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Solution NMR assignment of the C-terminal domain of human chTOG

Solution NMR assignment of the C-terminal domain of human chTOG The microtubule regulatory protein colonic and hepatic tumor overexpressed gene (chTOG), also known as cytoskeleleton associated protein 5 (CKAP5) plays an important role in organizing the cytoskeleton and in particular in the assembly of k-fibres in mitosis. Recently, we dissected the hitherto poorly understood C-terminus of this protein by discovering two new domains—a cryptic TOG domain (TOG6) and a smaller, helical domain at the very C-terminus. It was shown that the C-terminal domain is important for the interaction with the TACC domain in TACC3 during the assembly of k-fibres in a ternary complex that also includes clathrin. Here we now present the solution NMR assignment of the chTOG C-terminal 1 15 domain which confirms our earlier prediction that it is mainly made of α-helices. However, the appearance of the H– N HSQC spectrum is indicative of the presence of a considerable amount of unstructured and possibly flexible portions of protein in the domain. Keywords Mitosis · Kinetochore · TACC3 · ChTOG · Cell cycle Biological context it is through this part of the protein that a large number of interactions to other regulatory proteins occurs, defining the The human protein colonic and hepatic tumor overexpressed specific and distinct function of each family member. Mem- gene (chTOG), also known as CKAP5, and its Drosophila bers of this family function as tubulin polymerases and are melanogaster homologue, MSPS (mini spindles) are mem- thus important for remodelling of the microtubule cytoskel- bers of the XMAP215 protein family (Ohkura et al. 2001). eton (Al-Bassam and Chang 2011). They target microtubule These proteins vary considerably in size and consist of an plus ends and mutants in this protein family cause reduced N-terminal region comprising 2, 3 or 5 highly conserved microtubule growth rates and aberrant spindle morpholo- TOG domains followed by a C-terminal region that is much gies (Currie et al. 2011). In mitosis, they are found in at more diverse in sequence and varying in length. For most least three distinct pools: at centrosomes, associated with members of the XMAP215 protein family this C-terminal microtubule plus ends and in kinetochore fibres (Gutiérrez- region is poorly described and studied. Furthermore, while Caballero et  al. 2015). Indeed, XMAP215 proteins play the organisation of the N-terminal TOG domain array is an important role in the assembly of kinetochore fibres as highly conserved between yeast and higher eukaryotes, this they help to crosslink adjacent microtubules in a complex is not the case for the C-terminus (Gard et al. 2004). e.g., with clathrin and transforming acidic coiled-coil protein 3 the yeast homologue, Stu2p contains a C-terminal coiled (TACC3). Assembly of the complex is regulated by Aurora- coil dimerisation domain while family members in higher A phosphorylation of TACC3 as phosphorylation on Ser558 eukaryotes are monomeric (van Breugel et al. 2003). Yet (TACC3 numbering) is required for the subsequent interac- tion between clathrin and TACC3 (Hood et al. 2013). The specific distribution of all three proteins during mitosis dif- * Mark Pfuhl fers slightly with chTOG more prominent at centrosomes mark.pfuhl@kcl.ac.uk whereas TACC3 is more evident on spindle microtubules. Both clathrin and TACC3 are required for the correct locali- Cardiovascular and Randall Division, King’s College sation of the complex yet neither can independently bind London, Guy’s Campus, London SE1 1UL, UK to microtubules in vitro. To improve our understanding of Faculty of Biological Sciences, School of Molecular the events regulating complex assembly, there has been and Cellular Biology, Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK Vol.:(0123456789) 1 3 222 E. Rostkova et al. considerable work to better define the interaction sites of the three proteins. It has been shown that a short stretch of polypeptide chain near the TACC domain in TACC3 and the ankle region of clathrin combine to create a composite