam et al., 2006; Ayaz et al., 2012). We first performed rescue assays using a ‘full-length’ Stu2p construct that retains the ability to dimerize and thus that contains two identical TOG1-TOG2 segments linked through the coiled-coil dimerization interface. A construct (TOG2-TOG2) in which TOG1 was replaced by a second copy of TOG2 rescued as well as did the wild-type (TOG1-TOG2) (Figure 4A). We also tried to replace TOG2 with a second copy of TOG1, but control experiments indicated that this variant was not stable (data not shown) and we have not yet pursued it further. Instead, we used site-directed mutagenesis as an alternate way to ablate the αβ-tubulin binding activity of TOG1 or TOG2. Constructs in which either TOG was impaired for αβ-tubulin binding (TOG1(R200A)-TOG2 or TOG1-TOG2(R519A)) also gave full rescue (Figure 4A). Ablating the function of individual TOGs is not without functional consequences under more stringent conditions, because these same constructs do show compromised rescue under conditions of microtubule stress (Ayaz et al., 2012). These data indicate that under normal conditions, the polymerase can function with only TOG1 or only TOG2 domains. Thus, having different TOG domains does not appear to be essential for polymerase function. Figure 4 Download asset Open asset Two TOG domains are required for Stu2 function, but they do not have to be different. (A) Yeast carrying plasmid-based rescue constructs coding for dimerization-competent variants of Stu2p were plated at serial dilutions on media that was unmodified (control) or that contained 500 μM CuSO4 (to deplete endogenous Stu2p; see text). All constructs, including those with debilitated TOG1 or TOG2 domains, showed full rescue. TOG domains are shown in blue, and the basic region in red. The coiled-coil is cartooned as a zipper. (B) As in A but using rescue constructs that are dimerization-impaired because the coiled-coil dimerization domain was deleted. In this more stringent background insults to either TOG domain abolished rescue activity. Replacing TOG1 with a second copy of TOG2 does not have adverse effects. https://doi.org/10.7554/eLife.03069.007 Using dimeric rescue constructs did not allow us to test Stu2p variants that contained only a single functional TOG domain of either kind. We therefore introduced the same mutations into a previously characterized dimerization-impaired variant of Stu2p in which the coiled-coil dimerization element had been deleted (Stu2p-Δcc) (Al-Bassam et al., 2006; Figure 4B). Consistent with prior observations (Al-Bassam et al., 2006), dimerization impaired Stu2p only partially compensated for the depletion of endogenous Stu2p. Even in this sensitized background, however, we found that as long as there were two functional TOG domains they did not need to be different. Stu2p(TOG2-TOG2)-Δcc showed rescue efficiency very similar to that of Stu2p(TOG1-TOG2)-Δcc (Figure 4B). Dimerization impaired variants of Stu2p in which TOG1 or TOG2 was defective for αβ-tubulin binding did not show any rescue activity (Figure 4B), consistent with a prior in vitro study of XMAP215 that demonstrated a requirement for at least two TOG domains (Widlund et al., 2011). More importantly, the ability of TOG2 to substitute for TOG1 in this more stringent, dimerization-impaired background strengthens the conclusion that the polymerase does not require different TOG domains for its function. Stu2p function tolerates substantial variation in the sequence linking two TOG domains The dimeric Stu2p(TOG1-TOG2(R519A)) and Stu2p(TOG1(R200A)-TOG2) variants rescued the depletion of endogenous Stu2p in spite of the fact that the two functioning TOG domains were linked through the coiled-coil dimerization segment (and in the case of TOG1-TOG2(R519A) with a defective TOG domain in the linking sequence). This result suggested that how two TOG domains were linked was relatively unimportant, as long as they were linked. To explore this more systematically we determined how randomizing and/or shortening the TOG1-TOG2 linker in dimerization impaired Stu2p affected its rescue activity. To test if the primary sequence of the TOG1-TOG2 linker was important for function, we prepared two variants of Stu2pΔcc in which the order of the central 65 amino acids of the TOG1-TOG2 linker (residues 252–316) was randomized (Figure 5A). This ‘shuffling’ strategy preserves the overall amino acid composition but should disrupt any local features specific to the natural sequence. Both shuffled linkers gave rescue activity nearly indistinguishable from the wild-type linker (Figure 5A), consistent with the robust rescue activity of alternatively linked functional TOGs in dimeric Stu2p(TOG1-TOG2(R519A)) and Stu2p(TOG1(R200A)-TOG2) (Figure 4A). Thus, the sequence linking the two functional TOG domains tolerates significant variation. Figure 5 Download asset Open asset Stu2p function tolerates variation in the primary sequence and in the length of the TOG1-TOG2 linker. Rescue assays were performed as in Figure 4, using dimerization-impaired rescue constructs. (A) Stu2p variants with ‘shuffled’ (randomized) linker sequences rescue the depletion of endogenous Stu2p comparably to those with the natural linker. (B) Stu2p function is substantially abolished when the TOG1-TOG2 linker is truncated by 60 amino acids. Smaller truncations only show slightly compromised rescue activity. (C) Histogram illustrating the distribution of TOG1-TOG2 linker lengths in ∼300 orthologs. The distribution shows that the linker length can vary but has a minimum tolerable length of ∼40 amino acids. https://doi.org/10.7554/eLife.03069.008 We also made a series of internal deletions in the TOG1-TOG2 linker to determine if shortening the linker affected rescue activity. Deleting 14 amino acids had little effect on rescue activity (Figure 5B). Rescue activity was mildly compromised by deletions of 22, 32, 40, and 50 amino acids (Figure 5B). Deleting 60 amino acids from the TOG1-TOG2 linker substantially abolished rescue activity (Figure 5B). These data suggest that Stu2p function is compromised when the sequence linking two TOG domains becomes too short. To examine the question of linker length more generally, we analyzed the distribution of TOG1-TOG2 linker length in ∼300 Stu2p/XMAP215 orthologs. This analysis showed that linker lengths below ∼40 amino acids occur very rarely" @default.
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• W2984467720 title "Author response: A tethered delivery mechanism explains the catalytic action of a microtubule polymerase" @default.
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