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• W2003294736 abstract "Oncoprotein 18 (Op18) is a microtubule regulator that forms a ternary complex with two tubulin heterodimers. Dispersed regions of Op18 are involved in two-site cooperative binding and subsequent modulation of tubulin GTPase activity. Here we have analyzed specific determinants of Op18 that govern both stoichiometry and positive cooperativity in tubulin binding and consequent stimulatory and inhibitory effects on tubulin GTPase activity. The data revealed that the central and C-terminal regions of Op18 contain overlapping binding-motifs contacting both tubulin heterodimers, suggesting that these regions of Op18 are wedged into the previously noted 1-nm gap between the two longitudinally arranged tubulin heterodimers. Both the N- and C-terminal flanks adjacent to the central region are involved in stabilizing the ternary complex, but only the C-terminal flank does so by imposing positive binding cooperativity. Within the C-terminal flank, deletion of a 7-amino acid region attenuated positive binding cooperativity and resulted in a switch from stimulation to inhibition of tubulin GTP hydrolysis. This switch can be explained by attenuated binding cooperativity, because Op18 under these conditions may block longitudinal contact surfaces of single tubulins with consequent interference of tubulin-tubulin interaction-dependent GTP hydrolysis. Together, our results suggest that Op18 links two tubulin heterodimers via longitudinal contact surfaces to form a ternary GTPase productive complex. Oncoprotein 18 (Op18) is a microtubule regulator that forms a ternary complex with two tubulin heterodimers. Dispersed regions of Op18 are involved in two-site cooperative binding and subsequent modulation of tubulin GTPase activity. Here we have analyzed specific determinants of Op18 that govern both stoichiometry and positive cooperativity in tubulin binding and consequent stimulatory and inhibitory effects on tubulin GTPase activity. The data revealed that the central and C-terminal regions of Op18 contain overlapping binding-motifs contacting both tubulin heterodimers, suggesting that these regions of Op18 are wedged into the previously noted 1-nm gap between the two longitudinally arranged tubulin heterodimers. Both the N- and C-terminal flanks adjacent to the central region are involved in stabilizing the ternary complex, but only the C-terminal flank does so by imposing positive binding cooperativity. Within the C-terminal flank, deletion of a 7-amino acid region attenuated positive binding cooperativity and resulted in a switch from stimulation to inhibition of tubulin GTP hydrolysis. This switch can be explained by attenuated binding cooperativity, because Op18 under these conditions may block longitudinal contact surfaces of single tubulins with consequent interference of tubulin-tubulin interaction-dependent GTP hydrolysis. Together, our results suggest that Op18 links two tubulin heterodimers via longitudinal contact surfaces to form a ternary GTPase productive complex. microtubule adenyl-5′-yl imidodiphosphate glutathioneS-transferase Op18 fused at the N terminus with GST oncoprotein 18/stathmin Op18 tagged at the C terminus with the FLAG epitope polyacrylamide gel electrophoresis, PCR, polymerase chain reaction wild type Microtubules (MTs)1 are dynamic polymers of α/β-tubulin heterodimers and are required for a variety of cellular processes such as assembly of the mitotic spindle and vesicular transport. Op18 (also termed oncoprotein 18 or stathmin) is a cytosolic protein, which acts to destabilize the MT network. The activity of Op18 is turned off by phosphorylation at four Ser residues in response to multiple signal-transducing protein kinase systems during interphase and cell cycle-regulated protein kinases during mitosis (for review, see Ref. 1Lawler S. Current Biology. 