bind- ing site for microtubules, which is coordinated by phospho- rylation of TACC3 by Aurora-A (Hood et al. 2013). chTOG is recruited to this complex via its interaction with TACC3. Previous work has shown that the C-terminus of chTOG (residues 1517–1957) is sufficient for binding to TACC3 (Hood et al. 2013; Thakur et al. 2014). We initially charac- terised the equivalent region in MSPS (residues 1591–1941) by NMR spectroscopy. The NMR data showed that this fragment contains two distinct domains (Hood et al. 2013). Using a combination of more detailed NMR analysis and sequence similarity searches the first and larger of these was identified as a sixth, cryptic, TOG domain (TOG6; residues 1591–1850 in MSPS, residues 1517–1802 in chTOG) (Hood et  al. 2013; Burgess et al. 2015). The remaining, shorter domain at the very C-terminus of both proteins (residues 1860–1941 in MSPS, 1817–1957 in chTOG) did not show any similarity to a known fold. The preliminary NMR anal- ysis of this region in MSPS suggested the presence of 4 α-helices so that this fragment was termed provisionally the ‘4 helix domain’. Efforts to study this domain from MSPS on its own failed because of its tendency to degrade already in the E. coli cells during recombinant expression. Moreover, there were simi- lar problems of degradation with the TACC domain of the drosophila homologue of TACC3, dTACC. As the chTOG/ MSPS CTD is implicated in interactions with the TACC domain it was therefore decided to characterize the CTD of chTOG instead. The importance of the very C-terminal resi- dues in chTOG for binding to TACC3 was demonstrated by the loss of interaction upon deletion of residues 1932–1957 (Gutiérrez-Caballero et al. 2015). These results suggest that the binding site for the TACC domain resides in the CTD and not in the cryptic TOG6 domain. This makes a study of the chTOG CTD an important step in dissecting k-fibre assembly. Methods and experiments Residues 1817–1957 of chTOG were cloned into pETM6T1 for expression as a NusA fusion protein (Harrison 2000) with a N-terminal 6-His-tag and a TEV site between NusA and the chTOG fragment. Protein was expressed in E. coli 1 15 Fig. 1 H– N TROSY spectrum of 400 µM chTOG 1817–1957 compris- BL21 cells over night at a temperature of 18 °C. NMR sam- ing the C-terminal domain recorded at a temperature of 298 K and a field of 800 MHz. Well resolved peaks are labelled with residue type and num- ples were prepared in a buffer of 20 mM HEPES, 150 mM ber in part (a) while peaks in the crowded central region are labelled in Glutamic acid/Arginine, 2 mM DTT pH 7.2 with protein sub spectra (b, c). Positions of sub spectra (b, c) in the overall spectrum concentrations between 200 and 500 µM. Backbone assign- (a) are indicated by dashed boxes. Red peaks indicate peaks with negative intensity caused by aliasing of the ment of the CTD was performed using HNCA, HNCACB, N resonance frequency. They are the resonances of the arginine Hε/Nε groups HN(CO)CACB, HN(CO)CA, H(CCCO)NH, (H)C(CCO)NH 1 3 Solution NMR assignment of the C-terminal domain of human chTOG 223 and HNCO experiments recorded at 700, 800 and 950 MHz Assignments and data deposition on Bruker Avance spectrometers at 25 °C. Side chain reso- nance assignment was initialized using the backbone-side- The chTOG CTD expressed well in standard E. coli BL21 chain TOCSY experiments H(CCCO)NH and (H)C(CCO) cells with good yields of about 10 mg in 1 L of LB. The NH and completed using a combination of HCCH TOCSY domain gives good spectra (see Fig. 1) even though there and C NOESY-HSQC experiments. Spectra were pro- appears to be a substantial variation in peak intensity and cessed with Topspin 3.1 (Bruker) and all assignments were distribution: numerous peaks of very high intensity cluster performed with CCPN analysis 2.4 (Vranken et al. 2005). Fig. 2 Secondary chemi- cal shifts and chemical shift index (CSI) (Wishart and Sykes 1994). All values were calculated using CCPN analysis version 2.4 and figures were generated in apple numbers. a Cα, b Cβ, c C′, d Hα, e CSI. Positions of secondary structure elements based on the chemical shift analysis are indicated as bars 1 3 224 E. Rostkova et al. distribution, and reproduction in any medium, provided you give appro- around the random coil region while a slightly larger number priate credit to the original author(s) and the source, provide a link to are more widely distributed which suggests a considerable the Creative Commons license, and indicate if changes were made. amount of at least partially disordered protein in the domain. 