Structure Determination of Tubulin Binding Domains of +TIPs

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TOG domains from yeast Stu2p and Drosophila Mini spindles

We attempted to crystallize TOG1, TOG2, and TOG1-2 constructs, but only obtained diffraction-quality crystals from the TOG2 domains of yeast Stu2p and Drosophila Msps.  The TOG2 domain from Stu2p crystallized in the space group P2₁2₁2₁ with one molecule in the asymmetric unit.  The structure was determined using multi-wavelength anomalous dispersion (MAD) phasing from Selenomethionine (SeMet)-derivatized protein to a resolution of 1.7 Å.  The TOG2 domain from the Drosophila Msps was crystallized in the space group C222₁ (one molecule per asymmetric unit); the structure was determined to 2.1 Å resolution also using MAD phasing.  Data, phasing and refinement statistics for these and subsequently described structures are presented in Table 1.

The Stu2p and Msps TOG2 are elongated domains (~20 x 30 x 60 Å) formed by six HEAT-like repeats A thru F, each comprised of a pair of parallel helices (Figures 3A and B).  (We denote the helices using the TOG domain number followed by the HEAT-like repeat letter; fragmented helices are denoted numerically by subscripts and the parallel helix is denoted by a prime.)  Only HEAT repeats C and D of Msps TOG2 and C and F of Stu2p form canonical HEAT repeats, in which the first a helix is kinked by 90°, positioning the N-terminal segment of the helix orthogonal to helix a’ (Figure 3D), structurally similar to the third orthogonal helix found in armadillo repeats.  HEAT-like repeat E, particularly of Stu2p, is characterized by a segmented N-terminal a helix we denote as a₁ and a₂ (Figure 3D).  The TOG domain has a small helical twist along the axis of the HEAT-like repeats, compared with the more substantial twist of other HEAT-repeat structures such as importin-b (Cingolani et al., 1999).

Interesting and likely important features of the TOG domain are the structured loops between the helices. The intra-HEAT loops on one face of the molecule (face A) show a higher degree of structural identity between Stu2p and Msps TOG2 (r.m.s.d. of 1.3 Å over 60 mainchain atoms)(Figure 3C) than the loops on the opposite B face (r.m.s.d. of 3.7 Å over 87 homologous mainchain atoms, not factoring the larger Stu2p inserts that occur in face B) and the helices themselves (r.m.s.d. of 2.1 Å over 513 mainchain atoms). The loops in face A also contain the most highly conserved residues among TOG domains from many species (Figure 3E).  Among these are seven highly conserved lysines and an arginine, making the net charge on face A highly positive (Figure 3F, Figure S2).  Also exposed on face A is an invariant tryptophan residue, W292, found in TOG types A and B (type C has a conserved phenylalanine), situated on the intra-HEAT loop between a2A₂ and a2A’ (Figure 3G, Figure S2).  W292 along with valine V334 establishes a conserved hydrophobic character to face A.  Directly below W292, a highly conserved, buried salt bridge between R295 and D331 prohibits the tryptophan from torsional engagement with the core (Figure 3G).

The combination of highly conserved, exposed hydrophobics and positively charged residues makes face A a likely region for interacting with tubulin.  We tested this hypothesis through the single or double mutagenesis of the conserved tryptophans exposed on face A of Msps TOG1 and TOG2 (W21 and W292 respectively) to glutamates.  The ability of Msps TOG1-2 to shift a/b tubulin over gel filtration was diminished with either single point mutant (W21E and W292E) and completely abrogated in the double point mutant (Figure 3H).  This result indicates that the solvent-exposed conserved tryptophan is a critical determinant for tubulin binding on face A and that binding is a cooperative activity between tubulin heterodimers and the arrayed TOG domains.  While this paper was being submitted, Al-Bassam et al. reported the structure of the TOG3 domain from Zyg-9, the XMAP215 homolog in C. elegans.  This class B TOG domain displays an overall fold and tubulin binding activity that corroborates our Msps and Stu2p TOG2 domain results (Al-Bassam et al., 2007).

CH Domains of Yeast Bim1p and Human EB1

The CH domains of EB1 and Bim1p structures were determined using MAD phasing and SeMet derivatized protein.  The structure of human EB1 is very similar to a previously reported structure (Hayashi and Ikura, 2003), but ours was determined with two molecules in the asymmetric unit and to a higher resolution of 1.25 Å.  We also delineate two helices, a 3₁₀ helix (between a3 and a4) and a7, which were modeled as loop regions in the Hayashi et al. structure.  Bim1p was determined from a P2₁2₁2 lattice to a resolution of 1.9 Å with one molecule in the asymmetric unit.  The overall fold of the Bim1p CH domain is nearly identical to the human with an r.m.s.d. of 1.3 Å over 351 mainchain atoms  (Figure 4C, compared using EB1 protomer A) while the comparable r.m.s.d. between our human EB1 and the Hayashi et al. structure is 0.5 Å. The EB1/Bim1p CH domain is formed by eight helices that pack around a central conserved hydrophobic helix, a3. Helices 3,4 and 6 are aligned in parallel, with a4 flanked on one side by an extended loop dissected by the 3₁₀ helix and flanked on the other by the extended a4-5 loop (Figures 4A and B).

