Correspondence to Ronald D. Vale: vale{at}cmp.ucsf.edu
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abbreviations used in this paper: APC, adenomatous polyposis coli; ß-ME, ß-mercaptoethanol; MACF, microtubule actin cross-linking factor; MAD, multiwavelength anomalous diffraction; SeMet, selenomethionine.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The two best characterized microtubule plus end binding proteins are Clip-170 and EB1, which are conserved from yeast to humans. The Clip-170 protein family is characterized by NH2-terminal microtubule-interacting Cap-Gly domains and a COOH-terminal region that recruits various cargos, including the dyneindynactin complex (Lansbergen et al., 2004), and vesicles (Pierre et al., 1992). EB1 members have a similar bipartite composition: the NH2-terminal domain mediates microtubule plus end localization and a COOH-terminal cargo binding domain that captures cell polarity determinants. In Saccharomyces cerevisiae, an EB1 homologue, Bim1p, links microtubules to the cell cortex by interacting with the polarity determinant Kar9p (Korinek et al., 2000; L. Lee et al., 2000; Miller et al., 2000). In mammalian cells, the tumor suppressor adenomatous polyposis coli (APC) protein plays a functionally homologous role (Su et al., 1995; Lu et al., 2001). In addition to its role as a scaffold for the regulated degradation of ß-catenin (Su et al., 1993), APC plays a role in microtubule-based cell polarity (Rubinfeld et al., 1993; Lu et al., 2001). EB1 was originally identified as an APC binding protein, and this interaction appears to be important for the later pathway (Su et al., 1995; Lu et al., 2001). Several signaling pathways establish polarity via EB1APC recruitment including: (a) the sequential activation of CDC42, Par6-associated atypical PKC, and GSK-3ß, which in turn localizes APC to the leading edge of astrocytes in a wounded monolayer (Etienne-Manneville and Hall, 2003; Kodama et al., 2003), (b) an NGF-induced axon growth pathway (Zhou et al., 2004), (c) the Rho-activated mDia recruitment of EB1 and APC in fibroblast cell migration (Wen et al., 2004), and (d) a microtubule actin cross-linking factor (MACF)based recruitment of EB1 and APC to the muscletendon junction in Drosophila melanogaster (Subramanian et al., 2003). The EB1APC interaction also is likely to be critical for tumorigenesis/metastasis of intestinal epithelial, probably through a loss of the normal polarity of the epithelia. The majority of APC mutations in human tumors involve deletion of its EB1 binding domain (Powell et al., 1992; Su et al., 1993).
Previous biochemical studies have shown that the COOH-terminal half of EB1 is sufficient for binding APC (Askham et al., 2000; Bu and Su, 2003). Likewise, in yeast, the corresponding region of Bim1p is used for Kar9p binding (Miller et al., 2000). Overexpression of the EB1 COOH-terminal domain also produced a dominant-negative phenotype in cell polarization (Wen et al., 2004; Zhou et al., 2004). Collectively, these studies establish EB1's COOH terminus as a cargo binding domain and a key determinant in search and capture processes. However, the structural mechanism by which EB1 recognizes its cargo is unknown. Here, we have identified a repeat motif in APC that binds EB1 and found a similar motif in the MACF family of spectraplakins, which allows these proteins to bind to EB1 and track along microtubule plus ends in vivo. We also solved the X-ray crystal structure of EB1's cargo binding domain and mapped the residues involved in MACF binding by alanine scanning mutagenesis. These results provide a structural understanding of how EB1 recruits cargo to the microtubule plus end.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
We tested whether or not the dual MACF repeat is indeed capable of binding to EB1. We purified the human MACF2 repeat (MACF21595-1637) and tested its binding to GST fusions of full-length EB1 or its COOH-terminal (EB1185-255) domain prebound to glutathione-Sepharose. Both full-length GST-EB1 (not depicted) and GST-EB1185-255 retained the MACF21595-1637 peptide, whereas GST alone did not (Fig. 1 D). Thus, like APC, the MACF2 repeat motif is capable of specifically binding to the EB1 COOH-terminal domain in vitro. Additionally, competition experiments using GST-EB1185-255 prebound to glutathione-Sepharose, fixed levels of GFP-APC2744-2843, and increasing level of MACF21595-1637 revealed that MACF2 could compete with APC for binding to the EB1 COOH-terminal domain (Fig. 1 E).