13 1 This is confirmed by the analysis of C and H secondary chemical shifts where we can clearly confirm the existence References of the predicted four helices from our previous work on the MSPS homologue of the domain (Fig. 2). In addition, there Al-Bassam J, Chang F (2011) Regulation of microtubule dynamics is evidence for a fifth helix at the C-terminus even though by TOG-domain proteins XMAP215/Dis1 and CLASP. Trends Cell Biol 21:604–614. https ://doi.org/10.1016/j.tcb.2011.06.007 the secondary chemical shifts are slightly weaker than those Burgess SG, Bayliss R, Pfuhl M (2015) Solution NMR assignment of for the other four helices. In between we see very weak sec- the cryptic sixth TOG domain of mini spindles. Biomol NMR ondary chemical shifts suggestive of a largely disordered, Assign 9:1–3. https ://doi.org/10.1007/s1210 4-015-9620-4 flexible protein in good agreement with the appearance of Currie JD, Stewman S, Schimizzi G, Slep KC, Ma A, Rogers SL 1 15 (2011) The microtubule lattice and plus-end association of Dros- the H– N HSQC experiment. Finally, the presence of at ophila mini spindles is spatially regulated to fine-tune microtubule least 6 sidechain arginine He/Ne peaks (shown in red in dynamics. Mol Biol Cell 22:4343–4361. https ://doi.org/10.1091/ Fig. 1a)—out of a total of nine possible ones—should be mbc.E11-06-0520 noted given the relatively high pH value of 7.2. These sug- Gard DL, Becker BE, Romney SJ (2004) MAPping the eukaryotic tree of life: structure, function, and evolution of the MAP215/ gest the presence of a quite high proportion of arginines Dis1 family of microtubule-associated proteins. Int Rev Cytol in protective interactions such as salt bridges or hydrogen 239:179–272. https ://doi.org/10.1016/S0074 -7696(04)39004 -2 bonds. Gutiérrez-Caballero C, Burgess SG, Bayliss R, Royle SJ (2015) The CTD domain construct comprises 141 amino acids TACC3-ch-TOG track the growing tips of microtubules indepen- dently of clathrin and aurora-A phosphorylation. Biology Open (residues 1817–1957). It was possible to find assignments 4:170–179. https ://doi.org/10.1242/bio.20141 0843 for all of these even though backbone amide peaks were Harrison RG (2000) Expression of soluble heterologous proteins via missing for three of them (F1875, V1912, R1945). Out of fusion with NusA protein. Innovations 11:4–7 a total of 141 backbone nitrogens, 141 α carbons, 131 β Hood FE, Williams SJ, Burgess SG, Richards MW, Roth D, Straube A, Pfuhl M, Bayliss R, Royle SJ (2013) Coordination of adjacent carbons, 126 γ carbons, 86 δ carbons, 26 ε carbons and 141 domains mediates TACC3-ch-TOG-clathrin assembly and mitotic backbone carbonyls a total of 132 (94%), 141 (100%), 131 spindle binding. J Cell Biol 202:463–478. https://doi.or g/10.1083/ (100%), 102 (81%), 65 (76%), 25 (96%) and 132 (94%), jcb.20121 1127 respectively, could be assigned. 405 out of a total of 423 Ohkura H, Garcia MA, Toda T (2001) Dis1/TOG universal microtubule adaptors: one MAP for all? J Cell Sci 114:3805–3812. https://doi. backbone resonances (96%) and 543 out of a total of 647 org/10.1016/0012-1606(84)90117 -9 sidechain proton resonances (84%) were assigned. In terms Thakur HC, Singh M, Nagel-Steger L, Kremer J, Prumbaum D, Fansa of sidechain nitrogen containing groups, all six asparagine EK, Ezzahoini H, Nouri K, Gremer L, Abts A, Schmitt L, Raun- δ NH groups, three of five glutamine ε NH groups and ser S, Ahmadian MR, Piekorz RP (2014) The centrosomal adap- 2 2 tor TACC3 and the microtubule polymerase chTOG interact via two out of nine arginine ε groups could be assigned. The defined C-terminal subdomains in an aurora-A kinase-independent assignment has been deposited with the BMRB, accession manner. J Biol Chem 289:74–88. https ://doi.or g/10.1074/jbc. code 