Many of the highly conserved surface residues in EB1 proteins are found in a6, featuring a conserved hydrophobic groove created by Phe107, Trp110 and Phe114 (Figure 4D) and conserved electrostatic residues Gln102, Asp103, Glu106, Gln109 and Lys113 (not shown).  To explore the tubulin binding interface of EB1, we mutated several of the conserved surface residues using single or cluster mutations, transfected these EGFP-tagged constructs into HeLa cells, and examined plus end tracking by time lapse microscopy.  To prevent heterodimerization with native EB1, the endogenous dimerization domain was replaced with the GCN4 leucine zipper motif.  Mutations on one hemisphere of the domain (delineated as face A) resulted in ablation of in vivo plus end tracking activity (mapped in Figure 4E), yielding instead, diffuse cytosolic localization.  This region is delineated by a1 and the a3-a4 loop that encompasses the 3₁₀ helix described earlier. This tubulin binding zone partially overlaps with the CH domain’s actin binding zone delineated by the regions homologous to a1 and a5-a6 in EB1 (Figure S4)(Mino et al., 1998; Sutherland-Smith et al., 2003).  We note that the majority of EB1’s surface is highly conserved, likely reflecting its use in a variety of conserved protein-protein interactions in addition to tubulin binding (Ligon et al., 2006; Vaughan, 2005).

Cap-Gly Domain of Human CLIP-170

The first Cap-Gly domain from human CLIP-170 was crystallized in the space group P2₁2₁2 with one molecule in the asymmetric unit, and the structure was determined to 2.0 Å resolution by MAD phasing (using a SeMet-derivatized mutant, Q124M).  The fold, which has a weak similarity to a SH3 domain, is composed of seven b-strands that form two b-sheets (Figures 5A and B).  Strands are connected by long, extended loop regions with conserved glycine residues mediating key structural turns.  The central b-sheet bifurcates the conserved hydrophobic core and undergoes a sharp bend in b2 enabling the b2-1-7 region of the b-sheet to envelop the lower half of the conserved hydrophobic core.  The overall architecture is highly similar to the structure of the p150^Glued Cap-Gly domain and a Cap-Gly domain from a putative C. elegans a-tubulin folding chaperone, F53F4 (respective r.m.s.d. values of 0.9 Å and 1.1 Å over 213 mainchain atoms; Figure 5C)(Hayashi et al., 2005; Li et al., 2002).

While both halves of the hydrophobic core are highly conserved across Cap-Gly domains, the upper half’s constituency is conserved to a higher degree with core aromatic residues including Phe76, Phe82, Trp87, Tyr108, Phe109 and Phe118 and structural elements including the b4-b5 sheet and the extended b2- b3 loop.  Comparing Cap-Gly structures using only the highly conserved segment from b2 to b6 (CLIP-170_72-118), CLIP-170 exhibited an r.m.s.d. of only 0.4 Å with both p150^Glued and F53F4.3.

The most highly conserved face of the Cap-Gly domain consists of conserved, exposed hydrophobic residues of the upper core and an invariant region in the b3- b4 loop, containing the sequence GKNDG (residues 97-101)(Figure 6D). The GKNDG motif flanks the exposed hydrophobic core and is stabilized by two conserved salt bridge pairs: Arg63-Asp93 and Asp100-Arg107 (Figures 5A, B and D).  A co-crystal of the p150^Glued Cap-Gly domain with the C-terminal dimerization domain of EB1 has revealed that the GKNDG Cap-Gly sequence interacts with the sequence motif EEY/F-COO^- on EB1 (Honnappa et al., 2006).  This EEY/F-COO^- motif is also found in a-tubulin, and thus it was proposed that the same interface occurs between Cap-Gly domains and tubulin.  To test if this conserved GKNDG motif contributed to tubulin binding and plus-end localization, we altered the charge of this segment by mutating the motif to GEDDG.  Using the plus end tracking competent Drosophila construct CLIP-190_3-235-LZ-GFP, the GEDDG mutant failed to plus end track or localize to microtubules but was instead diffusely localized in the cytoplasm (Figure 6E; Movie S1).  This result substantiates the hypothesis of Honnappa et al. for the role of this motif in microtubule interactions and suggests that EB1 and tubulin compete for the same binding site.