1998; 8: R212-R214Abstract Full Text Full Text PDF PubMed Google Scholar). Prevention of phosphorylation-inactivation by mutations of kinase target sites of Op18 results in destabilization of the mitotic spindle and a consequent block in cell division (2Marklund U. Larsson N. Gradin H.M. Brattsand G. Gullberg M. EMBO J. 1996; 15: 5290-5298Crossref PubMed Scopus (248) Google Scholar, 3Larsson N. Marklund U. Gradin H.M. Brattsand G. Gullberg M. Mol. Cell. Biol. 1997; 17: 5530-5539Crossref PubMed Scopus (169) Google Scholar), which argues against an active role of Op18 during mitosis. Rather, regulated phosphorylation inactivation of Op18 in response to receptor-stimulated kinase systems suggests that the primary role of Op18 is to modulate the MT system in response to external signals during the interphase of the cell cycle (4Gradin H.M. Marklund U. Larsson N. Chatila T.A. Gullberg M. Mol. Cell. Biol. 1997; 17: 3459-3467Crossref PubMed Scopus (124) Google Scholar, 5Gradin H.M. Larsson N. Marklund U. Gullberg M. J. Cell Biol. 1998; 140: 131-141Crossref PubMed Scopus (124) Google Scholar). MTs are continuously rearranging, and most individual MTs are either growing or shrinking with transitions between the two states, a phenomena termed dynamic instability (for review, see Ref. 6Desai A. Mitchison T.J. Annu. Rev. Cell Dev. Biol. 1997; 13: 83-117Crossref PubMed Scopus (1950) Google Scholar). The β-tubulin subunit of the heterodimer contains an exchangeable GTP site (E-site), and MTs utilize polymerization-induced GTP hydrolysis to generate dynamic instability. The tip of a polymerizing MT is thought to contain a stabilizing cap of GTP-tubulin, the loss of which results in depolymerization, i.e. a catastrophe. Two families of MT destabilizing proteins, represented by XKCM1 and Op18, have been shown to promote MT catastrophes (7Walczak C.E. Mitchison T.J. Desai A. Cell. 1996; 84: 37-47Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar, 8Desai A. Verma S. Mitchison T.J. Walczak C.E. Cell. 1999; 96: 69-78Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar, 9Belmont L.D. Mitchison T.J. Cell. 1996; 84: 623-631Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar, 10Howell B. Larsson N. Gullberg M. Cassimeris L. Mol. Biol. Cell. 1999; 10: 105-118Crossref PubMed Scopus (158) Google Scholar). XKCM1 acts by physically disrupting the GTP-tubulin cap in an ATP-dependent cycle, but the mechanism by which Op18 promotes catastrophes is still undefined. However, based on the findings of (i) MT plus-end specificity of Op18 catastrophe promotion, (ii) blocking of catastrophe promotion by capping of MTs ends with a non-hydrolyzable GTP analog (10Howell B. Larsson N. Gullberg M. Cassimeris L. Mol. Biol. Cell. 1999; 10: 105-118Crossref PubMed Scopus (158) Google Scholar), and (iii) the ability of Op18 to stimulate GTPase activity of Op18-tubulin complexes (11Larsson N. Segerman B. Gradin H.M. Wandzioch E. Cassimeris L. Gullberg M. Mol. Cell. Biol. 1999; 19: 2242-2250Crossref PubMed Scopus (35) Google Scholar). A prevalent view is that Op18 may promote catastrophes by stimulating GTP hydrolysis at the plus-end of MTs (for review, see Refs. 12Walczak C.E. Curr. Opin. Cell Biol. 2000; 12: 52-56Crossref PubMed Scopus (150) Google Scholar, 13Andersen S.S. Trends Cell Biol. 2000; 10: 261-267Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Op18 forms a ternary complex with two tubulins and this has been thought to be critical for the mechanism by which Op18 destabilizes MTs (9Belmont L.D. Mitchison T.J. Cell. 1996; 84: 623-631Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar, 14Jourdain L. Curmi P. Sobel A. Pantaloni D. Carlier M.F. Biochemistry. 