DmEB1 associates with the Drosophila spectraplakin Shot
Next, we wished to determine if the MACF interaction with EB1 also occurs in vivo. We pursued this question using Drosophila S2 cells because DmEB1 can be very efficiently depleted in these cells by RNAi (Rogers et al., 2002). The solitary MACF homologue in Drosophila is Shot, which is expressed as many different isoforms. Shot was initially isolated in a screen for proteins involved in integrin-mediated adhesion and has subsequently been found to play a critical role during axon extension as a cytoskeletal cross-linker (Gregory and Brown, 1998; Lee and Kolodziej, 2002). Additional work demonstrated that Shot recruits DmEB1 and APC1 to the muscletendon junction to promote microtubule assembly, but it was not investigated whether the ShotDmEB1 association was direct or indirect (Subramanian et al., 2003). In S2 cells, DmEB1 localizes to the plus ends of microtubules in a "comet-tail" pattern throughout the cell cycle as well as to centrosomes and spindle poles (Rogers et al., 2002). When cells were double stained for DmEB1 and Shot, we found that the distribution of the proteins overlapped but were not identical (Fig. 2, AC). Shot antibodies labeled microtubules along their entire length but were enriched at their tips and also stained small punctae throughout the cell. However, close examination of the microtubules with double staining of Shot and DmEB1 revealed a terminal "cap" of DmEB1 (1 µm long) that lacked Shot followed by a region of overlap of both proteins in 106 out of 120 microtubules examined (Fig. 1 C). These results indicate that DmEB1 and Shot colocalize on microtubules but also maintain individual zones of localization, which is similar to observations made for EB1 and APC in mammalian cells (Juwana et al., 1999; Askham et al., 2000; Mimori-Kiyosue et al., 2000; Nakagawa et al., 2000; Barth et al., 2002).
|
We tested if EB1 was required for the plus end tracking of Shot-GFP by depleting EB1 (<1% of wild type) using RNAi (Rogers et al., 2002). RNAi-treated cells expressing GFP-tubulin under a constitutive actin promoter were double labeled for Shot and DmEB1 to identify unambiguously the DmEB1-depleted cells. Control cells showed colocalization of Shot at microtubule plus ends. In EB1 RNAi-treated cells, Shot never associated with microtubules (n = 900 cells), either at their tips or along their length (Fig. 2 E). Instead, Shot immunofluorescence staining revealed a punctuate pattern with no discernible microtubule association. In contrast, RNAi depletion of D-Clip-190 (another plus endtracking protein) did not alter Shot's microtubule localization (unpublished data). These results indicate that association of Shot with microtubules requires DmEB1; however, albeit less likely, it cannot be ruled out that Shot mislocalization in DmEB1 RNAi-treated cells could be due to the relative undynamic nature of microtubules after DmEB1 depletion (Rogers et al., 2002).
The crystal structure of EB1's COOH-terminal cargo binding domain
To better understand the structural basis of cargo recognition by EB1, we crystallized its conserved COOH-terminal cargo binding domain (residues 185255). Two crystal forms were obtained: P21 (two molecules per asymmetric unit, 2.0 Å resolution) and C2221 (one molecule per asymmetric unit, 1.8 Å resolution) (see Materials and methods). Phase information was obtained for both crystal forms by multiwavelength anomalous diffraction (MAD) using the selenomethionine (SeMet)-derivatized construct V243M, in which a methionine was incorporated at the homologous site of a methionine in the EB1 homologue RP1 (Fig. 3 D) to enhance MAD phasing power. The refinement of the structures yielded the following statistics: P21 crystal form, R = 21.9, Rfree = 25.1; C2221 crystal form, R = 22.1, Rfree = 25.9. Further details on the diffraction data, phasing, refinement, and model statistics are presented in Table I. Coordinates have been deposited in the Protein Data Bank under accession codes 1YIG (P21 crystal form) and 1YIB (C2221 crystal form).
|
|
To elucidate tentative sites responsible for cargo interaction, we examined the spatial positions of conserved residues (defined by invariance, 85% identity and 85% homology) across 13 representative EB1 family members from yeast to vertebrates (Fig. 3 D). Under this classification system, conserved residues within the NH2-terminal region of the coiled coil (A and
A') are predominantly buried and constitute the hydrophobic core of the coiled coil (Figs. 3 D and 4 B). The highest near-continuous stretch of conservation occurs along the second half of
A, spanning residues 211229 at the coiled coilfour-helix bundle junction (Fig. 3 D). Amidst this stretch of conservation are the invariant residues F216, Y217, and F218 (Fig. 3, B and D) that form symmetrical, stacked arrays of solvent-exposed hydrophobic side chains that flank each side of the coiled coil. The register of the coiled coil dictates that the spatial FYF' motif be comprised of F216 and Y217 from one chain and F218' from the homodimeric mate (Fig. 3 B). Additional conservation at this junction is provided by two solvent-exposed residues of the
B helix: I245 and Y247. This conserved surface is characterized by both a hydrophobic character and a net negative charge contributed by the acidic residues E211, E213, D215, and E225 (Fig. 4 D). The composite architecture of a conserved solvent-exposed region with hydrophobic and charged character implicated this zone as the tentative cargo binding site.