27235. M113.53233 3 van Breugel M, Drechsel D, Hyman A (2003) Stu2p, the budding yeast Acknowledgements This project is funded by a CRUK programme member of the conserved Dis1/XMAP215 family of microtubule- Grant C24461/A12772 to RB and a BBSRC project Grant BB/ associated proteins is a plus end–binding microtubule destabilizer. L023113/1 to RB and MP. NMR spectra were recorded in the Cen- J Cell Biol 161:359–369. https ://doi.org/10.1083/jcb.20021 1097 tre for Biomolecular Spectroscopy at King’s College London and at Vranken WF, Boucher W, Stevens TJ, Fogh RH, Pajon A, Llinas M, the Francis Crick Institute. We would like to thank Andrew Atkinson, Ulrich EL, Markley JL, Ionides J, Laue ED (2005) The CCPN Geoff Kelly, Tom Frankiel and Alain Oregioni for their help with data data model for NMR spectroscopy: development of a software collection. pipeline. Proteins 59:687–696. https://doi.or g/10.1002/prot.20449 Wishart DS, Sykes BD (1994) The C-13 chemical-shift index: a simple method for the identification of protein secondary structure using Open Access This article is distributed under the terms of the Crea- C-13 chemical-shift data. J Biomol NMR 4:171–180 tive Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, 1 3 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Biomolecular NMR Assignments Springer Journals

Solution NMR assignment of the C-terminal domain of human chTOG

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Springer Journals
Copyright
Copyright © 2018 by The Author(s)
Subject
Physics; Biological and Medical Physics, Biophysics; Polymer Sciences; Biochemistry, general
ISSN
1874-2718
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1874-270X
DOI
10.1007/s12104-018-9812-9
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Abstract

The microtubule regulatory protein colonic and hepatic tumor overexpressed gene (chTOG), also known as cytoskeleleton associated protein 5 (CKAP5) plays an important role in organizing the cytoskeleton and in particular in the assembly of k-fibres in mitosis. Recently, we dissected the hitherto poorly understood C-terminus of this protein by discovering two new domains—a cryptic TOG domain (TOG6) and a smaller, helical domain at the very C-terminus. It was shown that the C-terminal domain is important for the interaction with the TACC domain in TACC3 during the assembly of k-fibres in a ternary complex that also includes clathrin. Here we now present the solution NMR assignment of the chTOG C-terminal 1 15 domain which confirms our earlier prediction that it is mainly made of α-helices. However, the appearance of the H– N HSQC spectrum is indicative of the presence of a considerable amount of unstructured and possibly flexible portions of protein in the domain. Keywords Mitosis · Kinetochore · TACC3 · ChTOG · Cell cycle Biological context it is through this part of the protein that a large number of interactions to other regulatory proteins occurs, defining the The human protein colonic and hepatic tumor overexpressed specific and distinct function of each family member. Mem- gene (chTOG), also known as CKAP5, and its Drosophila bers of this family function as tubulin polymerases and are melanogaster homologue, MSPS (mini spindles) are mem- thus important for remodelling of the microtubule cytoskel- bers of the XMAP215 protein family (Ohkura et al. 2001). eton (Al-Bassam and Chang 2011). They target microtubule These proteins vary considerably in size and consist of an plus ends and mutants in this protein family cause reduced N-terminal region comprising 2, 3 or 5 highly conserved microtubule growth rates and aberrant spindle morpholo- TOG domains followed by a C-terminal region that is much gies (Currie et al. 2011). In mitosis, they are found in at more diverse in sequence and varying in length. For most least three distinct pools: at centrosomes, associated with members of the XMAP215 protein family this C-terminal microtubule plus ends and in kinetochore fibres (Gutiérrez- region is poorly described and studied. Furthermore, while Caballero et  al. 2015). Indeed, XMAP215 proteins play the organisation of the N-terminal TOG domain array is an important role in the