1997; 36: 10817-10821Crossref PubMed Scopus (213) Google Scholar). Two recent reports have given important insights into the structure of the tubulin-Op18 complex. First, digital image analysis of electron micrographs showed that Op18 links two longitudinally arranged but slightly separated tubulin heterodimers in a protofilament-like fashion (15Steinmetz M.O. Kammerer R.A. Jahnke W. Goldie K.N. Lustig A. van Oostrum J. EMBO J. 2000; 19: 572-580Crossref PubMed Scopus (87) Google Scholar). Second, mass spectroscopy analysis of chemically cross-linked Op18-tubulin peptides identified contact points between both the central and C-terminal part of Op18 and helix 10 on 32 α-tubulin, a helix located at the surface where longitudinal contacts are made in MTs (16Wallon G. Rappsilber J. Mann M. Serrano L. EMBO J. 2000; 19: 213-222Crossref PubMed Scopus (60) Google Scholar). In both reports it was proposed that Op18 is an extended α-helical protein with separated binding regions for each heterodimer; one in the N-terminal/central part and one in the C-terminal part of the protein. However, two mutually exclusive models of Op18 linkage of the two heterodimers were proposed. Thus, whereas Steinmetz et al. (15Steinmetz M.O. Kammerer R.A. Jahnke W. Goldie K.N. Lustig A. van Oostrum J. EMBO J. 2000; 19: 572-580Crossref PubMed Scopus (87) Google Scholar) proposed that Op18 linked the two longitudinally arranged heterodimers by extended binding along lateral surfaces, Wallon et al. (16Wallon G. Rappsilber J. Mann M. Serrano L. EMBO J. 2000; 19: 213-222Crossref PubMed Scopus (60) Google Scholar) proposed that each one of two distinct tubulin binding regions of Op18 binds one α-tubulin of the heterodimers via longitudinal contact surfaces, thereby linking the two tubulin heterodimers in a “head-to-head” fashion. To understand the mechanisms by which Op18 destabilizes MTs, it is important to test these two models and to elucidate how Op18-tubulin complex formation stimulates GTP hydrolysis. Here we present evidence that essential contact points for each one of the two tubulin heterodimers in the ternary complex are overlapping within the central region of Op18, and that these contact points are in the vicinity of longitudinally interacting surfaces of tubulin. Our data suggest a third model for Op18-tubulin interactions where Op18 lies between the two longitudinally arranged heterodimers. This model is consistent with the experimental evidence from the two reports discussed above and resolves the mutual incompatibility between the two previously proposed models. Moreover, taken together with previous studies, we present evidence that clearly dissociate stimulation of GTPase activity, which occurs autonomously within the ternary Op18-tubulin complex, from the catastrophe-promoting activity of Op18, which is likely to involve interactions with tubulin surfaces exposed at the MT tip. Construction of C-terminal-truncated Op18 derivatives with sequences encoding amino acids 100–147 deleted (Op18-Δ(100–147)) and 90–147 deleted (Op18-Δ(90–147)) have previously been described (11Larsson N. Segerman B. Gradin H.M. Wandzioch E. Cassimeris L. Gullberg M. Mol. Cell. Biol. 1999; 19: 2242-2250Crossref PubMed Scopus (35) Google Scholar, 17Marklund U. Osterman O. Melander H. Bergh A. Gullberg M. J. Biol. Chem. 1994; 269: 30626-30635Abstract Full Text PDF PubMed Google Scholar). Op18-Δ(139–147), Op18-Δ(130–147), Op18-Δ(116–147), and Op18-Δ(80–147) were constructed using an analogous PCR-based strategy and the unique primers: Δ(139–147), 5′-CCGCGGGCCTAGTCGGCTTCTTTGTTCTTCCGC-3′; Δ(130–147), 