Tubulin Binding and Polymerization Activity of +TIP Domains

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Tubulin Binding by Gel Filtration

Prior to initiating structural studies, we first sought to express and define regions of +TIP proteins that interact with tubulin.  For the XMAP215 family, sequence homology reveals a single common domain (termed the TOG domain) that is conserved across species (Andrade et al., 2001).  This domain is repeated twice in the S. cerevisiae homolog Stu2p, while higher eukaryotes have five arrayed TOG domains (Figure 1A).  Sequence analysis reveals that TOG domains can be further subclassified into two or possibly three types (termed A, B, and C TOG domains)(Figure S2).  The TOG domain types alternate in the polypeptide: for higher eukaryotes TOG domains 1 and 3 are type A, TOG domains 2 and 4 are type B, and TOG domain 5 is type C (mostly closely related to type A). The predicted pI of TOG domain A is negative at physiological pH, while type B displays a net positive charge.  C-terminal to the TOG domains, yeast Stu2p has a predicted coiled coil followed by a basic region that has been shown to bind to microtubules (Nakaseko et al., 2001), while higher eukaryotic homologues have an unique conserved C-terminal domain also implicated in microtubule association (Popov et al., 2001).  In contrast to Stu2p which dimerizes, higher eukaryotic family members are monomeric (Al-Bassam et al., 2006; Graf et al., 2000; van Breugel et al., 2003). 

We expressed the TOG1 and 2 domains from the Drosophila XMAP215 homolog (termed Mini spindles, Msps) and yeast Stu2p and tested whether they formed a complex with a/b tubulin heterodimers by co-elution in gel filtration chromatography.  Neither Msps TOG1 or TOG2 alone or added in trans formed a complex with tubulin, although a tandem TOG1-2 construct interacted with tubulin in this assay (Figures 1B and E; Figures S1A and B) corroborating preexisting evidence of an N-terminal tubulin binding domain in the XMAP215 family (Spittle et al., 2000).  Analysis of the shifted TOG1-2:tubulin peak via gel filtration and dynamic light scattering indicated a mass of 121 kDa,  which is less than the expected complex value of 167 kDa and suggests that the complex dissociates to some extent during the gel filtration run (Figures 1B and E; Figure S1B).  In contrast to individual Msps TOG domains, single TOG1 and TOG2 domains from Stu2p interacted with a/b tubulin heterodimers (Figure 1E; Figures S1D and E).  When TOG1 and TOG2 were added in trans in the presence of tubulin, the shift was identical to that produced by a single Stu2p TOG domain with tubulin, indicating that TOG1 and TOG2 were competing for identical or overlapping sites on a/b tubulin (Figure 1E; Figures S1D-H).  The Stu2p TOG1-2 construct was insoluble in E. coli and thus precluded further investigation.  The co-elution of Stu2p TOG1 with tubulin agrees with findings of Al-Bassam et al., but in contrast to our result, TOG2 did not bind to tubulin in the Al-Bassam study (Al-Bassam et al., 2006). 

The EB1 family of plus end binding proteins is characterized by an N-terminal calponin homology (CH) domains, a flexible linker region and a C-terminal dimerization/cargo recruitment domain composed of a coiled coil and four helix bundle (Figure 1A) (Honnappa et al., 2005; Slep et al., 2005).  Expressed CH domains (both single and dimerized) from human EB1 and yeast Bim1p did not produce a shift in tubulin elution in the gel filtration assay, suggesting that they do not interact strongly with tubulin (Figures 1C and E; Figures S1I-L).  These results agree with other in vitro binding assays with tubulin monomers (Niethammer et al., 2007), although other experiments suggest direct interactions between EB1 and microtubules (Hayashi and Ikura, 2003) and evidence for a microtubule lattice seam interaction has been obtained for the S. pombe homolog Mal3p (Sandblad et al., 2006).