|
To test the individual binding contribution of residues within the 211229 conserved span, we prepared single alanine mutations, with the exception of Y217 for which both alanine (Y217A) and phenylalanine (Y217F) mutants were generated. Mutant EB1 proteins were analyzed for MACF21595-1637 binding using the surface plasmon resonance assay at a fixed concentration of 2.5 µM and compared with the behavior of wild-type EB1 at the same concentration (Fig. 5 B). A spectrum of association rates was obtained for the various mutants and classified according to effect (altered association, yellow; no binding, red). Only one mutant of a nonconserved glycine (G219A) displayed wild-type association kinetics (Fig. 3 D). Several alanine mutations of charged/polar residues (E211A, K212A, E213A, R214A, D215A, R222A, N223A, and Q229A) displayed altered association. No binding was obtained for the F216A, Y217A, F218A, K220A, I224A, and E225A mutants. All mutants that showed no MACF2 binding were analyzed by gel filtration to determine if homodimerization had been compromised. All mutants eluted at the position of a homodimer at the concentrations used for the binding study (not depicted), except for I224A, which eluted at the position of a monomer (Fig. 5 C). I224 is the only residue mutated that is completely buried, situated at the core of the four-helix bundle. These results indicate that destabilization of the four-helix bundle compromises dimerization and that dimerization is essential for MACF2 binding.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Diverse cell polarity determinants share a common EB1 binding motif
We have elucidated a motif shared by mammalian APC and several mammalian spectraplakin MACFs. Although these two families bear no apparent sequence homology outside the identified EB1 binding site, they do share a similar composite domain architecture and cellular functions. Both proteins are extremely large (1,0005,000 amino acids) due to repeated domains in their central regions (ß-catenin and axin binding sites in APC [Su et al., 1993; Rubinfeld et al., 1993] and dystrophin-like spectrin repeats in MACFs MACFs [Leung et al., 1999]). Both proteins also are characterized by microtubule binding motifs (Smith et al., 1994; Deka et al., 1998; Sun et al., 2001) followed immediately after by the COOH-terminal EB1 recognition motif. In recent studies, both APC and the spectraplakins ACF7 and Shot have been shown to play roles in axon growth, cell polarity, and migration (S. Lee et al., 2000; Lu et al., 2001; Etienne-Manneville and Hall, 2003; Kodama et al., 2003; Subramanian et al., 2003; Wen et al., 2004; Zhou et al., 2004). In these studies, overexpression of the EB1 COOH-terminal domain resulted in a dominant-negative disruption of polarity signaling, indicative that an interaction with full-length EB1 is required for proper signal transduction/cytoskeletal stabilization by mammalian APC and spectraplakins. GSK-3ß, a kinase implicated in cell polarity signal transduction pathways, also has been implicated in the regulation of APC and MACFs (Etienne-Manneville and Hall, 2003; Kodama et al., 2003; Zhou et al., 2004). It is possible that this kinase might, at least in part, control polarity by regulating EB1 interaction because in vitro studies have shown that phosphorylation of APC abrogates binding to EB1 (Askham et al., 2000; Nakamura et al., 2001; Bu and Su, 2003).
Interestingly, the two APC genes in Drosophila have the ß-catenin/axin binding domains, but lack the COOH-terminal region in mammalian APC that contains the EB1 binding motif. To date, Drosophila APCs have not been observed tracking along microtubule tips, although DmAPC2 has been found to tether mitotic spindles to cortical actin (McCartney et al., 2001). Furthermore, the spectraplakin interaction with EB1 and microtubule tips appears to be widely conserved in all metazoans. Previous work has implicated the MACF Shot as an EB1-associated factor based on colocalization and coimmunoprecipitation, but a direct interaction was not shown (Subramanian et al., 2003). Here, we have definitively demonstrated that the in vivo association of Shot with the tips of microtubules is mediated by EB1. Thus, we speculate that the spectraplakins may have represented the original metazoan invention of a microtubule tracking, cell polarity factor and that vertebrates subsequently incorporated this feature into APC by duplication and gene integration. Consistent with this idea, the EB1 binding motifs are more similar between vertebrate MACF and APC than they are between MACFs from vertebrates and invertebrates.
In addition to the EB1 interaction, our localization studies suggest other features about the mechanism by which spectraplakins and APC interact with microtubules. Shot colocalizes only with the third of EB1 furthest from the microtubule plus end (Fig. 2 C). This pattern, which is not observed for other proteins such as RhoGEFs (Rogers et al., 2004), suggests that Shot does not coassemble with EB1 on the growing end of the microtubule but recognizes EB1 after a temporal delay, perhaps also suggesting some additional mechanism of regulation.
The EB1 COOH-terminal domain is a structural scaffold for dimerization and cargo binding
Our X-ray crystallographic studies show that the conserved EB1 COOH-terminal domain constitutes a unique coiled coilfour-helix bundle motif that confers both dimerization and cargo binding. Although EB1 and Bim1p were suggested to have a coiled coil domain based on sequence analysis (Rehberg and Graf, 2002), this is the first physical evidence that EB1 proteins are indeed dimers. Dimerization appears to rely not only on the coiled coil but also on the four-helix bundle motif, which provides nearly half of the buried surface area formed in dimerization. Consistent with this idea, destabilization of the four-helix bundle with the I224A mutation abolishes dimerization.
We also used alanine scanning mutagenesis, based on the crystal structure, to uncover a bivalent cargo binding site in the EB1 COOH-terminal domain. Both subunits of the homodimer appear to contribute residues to the cargo binding site, which is also supported by the finding that abrogating dimerization prevents cargo binding. Although the EB1 COOH-terminal domain is highly negatively charged, many of the key determinants in MACF2 binding are hydrophobic and form a discrete exposed hydrophobic pocket in the center of the domain. In comparison to the high conservation of EB1's COOH-terminal domain (from yeast to man), the repeat binding motifs found in APC and MACF2 are less well conserved, especially if one compares Drosophila to human. However, general features are the presence of several hydrophobic residues (mainly prolines) and a net positive charge. We speculate that these prolines and other hydrophobics dock into EB1's hydrophobic pocket, whereas the charged residues may augment the binding interaction by providing a peripheral gasket.