5′-CCGCGGGCCTAGTCGGCGTGCTTATCCTTCTCTCG-3′; Δ(116–147), 5′-CCGCGGGCCTAGTCGGCTTGTGCCTCTCGGTTCTC-3′; and Δ(80–147), 5′-CCGCGGGCCTAGTCGGC-3′. Each primer contains a silent bglI/SfiI site at the 5′-end and was used in conjunction with the T7 primer from pBluescript SK(+) (Stratagene). Full-length Op18 cDNA carried on pBluescript was used as template to amplify PCR fragments corresponding to each of the truncated derivatives. The PCR products were then used to replace theHindIII-SfiI fragment of Op18 cDNA with a silent SfiI site introduced at a position overlapping the stop codon after Asp-149 of Op18 (17Marklund U. Osterman O. Melander H. Bergh A. Gullberg M. J. Biol. Chem. 1994; 269: 30626-30635Abstract Full Text PDF PubMed Google Scholar). To construct stepwise C-terminal deletions starting from amino acid residues 116 to 130 of Op18, a SfiI site was introduced in Op18 at a position overlapping the Met-116 codon. This derivative, termed Op18-(SfiI116), was constructed by overlap-PCR using the primers 5′-GCCCAAGCGGCCGCCAAAGCGGAACGTGCTCGCGAGAAGG-3′and 5′-TTCCGCTTTGGCGGCCGCTTGGGCCTCGCGATTCTCTTTA-3′, together with the T7 and T3 primers of pBluescript. Op18-Δ(119–147), Op18-Δ(123–147), and Op18-Δ(127–147) were constructed by extending Op18-(SfiI116) from theSfiI site with annealed oligonucleotides encoding C-terminal amino acids followed by two consecutive stop codons: Δ(119–147), 5′-CGGCTGCCGCCGACTAATAGCCAAG-3′ and 5′-GGCTATTAGTCGGCGGCAGCCGCTT-3′; Δ(123–147), 5′-CGGCTGCCAAACTGGAACGTGCCGACTAATAGCCAAG-3′ and 5′-GGCTATTAGTCGGCACGTTCCAGTTTGGCAGCCGCTT-3′; Δ(127–147), 5′-CGGCTGCCAAACTGGAACGTTTGCGAGAGAAGGCCGACTAATAGCCAAG-3′ and 5′-GGCTATTAGTCGGCCTTCTCTCGCAAACGTTCCAGTTAGGCAGCCGCTT-3′. Coding regions of C-terminal-truncated Op18 derivatives were excised asNcoI to NotI fragments and used to replaced the corresponding fragment of Op18 cDNA in pGEX 4T-1 (18Larsson N. Segerman B. Howell B. Fridell K. Cassimeris L. Gullberg M. J. Cell Biol. 1999; 146: 1289-1302Crossref PubMed Scopus (60) Google Scholar). Construction of the N-terminal-truncated derivatives Op18-Δ(4–45) and Op18-Δ(5–55) have previously been described (10Howell B. Larsson N. Gullberg M. Cassimeris L. Mol. Biol. Cell. 1999; 10: 105-118Crossref PubMed Scopus (158) Google Scholar). It should be noted that Op18-Δ(4–45) was termed Op18-Δ(5–46) in its original description, but these derivatives are identical because both residues 4 and 46 are serines. Op18-Δ(4–62) and Op18-Δ(5–72) were constructed by PCR using the primers Δ(4–62), 5′-TCCCGAGCTCACATGAAGCTGAGGTCTTG-3′ and Δ(5–72), 5′-TCCCGAGCTCAGCTGAGAAACGAGAGCACG-3′ with the T3 primer from pBluescript SK(+) as second primer and FLAG epitope-tagged Op18 as template (17Marklund U. Osterman O. Melander H. Bergh A. Gullberg M. J. Biol. Chem. 1994; 269: 30626-30635Abstract Full Text PDF PubMed Google Scholar). The primers introduce a silent SacI site in the 5′-end. The PCR fragments were excised as SacI toBamHI fragments and inserted into the corresponding sites of pET3d Op18-Δ(5–149) (10Howell B. Larsson N. Gullberg M. Cassimeris L. Mol. Biol. Cell. 1999; 10: 105-118Crossref PubMed Scopus (158) Google Scholar). The coding sequence of PCR-generated fragments were confirmed by nucleotide sequence analysis using ABI PRISM dye terminator cycle sequencing kit from PE Biosystems. GST fusion proteins were expressed and purified on glutathione-Sepharose 4B beads as recommended by the manufacturer (Amersham Pharmacia Biotech). FLAG-tagged Op18 derivatives were expressed and purified as described previously (19Marklund U. Brattsand G. Shingler V. Gullberg M. J. Biol. Chem. 1993; 268: 15039-15047Abstract Full Text PDF PubMed Google Scholar). Purified recombinant proteins were