Structural basis of microtubule plus end tracking by XMAP215, CLIP-170, and EB1 - PubMed (original) (raw)

Structural basis of microtubule plus end tracking by XMAP215, CLIP-170, and EB1

Kevin C Slep et al. Mol Cell. 2007.

Abstract

Microtubule plus end binding proteins (+TIPs) localize to the dynamic plus ends of microtubules, where they stimulate microtubule growth and recruit signaling molecules. Three main +TIP classes have been identified (XMAP215, EB1, and CLIP-170), but whether they act upon microtubule plus ends through a similar mechanism has not been resolved. Here, we report crystal structures of the tubulin binding domains of XMAP215 (yeast Stu2p and Drosophila Msps), EB1 (yeast Bim1p and human EB1), and CLIP-170 (human), which reveal diverse tubulin binding interfaces. Functional studies, however, reveal a common property that native or artificial dimerization of tubulin binding domains (including chemically induced heterodimers of EB1 and CLIP-170) induces tubulin nucleation/assembly in vitro and, in most cases, plus end tracking in living cells. We propose that +TIPs, although diverse in structure, share a common property of multimerizing tubulin, thus acting as polymerization chaperones that aid in subunit addition to the microtubule plus end.

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Figures

Figure 1

Figure 1. Delineation of tubulin binding domains

A) Domain structure diagrams of XMAP215 family members Msps and Stu2p, EB1 family members EB1 and Bim1p and CLIP-170. Domains indicated are TOG domains, CTD (C-Terminal Domain), CC (Coiled Coil), CH (Calponin Homology domain), DD (Dimerization Domain), CG (Cap-Gly) domain, Zn (Zinc-Finger Domain). Scale bar indicates 1000 amino acids. Gel filtrations studies testing the interaction of +TIP domains with tubulin (B-E). Traces are colored as: tubulin alone, black; +TIP domain(s) alone, red; tubulin plus +TIP, green. Plots indicate absorption at 280 nm on the y-axis and elution volume in ml along the x-axis. B) Msps TOG1-2. C) EB1 full length. D) CLIP-1701-350. E) Summary of gel filtration data presented in B-D and Supplementary fig. 1. where ++, + and - indicate strong, moderate and no tubulin shift respectively.

Figure 2

Figure 2. +TIP tubulin binding domains promote microtubule nucleation

(A-C) Polymerization of tubulin determined via turbidity (absorbance (AU) measured at 350 nm). Tubulin alone (12.5 μM), black curve, tubulin (12.5 μM) plus +TIP construct (1 μM) indicated by color-coded legend above the curves. All +TIPs alone showed no change in turbidity over time and a GST control showed no effect on tubulin’s polymerization rate (data not shown). D) Analysis of TMR-labeled tubulin polymerized for 100 or 300 sec as in (A-C), fixed in glutaraldehyde and pelleted onto coverslips for imaging. Scale bar, 50 μm.

Figure 3

Figure 3. Structure of the second TOG domains from Mini spindles and Stu2p

Ribbon diagram of the TOG2 domain from Msps (A) and Stu2p (B) with the six HEAT-like repeats represented in shades of similar color and labeled A-F. The conserved and non-conserved regions (Faces A and B respectively) are indicated. C) Least-squares fit of Msps (color) and Stu2p (grey) with TOG 2 domains shown in cylindrical helix representation. D) Individual TOG2 HEAT-like repeats are shown for Msps and Stu2p in ribbons format in similar orientations after global least squares fit of each TOG2 domain. The definitive α helix kink that defines HEAT repeats is evident in the α2 helices of Msps HEAT-like repeats C and D and Stu2p HEAT-like repeats C and F. E) 90° rotations of the Msps TOG2 domain about its long axis shown at left in ribbons for orientation and at right in CPK representation for conservation mapping. TOG2 residues with 80% identity across species are represented in green, 80% conservation in yellow (see Figure S2). E) 2Fo-Fc electron density map at 1.7 Å resolution of the Stu2p TOG2 structure contoured at 1.0 σ showing the surface exposed and highly conserved KEKK loop of HEAT-like repeat C. G) 2Fo-Fc electron density map at 2.1 Å resolution of the Msps TOG2 structure contoured at 1.0 σ showing the surface exposed W292 residue and the buried R295 - D331 salt bridge. Inset (upper left) indicates the relative orientation of the TOG domain (F and G). H) Gel filtration tubulin binding assays for wild type (WT) and mutant Msps TOG1-2. Single or double mutations of the conserved TOG domain tryptophan (TOG1: W21E, TOG2: W292E) are indicated above the chromatogram. Tubulin alone, black; Msps TOG1-2 WT alone, red. The plot indicates absorption at 280 nm on the y-axis (mAU) and elution volume in ml along the x-axis.