The conservation of solvent-exposed residues in EB1's COOH-terminal domain extends from vertebrates down to the yeast homologues Bim1 and Mal3. Kar9 is functionally homologous to APC and Shot in its general role of microtubule search and capture (Miller et al., 2000); however, Kar9 does not have a repeat motif architecture similar to APC/spectraplakins (Miller and Rose, 1998). A weak segment of homology with hydrophobic character was reported between regions of Kar9 and the EB1 binding motif (Bienz, 2001), but this site has not been experimentally confirmed. It will be interesting to compare and contrast the molecular basis for the EB1APC interaction and the Bim1Kar9 interaction to determine if a similar or unique set of determinants govern these binding interactions.
Our results also help to explain previous mutagenesis results of EB1 that were performed before the availability of a crystal structure. Wen et al. (2004) mutated conserved hydrophilic residues within aa 211229 in EB1 and found that the mutations E211A/E213A, E211A/D215A, K220A/R222A, and E211A/E213A/K220A/R222A inhibited EB1APC interaction in vitro and yielded supporting phenotypic behavior in vivo in an mDia-mediated cell polarity pathway. Our results predict the strongest effect for constructs containing the K220A mutation and milder effects for the one with the E213A mutation, which agrees with the observations by Wen et al. (2004). This correlation between EB1 mutants binding to MACF21595-1637 and APC also substantiates the likelihood of a similar binding architecture between these two classes of proteins and the EB1 COOH-terminal domain.
Implications of the EB1 COOH-terminal domain structure for other cargo binding interactions
Several EB1 family members exist in metazoan genomes (four in Drosophila [Rogers et al., 2002] and three in humans [Su and Qi, 2001]). Although the crystal structures reported in this paper describe a homodimeric structure, we cannot rule out the possibility that EB1 heterodimers exist and are used for diverse cargo recognition. The length of the coiled coil domains and the residues in the COOH-terminal domain that constitute the hydrophobic core and mediate dimer contacts are highly conserved across family members, especially within species (Fig. 3 D). Because each bivalent cargo binding site is formed from residues contributed by each of the EB1 molecules, heterodimers could confer complex, pair-wise cargo recognition motifs. Thus, the possibility of whether or not heterodimers of EB1 can form in vitro and within cells merits investigation.
The EB1 COOH-terminal domain also may contain cargo binding sites other than the one that we defined for APC and MACF at the coiled coilfour-helix bundle junction. Several proteins, including the dyneindynactin complex (Valetti et al., 1999; Vaughan et al., 2002), Ncd (unpublished data; Ghoshima, G, personal communication), the microtubule-destabilizing kinesin Klp10A (Mennella et al., 2005), and RhoGEF2 (Rogers et al., 2004), have been reported to track along microtubule tips and associate with EB1. However, only in the cases of the p150Glued dynactin subunit (Askham et al., 2002; Tirnauer et al., 2002; Bu and Su, 2003; Wen et al., 2004) and Klp10A (Mennella et al., 2005) has a direct interaction with EB1 been shown. In the case of p150Glued, deletion studies have suggested that p150Glued and APC use overlapping yet unique binding sites on EB1's COOH-terminal domain (Askham et al., 2002; Bu and Su, 2003; Wen et al., 2004). The p150Glued binding site maps to EB1's coiled-coilfour-helix bundle but also requires EB1's COOH terminus (13 residues not included in our crystallization construct). Thus, it is possible that EB1 contains multiple cargo binding sites, some of which might be independent and others which might be mutually exclusive. The crystal structure reported here should provide a valuable tool for probing these future questions concerning EB1cargo interactions.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
All aforementioned constructs were transformed into BL21 (DE3) E. coli for recombinant expression (excluding a SeMet growth of the pGEX-6P2 EB1185-255 V243M mutant in which the construct was transformed into B834 E. coli and grown as previously described [Leahy et al., 1994]). Cultures were grown in 2x YT at 37°C to an optical density at 600 nm of 0.6, temperature was decreased to 20°C, and IPTG was added to a final concentration of 100 µM. Cultures were induced for 16 h, harvested, and frozen at 20°C. Bacterial pellets of pGEX-6P2 EB1 constructs were resuspended in lysis buffer (25 mM Tris, pH 8.0, 200 mM NaCl, and 0.1% ß-mercaptoethanol [ß-ME]) and lysed using a microfluidizer (Microfluidics, Inc.). 