routinely analyzed by SDS-polyacrylamide gel electrophoresis to confirm purity and to confirm that each truncated Op18 derivative migrated corresponding to the predicted molecular weight. Analysis of tubulin GTPase activity was performed in PEM buffer adjusted to pH 6.5 with KOH (80 mmpiperazine-N,N′-bis[2-ethanesulfonic acid], 1 mm EGTA, 4 mm Mg2+) containing 5 mm AMP-PNP, to inhibit nonspecific ATPase activity as described (11Larsson N. Segerman B. Gradin H.M. Wandzioch E. Cassimeris L. Gullberg M. Mol. Cell. Biol. 1999; 19: 2242-2250Crossref PubMed Scopus (35) Google Scholar). In brief, tubulin was incubated with [α-32P]GTP, tubulin-[α-32P]GTP complexes recovered by centrifugation through a desalting column (P-30 Micro Bio-Spin, Bio-Rad) and single-turnover GTP hydrolysis was followed at 37 °C. Control experiments showed that the Op18 preparations used neither bound nor hydrolyzed [α-32P]GTP. Nucleotide hydrolysis was quantitated by ascending chromatography as described (20Austin S. Dixon R. EMBO J. 1992; 11: 2219-2228Crossref PubMed Scopus (124) Google Scholar). Op18-tubulin equilibrium binding experiments were in principle performed as previously reported (18Larsson N. Segerman B. Howell B. Fridell K. Cassimeris L. Gullberg M. J. Cell Biol. 1999; 146: 1289-1302Crossref PubMed Scopus (60) Google Scholar) but with modifications to increase detection of low-affinity binding. In brief, N-terminal GST-tagged or C-terminal FLAG-tagged Op18 derivatives (2 μm) and tubulin (0.8–42 μm) were mixed and allowed to associate for 15 min on ice. Op18-tubulin mixes (48 μl) were added to glutathione or anti-FLAG antibody coupled-Sepharose beads (12 μl) and incubated for 15 min at 8 °C to capture Op18-tubulin complexes. Rapid separation of Op18-tubulin complexes bound to glutathione or anti-FLAG antibody-coupled beads was obtained by applying the bead suspension into the caps of 1.5-ml Eppendorf tubes prepared with a bottom layer of 150 μl of PEM complemented with 27% sucrose, 17% glycerol, pH 6.0 and a top layer of 100 μl of PEM with 17% glycerol, pH 6.0. Samples were centrifuged shortly (30 s, 21,000 × g) to separate bead-bound from -free material. The buffers, volumes, and centrifugation steps described above allowed increased detection of low-affinity binding between truncated Op18 and tubulin as compared with our previous protocol (18Larsson N. Segerman B. Howell B. Fridell K. Cassimeris L. Gullberg M. J. Cell Biol. 1999; 146: 1289-1302Crossref PubMed Scopus (60) Google Scholar). To allow simultaneous quantification of Op18 and tubulin in the same sample, tubulin was labeled with [α-32P]GTP and Op18 was labeled with 125I. There are two major benefits to this strategy. First, the amount of GST·Op18/Op18·F present in the fraction of free tubulin after separation from the beads (about 30% of total Op18) can be compensated for in each data point, and second, only biologically active (i.e. GTP-bound) tubulin was detected in the fraction of free tubulin. The contribution of nonspecific tubulin binding was around 5% of the amount of free tubulin and was subtracted from the data presented. To calculate Op18-tubulin binding parameters, data points from equilibrium binding experiments were fitted either to a one affinity binding model (hyperbola) or to a previously described model assuming two-site positive cooperativity in binding (21Koshland Jr D.E. Nemethy G. Filmer D. Biochemistry. 