Figure 4

Figure 4. Structure of the calponin homology domains of EB1 and Bim1p

Ribbon diagram of the N-terminal CH domain from EB1 (A) and Bim1p (B) centered on the highly conserved α6 helix. C) Least-squares fit of EB1 (color) and Bim1p (grey) CH domains with cylindrical helix representation showing the overall structural conservation between these two members. D) 2Fo-Fc electron density map at 1.25 Å resolution of the EB1 CH domain structure contoured at 1.0 σ showing the highly conserved aromatic core. E) 90° rotation of the EB1 CH domain about the y-axis shown above in ribbon format for orientation and below in CPK representation for conservation mapping and to summarize mutagenesis results. Center: CH domain residues with 80% identity across species are represented in green, 80% conservation in yellow (D-E)(see Figure S3). Bottom row: results of CH domain mutagenesis on the ability of EB11-187-LZ to plus end track are mapped: ablation of microtubule association: brick red (three cluster mutants indicated: [S16A, R17E, H18E, D19R], [K59E, K60E], [K66E, L67D]); no effect on microtubule plus end tracking: slate.

Figure 5

Figure 5. Structure of the first Cap-Gly domain of CLIP-170

A) 90° rotation of CLIP-170′s first Cap-Gly domain about the y-axis shown above in ribbon format for orientation (left) and in CPK representation for conservation mapping (right) with 80% identity shown in green, 80% conservation in yellow (see Figure S4). B) Ribbon diagram of the first Cap-Gly domain from CLIP-170 with β-strands indicated in light green. Conserved glycine residues that constitute the Cap-Gly domain are indicated along the ribbon in magenta. Conserved residues across Cap-Gly domains (excluding glycines) are indicated in stick format (colored as in A). The D100-R107 and R63-D93 salt bridges are indicated. C) Least squares fit of the CLIP-170 Cap-Gly 1 domain (colored) and the human p150Glued Cap-Gly domain (grey, left)(Honnappa et al., 2006) and the C. elegans F53F4.3 Cap-Gly domain (grey, right) (Li et al., 2002). The loop insert present in F53F4.3 is indicated in red. D) 2Fo-Fc electron density map at 2.0 Å resolution, contoured at 1.0 σ showing a segment of the conserved β3-β4 loop’s GKNDG motif with D100 torsionally fixed via hydrogen bonds to R107 and S102 and the N99 rotamer fixed via a hydrogen bond between its δO and G101’s backbone amine. The GKNDG motif flanks the conserved hydrophobic region F82, W87, V103 and F118 as shown, that together comprise the tentative tubulin C-terminal binding site. Inset (upper left) indicates the relative orientation of the Cap-Gly domain. E) Images of Drosophila S2 cells transfected with CLIP-1903-235-LZ-EGFP (above) and the K98E, N99D mutant (below). Boxed regions at left are shown as time series at right (time in sec. indicated in the upper left). Arrows track a single microtubule tip for the CLIP-1903-235-LZ-EGFP construct across the time series, color-coded according to the time point. Scale bar, 5 μm.

Figure 6

Figure 6. In vivo analysis of +TIP tubulin binding domains and the plus end tracking correlate

In vivo analysis of Drosophila S2 cells transfected with EGFP fusions of +TIP domains or +TIP domains fused to the GCN4 leucine zipper (LZ) motif. Domains from top to bottom: DmEB11-191, CLIP-1903-235 and Msps TOG1-4 (residues 3-1175). Images at right correspond to a magnification of the boxed region in the first column and represent a time series at 4 sec intervals denoted at the top of each column. Arrows, color-coded to the respective time point, track individual microtubule plus ends. Scale bar in left column, 5 μm.

Figure 7

Figure 7. In vivo chemical dimerization facilitates microtubule plus end tracking in real time

HeLa cells dually transfected with FRB-EGFP and FKBP-EGFP fusions of EB1 and/or CLIP-170. EB1 constructs embody residues 1-185, CLIP-170 constructs residues 3-210 and 129-350. After one minute of imaging, rapamycin was added to the media for a final concentration of 50 nM. Magnified images at right correspond to the boxed region in the first column of the respective row. Images represent a single time point pre-rapamycin treatment and three consecutive time points, taken at 2 sec intervals, post-rapamycin treatment. Arrows, color-coded to the respective time point, track individual microtubule plus ends. Time in sec is indicated relative to rapamycin addition (t = 0 sec). Scale bar in left column, 5 μm.

Figure 8

Figure 8. Dimerization model for plus end tracking and chaperoned tubulin polymerization

Model for +TIP chaperoned tubulin polymerization. Above tubulin’s critical concentration, αβ tubulin heterodimers preferentially bind the microtubule plus end resulting in microtubule polymerization and growth. Below tubulin’s critical concentration, +TIP-mediated multimerization of αβ tubulin effectively raises the affinity for the microtubule plus end enabling multimer subunit addition to the microtubule plus end. Chaperone-mediated co-polymerization of +TIPs facilitates localization of +TIPs at the microtubule plus end. A lower affinity for the microtubule than for free multimerized tubulin subunits leads to +TIP dissociation and the apparent plus end tracking behavior noted for fluorescent-tagged +TIPs. XMAP215, EB1 and CLIP-170 family members are modeled as tethered tubulin binding domains (colored yellow, orange and purple respectively). A diverse array of oligomeric tubulin binding strategies (lateral vs. longitudinal) is likely for each +TIP family given the tethered nature of their domains. For simplicity, a single oligomeric tubulin interaction is modeled for each +TIP family. See Discussion for details.

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