2 mM PMSF was added, and the lysate was clarified by centrifugation at 36,000 g for 45 min. Supernatant was incubated with glutathione-Sepharose beads for 30 min at 4°C with rotation. Resin was collected by centrifugation at 200 g for 10 min, loaded onto a column, washed with 100 column volumes of lysis buffer, and then protein was applied to the column to test for retention. Additional purification of GST-EB1185-255 and GST-EB1185-255 V243M proceeded as follows. GST fusion protein was step eluted using 5 column volumes of lysis buffer supplemented with 50 mM glutathione, pH 8.0. 30 µl of PreScission protease (Amersham Biosciences; 2 mg/ml stock) was added and incubated at 4°C for 12 h, which yielded near complete cleavage. Cleaved fusion protein was concentrated in a Centriprep YM-3 (Millipore) and diluted 100-fold into 25 mM Tris, pH 7.0, 0.1% ß-ME, and loaded onto SP Sepharose Fast Flow resin (Amersham Biosciences) and eluted using a NaCl gradient of 01 M over 20 column volumes. Protein fractions were pooled, concentrated in a Centriprep YM-3, and exchanged into storage buffer (10 mM Tris, pH 7.0, 25 mM NaCl, and 0.1% ß-ME). All buffers used for the EB1185-255 V243M SeMet purification also contained 5 mM methionine as a supplemental reducing agent. Additional purification of GST-H6-MACF21595-1637-Biotinylation Tag entailed PreScission protease cleavage, followed by binding to Ni2+-nitrilotriacetic acid resin, washing in 25 mM Tris, pH 8.0, 15 mM imidazole, 300 mM NaCl, 0.1% ß-ME, and step elution using washing buffer supplemented with 285 mM imidazole. Eluted protein was concentrated and exchanged into 10 mM Tris, pH 8.0, 150 mM NaCl, and 0.1% ß-ME using a Centriprep YM-3. All H7-GFP fusion proteins were purified as follows. Bacterial cell pellets were thawed and resuspended in lysis buffer (25 mM Tris, pH 8.0, 300 mM NaCl, 15 mM imidazole, and 0.1% ß-ME), lysed in a microfluidizer, supplemented with 2 mM PMSF, and centrifuged at 36,000 g for 45 min at 4°C. Supernatant was incubated in batch with Ni2+-nitrilotriacetic acid resin for 30 min at 4°C. Resin was harvested by centrifugation at 200 g at 4°C, loaded into a column, and washed with 100 column volumes of lysis buffer. EB1 mutants were washed with 10 column volumes of lysis buffer supplemented with 15 mM imidazole, and step eluted with lysis buffer supplemented with 285 mM imidazole. All other H7-GFP fusion proteins were eluted using a gradient of 15300 mM imidazole over 20 column volumes. Eluted H7-GFP fusion proteins were concentrated in a Centriprep YM-3, exchanged into 10 mM Tris, pH 8.0, 150 mM NaCl, and 0.1% ß-ME, frozen in liquid nitrogen, and maintained at 80°C until used.
Drosophila tissue culture and immunofluorescence
Drosophila S2 cells were cultured, treated for RNAi, and processed for immunofluorescence as described previously (Rogers et al., 2002). Primary antibodies used for these studies were murine monoclonal anti-Shot (raised using the antigen Shot L(A) 14541909, which was prepared as a PreScission protease cleavage product from a GST fusion [Amersham Biosciences]), rabbit antiClip-190 (Lantz and Miller, 1998), and rabbit anti-DmEB1 (Rogers et al., 2002). Secondary antibodies (TMR anti-rabbit, Cy5 antimouse, Cy2 antirabbit, and Cy3 antiguinea pig; Jackson ImmunoResearch Laboratories) were used at a dilution of 1:300.
Time-lapse observation of Shot-GFP in living cells was performed by cotransfection of S2 cells with pUAS-Shot (L) B-GFP (Lee and Kolodziej, 2002) and pMT-GAL4 (Rogers et al., 2004) using the Cellfectin transfection reagent (Invitrogen) following the manufacturer's protocols. GAL4-mediated Shot-GFP was induced with a 2-h induction using 1 µM copper sulfate and imaged using a spinning disk confocal microscope (Solamere Technology Group) mounted on an Axiovert 200 (Carl Zeiss MicroImaging, Inc.) equipped with an XR/Mega10 intensified charge coupled device camera (Stanford Photonics) driven by QED In Vivo software (QED Imaging).
Crystallization, data collection, data processing, model building, and refinement
The EB1 COOH-terminal domain (residues 185255) was crystallized using the hanging drop vapor diffusion method at 20°C. 2 µl EB1 COOH-terminal domain at 15 mg/ml was added to 2 µl of mother liquor containing 22% PEG 200, 100 mM ammonium acetate, pH 4.6, and equilibrated over 1 ml of the mother liquor. Native crystals were crushed and used to streak-seed crystals of the EB1 COOH-terminal domain mutant V243M SeMet. The V243M SeMet protein crystallized in the space group P21 with two molecules in the asymmetric unit. Attempts to cocrystallize a complex of EB1185-255 with MACF21595-1637 using similar crystallization conditions yielded native and SeMet crystals of EB1 (without MACF21595-1637 present) in the space group C2221 with one molecule in the asymmetric unit. Crystals were transferred to Paratone-N and flash frozen in liquid nitrogen.