1966; 5: 365-385Crossref PubMed Scopus (2193) Google Scholar). Written in the form of a binding curve, a two-site positive cooperativity model has the form,B=Bmax2×11+2FKd­1+F2Kd­1×Kd­2×2FKd­11+FKd­2Equation 1 where K d-1 and K d-2 are the equilibrium dissociation constants for Op18 binding of the first and second tubulin heterodimer respectively, B is the Op18-tubulin molar ratio of complex-bound proteins, and F is the free tubulin concentration. Comparison of fits was performed using the F-test provided by GraphPad Prism (GraphPad Software, Inc., San Diego, CA). To validate the ratio of tubulin bound to Op18, samples were separated by 10–20% gradient SDS-PAGE followed by Coomassie Blue staining and scanning of protein bands using a personal densitometer (Molecular Dynamics) as described previously (18Larsson N. Segerman B. Howell B. Fridell K. Cassimeris L. Gullberg M. J. Cell Biol. 1999; 146: 1289-1302Crossref PubMed Scopus (60) Google Scholar). This procedure and previous work by others (14Jourdain L. Curmi P. Sobel A. Pantaloni D. Carlier M.F. Biochemistry. 1997; 36: 10817-10821Crossref PubMed Scopus (213) Google Scholar, 22Curmi P.A. Andersen S.S. Lachkar S. Gavet O. Karsenti E. Knossow M. Sobel A. J. Biol. Chem. 1997; 272: 25029-25036Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar) has established that the molar ratio of tubulin-wild-type Op18 complexes is 2:1 (i.e. B max = 2). This ratio is not altered by introduction of GST- or FLAG-epitope tags (18Larsson N. Segerman B. Howell B. Fridell K. Cassimeris L. Gullberg M. J. Cell Biol. 1999; 146: 1289-1302Crossref PubMed Scopus (60) Google Scholar). Therefore, the specific activity of [α-32P]GTP-labeled tubulin could be corrected within each experiment by assuming aB max value of 2.0 tubulin per GST·Op18-wt or Op18-wt·F. This correction increased the confidence by which the specific tubulin-Op18 stoichiometry in complexes of low-affinity binding truncated Op18 derivatives could be determined. Recent visualization of the ternary tubulin2-Op18 complex by electron microscopy shows that the two heterodimers are arranged longitudinally, similar to that of a protofilament (15Steinmetz M.O. Kammerer R.A. Jahnke W. Goldie K.N. Lustig A. van Oostrum J. EMBO J. 2000; 19: 572-580Crossref PubMed Scopus (87) Google Scholar). The images did not reveal how Op18 connected the two heterodimers; however, there appeared to be a 1-nm gap between the heterodimers, which corresponds to the approximate diameter of an α-helix. To determine whether Op18 links two tubulin heterodimers by independent or overlapping binding regions, we determined the stoichiometry of tubulin binding after consecutive truncations of Op18 from either the N or the C terminus. To facilitate quantitation of binding stoichiometry of truncated Op18 derivatives with impaired tubulin affinity, binding conditions were set to maximize affinity and to minimize dissociation during separation of bound complexes (see “Materials and Methods”). The truncation mutants generated for this purpose are outlined in Fig. 1 together with a secondary structure representation of Op18 according to Wallonet al. (16Wallon G. Rappsilber J. Mann M. Serrano L. EMBO J. 2000; 19: 213-222Crossref PubMed Scopus (60) Google Scholar). To avoid the fact that N- and C-terminal truncations indirectly obstruct binding by altering the position of the affinity tag used for separation of Op18-tubulin complexes, either the N-terminal GST-tag or the C-terminal FLAG-tag was introduced at opposite ends relative to truncations (see Fig. 1). Tubulin binding affinity and stoichiometry of full-length Op18 (Op18-wt) fused to either of these two affinity tags were essentially the same (Fig.2, insets in B andD). In agreement with our previous study (18Larsson N. Segerman B. Howell B. Fridell K. Cassimeris L. Gullberg M. J. Cell Biol. 1999; 146: 1289-1302Crossref PubMed Scopus (60) Google Scholar), Scatchard conversion of binding data showed that tubulin binding to Op18-wt is a complex process with nonlinear