The CLIP-170 family of +TIP proteins is characterized by an N-terminal, conserved Cap-Gly domain (one in yeast and two in higher eukaryotes), a long central coiled-coil, and C-terminal, conserved zinc-finger motifs utilized for cargo attachment (Figure 1A) (Pierre et al., 1992). Functional plus end tracking has been reported for a monomeric construct lacking the dimerization domain, but containing both tandem Cap-Gly domains (Pierre et al., 1994).  We found that both a single Cap-Gly domain (CLIP-170_57-210) and the tandem Cap-Gly domains from human CLIP-170 (CLIP-170_1-350) formed a clear complex with tubulin by gel filtration.  However, the distinct elutions for CLIP-170_57-210 versus CLIP-170_1-350 suggest that tubulin forms a 1:1 and a 2:1 complex with these two constructs respectively.  Thus tandem Cap-Gly domains may be capable of multimerizing tubulins (Figures 1D and E, Figures S1M), consistent with previously reported sedimentation behavior of CLIP-170 and tubulin (Arnal et al., 2004; Diamantopoulos et al., 1999).

In summary, the gel filtration tubulin binding studies indicate that TOG and Cap-Gly domains interact directly with a/b tubulin but with varying affinities dependent upon the specific species and the number of tubulin binding domains arrayed or homodimerized.  EB1 and Bim1p, in contrast, have sufficiently low affinity for a/b tubulin heterodimers so as to preclude measurement by gel filtration binding studies.  However, these results do not rule out a direct interaction of these domains with tubulin in solution or in the microtubule.

 Microtubule Nucleation Activity

To investigate functional roles of the +TIP tubulin binding domains, we examined their effects on tubulin polymerization in vitro using turbidity and microscopy as readouts of microtubule formation.  We compared and contrasted the effect of single +TIP domains versus tandem domains or artificially homodimerized (fusion to Glutatione S-Transferase (GST) or the GCN4 leucine zipper (LZ) motif) domains.

Msps TOG1-2, the minimum domain that binds to tubulin by gel filtration assays, failed to promote microtubule nucleation (Figure 2A). In contrast, GST-Msps TOG1-2 and an arrayed construct, Msps TOG1-2-1-2, potently promoted microtubule nucleation, greatly reducing the minimal lag time and increasing polymer mass (Figure 2A and D; note, TOG1-2-3-4 could not be expressed in bacteria).  Single Stu2p TOG domains failed to promote microtubule polymerization, and the addition of Stu2p TOG1 was even slightly inhibitory (Figure 2A).  EB1 CH domain constructs including native homodimer (EB1_FL), monomeric (EB1_1-133) and a truncated homodimer (EB1_12-255, which lacks a potential autoinhibitory tail sequence and a non-conserved N-terminal segment) failed to affect nucleation rates (Figure 2B).  Similar behavior was noted with monomeric Bim1p (Bim1p_1-187); however the homodimerized counterpart (Bim1p_1-187-LZ) potently promoted microtubule nucleation (Figures 2B and D).  Analysis of CLIP-170 constructs showed that a single Cap-Gly domain (CLIP-170_57-210) caused slight inhibition of microtubule nucleation, but the homodimerized counterpart (GST-CLIP-170_57-210) or a construct containing tandem Cap-Gly domains 1 and 2  (CLIP-170_1-350) promoted microtubule formation (similar to findings by Arnal et al. (Arnal et al., 2004) using CLIP-170_1-481) albeit with a lag time similar to that observed for tubulin alone (Figures 2C and D).  Microscopic analysis (Figure 2D) reveals that +TIPs, particularly CLIP-170, also induce microtubule bundling in vitro, an effect we note will augment the bulk turbidity readings in Figures A-C beyond a comparable concentration of non-bundled microtubules.  In summary, our results show that a single +TIP domain has no effect or an inhibitory effect on microtubule nucleation/polymerization, while multimerized domains strongly promote polymerization and often nucleation, with human EB1 being the only exception.  Multimerized +TIP domains that promoted microtubule polymerization also promoted polymerization at tubulin’s in vitro critical concentration (data not shown).