Diffraction data were measured at the Lawrence Berkeley National Laboratory Advanced Light Source beamline 8.3.1 using an ADSC 210 charge coupled device detector and maintained at 100°K using a nitrogen cryo stream. Data were processed and scaled using DENZO and SCALEPACK (Otwinowski, 1993). Heavy atom searches were performed using the CNS package (Brunger et al., 1998). Two selenium sites were found in each crystal form using an automated Patterson heavy atom search method and peak (P21 crystal form) or inflection (C2221 crystal form) anomalous wavelength data. The additional two selenium sites for the P21 crystal form were found using an anomalous difference Fourier map and an experimental density modified map that used phases from the two selenium sites found in the initial automated search. The coordinates for these two additional selenium sites were refined using the CNS package and used for subsequent phase determination and map building. MAD phasing used the selenium coordinates and the low energy remote wavelength as reference. Phases were improved by solvent flipping and histogram matching. Model building was done with O (Jones et al., 1991). Refinement was preformed with the CNS package and monitored with a random 10% of the data used for cross-validation. Model refinement used the MLHL target function, torsion angle molecular dynamics simulated annealing, B-factor refinement, and rebuilding in O with A-weighted difference Fourier maps. The C2221 crystal form structure refinement was extended to the high resolution of the native data set using the MLF target function. Figures were prepared with PyMOL (DeLano Scientific) and GRASP (Nicholls et al., 1991). Coordinates have been deposited in the Protein Data Bank under accession codes 1YIG (P21 crystal form) and 1YIB (C2221 crystal form).
Surface plasmon resonance studies
Surface plasmon resonance experiments were conducted using a Biacore 1000 and Streptavidin sensor chips (Biacore). Flow cells were pretreated with 2 x 1 min injections of 1 M NaCl, 50 mM NaOH, and 3 x 1 min injections of regeneration solution (0.25% SDS). Flow cells were normalized and one flow cell was injected with 50 µl of 10 µM H6-MACF21595-1637-Biotinylation Tag protein at a 5 µl/min flow rate to yield an increase of 600 response units. The second flow cell was used as a control surface over which identical analyte injections were performed in triplicate. All runs were conducted at a flow rate of 20 µl/min in running buffer (10 mM Tris, pH 8.0, 200 mM NaCl, 0.1% ß-ME, and 0.05% Tween-20) at 25°C. Sensor chips were prestabilized for 2 min with running buffer. Analyte was injected for 2.5 min followed by 6 min of running buffer for the analysis of dissociation. The sensor chip was regenerated by injection of 0.25% SDS for 1 min followed by a 5.5-min stabilization of running buffer before the start of the next sensogram. For full-length EB1 analysis, eight different concentrations of H6-EB1 were injected onto the sensor chip in triplicate (2.4 nM, 10 nM, 39 nM, 160 nM, 630 nM, 2.5 µM, 10 µM, and 40 µM). Binding studies using wild-type and mutant EB1 proteins were conducted at an analyte concentration of 2.5 µM and conducted in triplicate. Data was processed using BIAevaluation 3.2 RC1 software. Control analyte runs were subtracted from corresponding analyte plus MACF2 peptide runs and calibrated for comparison via baseline adjustment before analyte injection.
Gel filtration assay
Full-length human EB1 and EB1 point mutants (F216A, Y217A, F218A, K220A, I224A, and E225A) were diluted in running buffer (25 mM Tris, pH 8.0, 200 mM NaCl, and 0.1% ß-ME) to a final concentration of 32 µM. 100 µl of each protein was injected onto a Superdex200 Tricorn analytical gel filtration column (Amersham Biosciences) equilibrated in running buffer and analyzed at a flow rate of 0.3 ml/min, measuring the absorbance at 280 nm.
Online supplemental material
GFP-Shot in interphase S2 cells shows microtubule plus end localization (Video 1). Frames for Video 1 were acquired at a rate of one frame per 5 s. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200410114/DC1.
![]() |
Acknowledgments |
---|
The work was supported by the Agouron Institute Paul B. Sigler Fellowship of the Helen Hay Whitney Foundation (to K.C. Slep), the Wellcome Trust (to H. Ohkura), and the National Institutes of Health (grant NIH38499to R.D. Vale).
Submitted: 22 October 2004
Accepted: 5 January 2005
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Altschul, S.F., W. Gish, W. Miller, E.W. Myers, and D.J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403410.[CrossRef][Medline]
Askham, J.M., P. Moncur, A.F. Markham, and E.E. Morrison. 2000. Regulation and function of the interaction between the APC tumour suppressor protein and EB1. Oncogene. 19:19501958.[CrossRef][Medline]
Askham, J.M., K.T. Vaughan, H.V. Goodson, and E.E. Morrison. 2002. Evidence that an interaction between EB1 and p150(Glued) is required for the formation and maintenance of a radial microtubule array anchored at the centrosome. Mol. Biol. Cell. 13:36273645.
Barth, A.I., K.A. Siemers, and W.J. Nelson. 2002. Dissecting interactions between EB1, microtubules and APC in cortical clusters at the plasma membrane. J. Cell Sci. 115:15831590.
Beckett, D., E. Kovaleva, and P.J. Schatz. 1999. A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation. Protein Sci. 8:921929.[Abstract]
Bienz, M. 2001. Spindles cotton on to junctions, APC and EB1. Nat. Cell Biol. 3:E67E68.[CrossRef][Medline]
Brunger, A.T., P.D. Adams, G.M. Clore, W.L. DeLano, P. Gros, R.W. Grosse-Kunstleve, J.S. Jiang, J. Kuszewski, M. Nilges, N.S. Pannu, et al. 1998. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54:905921.[CrossRef][Medline]
Bu, W., and L.K. Su. 2003. Characterization of functional domains of human EB1 family proteins. J. Biol. Chem. 278:4972149731.