data-point distribution typical for positive cooperativity in binding. Analysis of C-terminal-truncated Op18 revealed that truncation up to residue 90 still allowed tubulin binding, albeit with poor affinity. Importantly, Scatchard analysis of binding data indicated that both Op18-Δ(90–147) and Op18-Δ(100–147) are still capable of binding two tubulin heterodimers, but the data-point distributions suggest non-cooperative binding (see Fig. 2 figure legend for B maxvalues). However, if C-terminal truncations extend into helix 1 (see Op18-Δ(80–147)), the binding affinity is too low for interpretable binding data. We conclude that all C-terminal-truncated Op18 derivatives that bind tubulin with estimable affinities are capable of binding two heterodimers.Figure 4The cooperativity imposing region of Op18 determines the switch from tubulin GTPase stimulatory to inhibitory activity. A, tubulin (10 μm in PEM, pH 6.5) preloaded with [α-32P]GTP was incubated at 37 °C with 36 μm of the indicated GST-tagged Op18 derivatives. B, tubulin was incubated as in Awith graded concentrations of the indicated GST-tagged Op18 derivatives. Initial single-turnover hydrolysis rates were evaluated as described under “Materials and Methods.” Data are mean ± S.E. of duplicate determinationsView Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 1Op18 truncation derivatives. Helices are denoted according to Wallon et al. (16Wallon G. Rappsilber J. Mann M. Serrano L. EMBO J. 2000; 19: 213-222Crossref PubMed Scopus (60) Google Scholar); helix 1 (residues 46–87), helix 2 (residues 96–134), a putative N-terminal α-helical region (?) and a polyproline helix (PPII, residues 33–43). Phosphorylation sites are indicated with P (Ser-16, -25, -38 and -63). For stability of truncated proteins, 3–4 amino acids of the N terminus and 2 amino acids of the C terminus of Op18 were preserved. The derivatives are epitope-tagged in the opposite end relative to the truncation. C-terminal truncations are tagged with GST and N-terminal truncations with the FLAG epitope.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 2Op18 contains overlapping regions within helix 1 involved in the binding of each of two tubulins in a ternary complex. Op18-tubulin equilibrium binding curves were determined using high-affinity conditions (PEM buffer with 17% glycerol, pH 6.5) for the indicated C-terminal-truncated GST-tagged (A) and N-terminal-truncated FLAG-tagged (C) Op18 derivatives (2 μm). B and D, Scatchard conversions of the binding curves. Note differences in scales. When applicable, the maximum specific tubulin-Op18 stoichiometry was determined by fitting the data points either to a one affinity or to a two-site positive cooperativity model, depending on which equation provided the best fit according to the F-test. The calculated maximal stoichiometry for each Op18 truncation was: GST·Op18-wt, 2.0 ± 0.1; GST·Op18-Δ(100–147), 2.2 ± 0.1; GST·Op18-Δ(90–147), 2.3 ± 0.4; Op18-wt·F, 2.0 ± 0.1; Op18-Δ(4–45)·F, 1.8 ± 0.1; Op18-Δ(5–55)·F, 2.6 ± 0.3. Straight lines in the Scatchard plots imply best fit to a one affinity model and curved lines imply best fit to a two-site positive cooperativity model. As outlined under “Materials and Methods,” the specific activity of [α-32P]GTP-labeled tubulin was routinely corrected to give a binding stoichiometry of 2:1 for tubulin and wild-type derivatives of Op18.