Deka, J., J. Kuhlmann, and O. Muller. 1998. A domain within the tumor suppressor protein APC shows very similar biochemical properties as the microtubule-associated protein tau. Eur. J. Biochem. 253:591597.[Abstract]
Desai, A., and T.J. Mitchison. 1997. Microtubule polymerization dynamics. Annu. Rev. Cell Dev. Biol. 13:83117.[CrossRef][Medline]
Etienne-Manneville, S., and A. Hall. 2003. Cdc42 regulates GSK-3beta and adenomatous polyposis coli to control cell polarity. Nature. 421:753756.[CrossRef][Medline]
Gregory, S.L., and N.H. Brown. 1998. kakapo, a gene required for adhesion between and within cell layers in Drosophila, encodes a large cytoskeletal linker protein related to plectin and dystrophin. J. Cell Biol. 143:12711282.
Jones, T.A., J.Y. Zou, S.W. Cowan, and Kjeldgaard. 1991. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A. 47:110-9.[CrossRef][Medline]
Juwana, J.P., P. Henderikx, A. Mischo, A. Wadle, N. Fadle, K. Gerlach, J.W. Arends, H. Hoogenboom, M. Pfreundschuh, and C. Renner. 1999. EB/RP gene family encodes tubulin binding proteins. Int. J. Cancer. 81:275284.[CrossRef][Medline]
Karcher, R.L., S.W. Deacon, and V.I. Gelfand. 2002. Motor-cargo interactions: the key to transport specificity. Trends Cell Biol. 12:2127.[CrossRef][Medline]
Kodama, A., I. Karakesisoglou, E. Wong, A. Vaezi, and E. Fuchs. 2003. ACF7: an essential integrator of microtubule dynamics. Cell. 115:343354.[CrossRef][Medline]
Korinek, W.S., M.J. Copeland, A. Chaudhuri, and J. Chant. 2000. Molecular linkage underlying microtubule orientation toward cortical sites in yeast. Science. 287:22572259.
Lansbergen, G., Y. Komarova, M. Modesti, C. Wyman, C.C. Hoogenraad, H.V. Goodson, R.P. Lemaitre, D.N. Drechsel, E. Van Munster, T.W. Gadella Jr., et al. 2004. Conformational changes in CLIP-170 regulate its binding to microtubules and dynactin localization. J. Cell Biol. 166:10031014.
Lantz, V.A., and K.G. Miller. 1998. A class VI unconventional myosin is associated with a homologue of a microtubule-binding protein, cytoplasmic linker protein-170, in neurons and at the posterior pole of Drosophila embryos. J. Cell Biol. 140:897910.
Leahy, D.J., H.P. Erickson, I. Aukhil, P. Joshi, and W.A. Hendrickson. 1994. Crystallization of a fragment of human fibronectin: introduction of methionine by site-directed mutagenesis to allow phasing via selenomethionine. Proteins. 19:4854.[Medline]
Lee, L., J.S. Tirnauer, J. Li, S.C. Schuyler, J.Y. Liu, and D. Pellman. 2000. Positioning of the mitotic spindle by a cortical-microtubule capture mechanism. Science. 287:22602262.
Lee, S., and P.A. Kolodziej. 2002. Short Stop provides an essential link between F-actin and microtubules during axon extension. Development. 129:11951204.
Lee, S., K.L. Harris, P.M. Whitington, and P.A. Kolodziej. 2000. short stop is allelic to kakapo, and encodes rod-like cytoskeletal-associated proteins required for axon extension. J. Neurosci. 20:10961108.
Leung, C.L., D. Sun, M. Zheng, D.R. Knowles, and R.K. Liem. 1999. Microtubule actin cross-linking factor (MACF): a hybrid of dystonin and dystrophin that can interact with the actin and microtubule cytoskeletons. J. Cell Biol. 147:12751286.
Lu, B., F. Roegiers, L.Y. Jan, and Y.N. Jan. 2001. Adherens junctions inhibit asymmetric division in the Drosophila epithelium. Nature. 409:522525.[CrossRef][Medline]
McCartney, B.M., D.G. McEwen, E. Grevengoed, P. Maddox, A. Bejsovec, and M. Peifer. 2001. Drosophila APC2 and Armadillo participate in tethering mitotic spindles to cortical actin. Nat. Cell Biol. 3:933938.[CrossRef][Medline]
Mennella, V.C., G.C. Rogers, S.L. Rogers, D.W. Buster, R.D. Vale, and D.J. Sharp. 2005. Functionally distinct Kinesin13 family members cooperate to regulate microtubule dynamics during interphase. Nat. Cell Biol. In press.
Miller, R.K., and M.D. Rose. 1998. Kar9p is a novel cortical protein required for cytoplasmic microtubule orientation in yeast. J. Cell Biol. 140:377390.
Miller, R.K., S.C. Cheng, and M.D. Rose. 2000. Bim1p/Yeb1p mediates the Kar9p-dependent cortical attachment of cytoplasmic microtubules. Mol. Biol. Cell. 11:29492959.
Mimori-Kiyosue, Y., N. Shiina, and S. Tsukita. 2000. Adenomatous polyposis coli (APC) protein moves along microtubules and concentrates at their growing ends in epithelial cells. J. Cell Biol. 148:505518.
Nakagawa, H., K. Koyama, Y. Murata, M. Morito, T. Akiyama, and Y. Nakamura. 2000. EB3, a novel member of the EB1 family preferentially expressed in the central nervous system, binds to a CNS-specific APC homologue. Oncogene. 19:210216.[CrossRef][Medline]
Nakamura, M., X.Z. Zhou, and K.P. Lu. 2001. Critical role for the EB1 and APC interaction in the regulation of microtubule polymerization. Curr. Biol. 11:10621067.[CrossRef][Medline]
Nicholls, A., K.A. Sharp, and B. Honig. 1991. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins. 11:281296.[Medline]
Otwinowski, Z. 1993. Oscillation data reduction program. Data Collection and Processing. N.I.L. Sawyer and S. Bailey, editors. Science and Engineering Research Council Daresbury Laboratory/Daresbury, Cheshire, UK. 5662.
Pierre, P., J. Scheel, J.E. Rickard, and T.E. Kreis. 1992. CLIP-170 links endocytic vesicles to microtubules. Cell. 70:887900.[Medline]
Powell, S.M., N. Zilz, Y. Beazer-Barclay, T.M. Bryan, S.R. Hamilton, S.N. Thibodeau, B. Vogelstein, and K.W. Kinzler. 1992. APC mutations occur early during colorectal tumorigenesis. Nature. 359:235237.[CrossRef][Medline]
Rehberg, M., and R. Graf. 2002. Dictyostelium EB1 is a genuine centrosomal component required for proper spindle formation. Mol. Biol. Cell. 13:23012310.
Rogers, S.L., G.C. Rogers, D.J. Sharp, and R.D. Vale. 2002. Drosophila EB1 is important for proper assembly, dynamics, and positioning of the mitotic spindle. J. Cell Biol. 158:873884.
Rogers, S.L., U. Wiedemann, U. Hacker, C. Turck, and R.D. Vale. 2004. Drosophila RhoGEF2 associates with microtubule plus ends in an EB1-dependent manner. Curr. Biol. 14:18271833.[CrossRef][Medline]
Rubinfeld, B., B. Souza, I. Albert, O. Muller, S.H. Chamberlain, F.R. Masiarz, S. Munemitsu, and P. Polakis. 1993. Association of the APC gene product with beta-catenin. Science. 262:17311734.[Medline]
Smith, K.J., D.B. Levy, P. Maupin, T.D. Pollard, B. Vogelstein, and K.W. Kinzler. 1994. Wild-type but not mutant APC associates with the microtubule cytoskeleton. Cancer Res. 54:36723675.[Abstract]
Su, L.K., and Y. Qi. 2001. Characterization of human MAPRE genes and their proteins. Genomics. 71:142149.[CrossRef][Medline]
Su, L.K., B. Vogelstein, and K.W. Kinzler. 1993. Association of the APC tumor suppressor protein with catenins. Science. 262:17341737.[Medline]
Su, L.K., M. Burrell, D.E. Hill, J. Gyuris, R. Brent, R. Wiltshire, J. Trent, B. Vogelstein, and K.W. Kinzler. 1995. APC binds to the novel protein EB1. Cancer Res. 55:29722977.[Abstract]
Subramanian, A., A. Prokop, M. Yamamoto, K. Sugimura, T. Uemura, J. Betschinger, J.A. Knoblich, and T. Volk. 2003. Shortstop recruits EB1/APC1 and promotes microtubule assembly at the muscle-tendon junction. Curr. Biol. 13:10861095.[CrossRef][Medline]
Sun, D., C.L. Leung, and R.K. Liem. 2001. Characterization of the microtubule binding domain of microtubule actin crosslinking factor (MACF): identification of a novel group of microtubule associated proteins. J. Cell Sci. 114:161172.
Tirnauer, J.S., S. Grego, E.D. Salmon, and T.J. Mitchison. 2002. EB1-microtubule interactions in Xenopus egg extracts: role of EB1 in microtubule stabilization and mechanisms of targeting to microtubules. Mol. Biol. Cell. 13:36143626.
Valetti, C., D.M. Wetzel, M. Schrader, M.J. Hasbani, S.R. Gill, T.E. Kreis, and T.A. Schroer. 1999. Role of dynactin in endocytic traffic: effects of dynamitin overexpression and colocalization with CLIP-170. Mol. Biol. Cell. 10:41074120.
Vaughan, P.S., P. Miura, M. Henderson, B. Byrne, and K.T. Vaughan. 2002. A role for regulated binding of p150(Glued) to microtubule plus ends in organelle transport. J. Cell Biol. 158:305319.
Wen, Y., C.H. Eng, J. Schmoranzer, N. Cabrera-Poch, E.J. Morris, M. Chen, B.J. Wallar, A.S. Alberts, and G.G. Gundersen. 2004. EB1 and APC bind to mDia to stabilize microtubules downstream of Rho and promote cell migration. Nat. Cell Biol. 6:820830.[CrossRef][Medline]
Zhou, F.Q., J. Zhou, S. Dedhar, Y.H. Wu, and W.D. Snider. 2004. NGF-induced axon growth is mediated by localized inactivation of GSK-3beta and functions of the microtubule plus end binding protein APC. Neuron. 42:897912.[CrossRef][Medline]