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The corresponding deletion analysis from the N terminus reveals that removal of more than 45 amino acids resulted in a major loss of tubulin binding affinity (compare Op18-Δ(4–45) and Op18-Δ(5–55) in Fig.2, C and D). However, the Op18-Δ(5–55) mutant, which has 10 amino acids of helix 1 deleted, was still capable of binding two tubulin heterodimers. Upon removal of an additional 7 or 17 amino acids from the N terminus, binding affinities became too low for interpretable binding data. Scatchard conversion of Op18-Δ(4–45) binding data revealed data-point distribution typical for positive cooperativity in binding, indicating that the N terminus is not important for the observed cooperativity in tubulin binding (Fig.2 D). The data above show that N- or C-terminal truncations that extend into helix 1 cause the most drastic drops in tubulin binding affinity. However, as shown in Fig. 2 C, helix 1 is clearly a very poor tubulin binder by itself as revealed by analysis of Op18-Δ(4–45), Δ(100–147)-F (termed H1-F). It seems unlikely that this result was biased by the use of the C-terminal FLAG-tag for separation of complexes, because the FLAG-tag can be efficiently used for separation of tubulin-Op18-Δ(100–147)·F complexes (data not shown and Ref.18Larsson N. Segerman B. Howell B. Fridell K. Cassimeris L. Gullberg M. J. Cell Biol. 1999; 146: 1289-1302Crossref PubMed Scopus (60) Google Scholar). Moreover, our result is in line with a recent report demonstrating that truncation of both the N- and C-terminal flanks of Op18 abolishes tubulin binding as determined by size exclusion chromatography (23Redeker V. Lachkar S. Siavoshian S. Charbaut E. Rossier J. Sobel A. Curmi P.A. J. Biol. Chem. 2000; 275: 6841-6849Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Taken together, because N- and C-terminal- truncated Op18 either bound two heterodimers of tubulin or was incapable of binding tubulin with an estimable affinity, the data demonstrate that essential parts of both tubulin binding sites of Op18 are overlapping within the central α-helical part of Op18. Given overlapping binding sites and the previously noted 1-nm gap between longitudinally arranged tubulin heterodimers in complex with Op18 (15Steinmetz M.O. Kammerer R.A. Jahnke W. Goldie K.N. Lustig A. van Oostrum J. EMBO J. 2000; 19: 572-580Crossref PubMed Scopus (87) Google Scholar), the simplest interpretation of binding data is that at least helix 1 of Op18 is inserted between the heterodimers. We have recently shown that Op18 binds tubulin according to a two-site positive cooperative model, i.e. Op18 binding to the first tubulin creates a second tubulin binding site of much higher affinity (18Larsson N. Segerman B. Howell B. Fridell K. Cassimeris L. Gullberg M. J. Cell Biol. 1999; 146: 1289-1302Crossref PubMed Scopus (60) Google Scholar). The binding data above show that both the N- and C-terminal regions flanking helix 1 are important for stabilization of the ternary Op18-tubulin complex. Interestingly, whereas derivatives with N-terminal truncations retained positive cooperativity, C-terminal truncations appear to bind tubulin without appreciable cooperativity. It was therefore of interest to search for a specific region within the C terminus that may impose positive cooperativity. To determine positive cooperativity in a series of successive C-terminal truncation derivatives of Op18, we excluded glycerol from the binding buffer. This results in lower tubulin binding affinities as compared with the data shown in Fig. 2, but two-site positive co" @default.
• W2003294736 created "2016-06-24" @default.
• W2003294736 creator A5021833592 @default.
• W2003294736 creator A5031974572 @default.
• W2003294736 creator A5038790904 @default.
• W2003294736 creator A5038967435 @default.
• W2003294736 date "2000-11-01" @default.
• W2003294736 modified "2023-09-30" @default.
• W2003294736 title "Mutational Analysis of Op18/Stathmin-Tubulin-interacting Surfaces" @default.
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