assembly of kinetochore fibres as highly conserved between yeast and higher eukaryotes, this they help to crosslink adjacent microtubules in a complex is not the case for the C-terminus (Gard et al. 2004). e.g., with clathrin and transforming acidic coiled-coil protein 3 the yeast homologue, Stu2p contains a C-terminal coiled (TACC3). Assembly of the complex is regulated by Aurora- coil dimerisation domain while family members in higher A phosphorylation of TACC3 as phosphorylation on Ser558 eukaryotes are monomeric (van Breugel et al. 2003). Yet (TACC3 numbering) is required for the subsequent interac- tion between clathrin and TACC3 (Hood et al. 2013). The specific distribution of all three proteins during mitosis dif- * Mark Pfuhl fers slightly with chTOG more prominent at centrosomes mark.pfuhl@kcl.ac.uk whereas TACC3 is more evident on spindle microtubules. Both clathrin and TACC3 are required for the correct locali- Cardiovascular and Randall Division, King’s College sation of the complex yet neither can independently bind London, Guy’s Campus, London SE1 1UL, UK to microtubules in vitro. To improve our understanding of Faculty of Biological Sciences, School of Molecular the events regulating complex assembly, there has been and Cellular Biology, Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK Vol.:(0123456789) 1 3 222 E. Rostkova et al. considerable work to better define the interaction sites of the three proteins. It has been shown that a short stretch of polypeptide chain near the TACC domain in TACC3 and the ankle region of clathrin combine to create a composite bind- ing site for microtubules, which is coordinated by phospho- rylation of TACC3 by Aurora-A (Hood et al. 2013). chTOG is recruited to this complex via its interaction with TACC3. Previous work has shown that the C-terminus of chTOG (residues 1517–1957) is sufficient for binding to TACC3 (Hood et al. 2013; Thakur et al. 2014). We initially charac- terised the equivalent region in MSPS (residues 1591–1941) by NMR spectroscopy. The NMR data showed that this fragment contains two distinct domains (Hood et al. 2013). Using a combination of more detailed NMR analysis and sequence similarity searches the first and larger of these was identified as a sixth, cryptic, TOG domain (TOG6; residues 1591–1850 in MSPS, residues 1517–1802 in chTOG) (Hood et  al. 2013; Burgess et al. 2015). The remaining, shorter domain at the very C-terminus of both proteins (residues 1860–1941 in MSPS, 1817–1957 in chTOG) did not show any similarity to a known fold. The preliminary NMR anal- ysis of this region in MSPS suggested the presence of 4 α-helices so that this fragment was termed provisionally the ‘4 helix domain’. Efforts to study this domain from MSPS on its own failed because of its tendency to degrade already in the E. coli cells during recombinant expression. Moreover, there were simi- lar problems of degradation with the TACC domain of the drosophila homologue of TACC3, dTACC. As the chTOG/ MSPS CTD is implicated in interactions with the TACC domain it was therefore decided to characterize the CTD of chTOG instead. The importance of the very C-terminal resi- dues in chTOG for binding to TACC3 was demonstrated by the loss of interaction upon deletion of residues 1932–1957 (Gutiérrez-Caballero et al. 2015). These results suggest that the binding site for the TACC domain resides in the CTD and not in the cryptic TOG6 domain. This makes a study of the chTOG CTD an important step in dissecting k-fibre assembly. Methods and experiments Residues 1817–1957 of chTOG were cloned into pETM6T1 for expression as a NusA fusion protein (Harrison 2000) with a N-terminal 6-His-tag and a TEV site between NusA and the chTOG fragment. Protein was expressed in E. coli 1 15 Fig. 1 H– N TROSY spectrum of 400 µM chTOG 1817–1957 compris- BL21 cells over night at a temperature of 18 °C. NMR sam- ing the C-terminal domain recorded at a temperature of 298 K and a field of 800 MHz. Well resolved peaks are labelled with residue type and num- ples were prepared in a buffer of 20 mM HEPES, 150 mM ber in part (a) while peaks in the crowded central region are labelled in Glutamic acid/Arginine, 2 mM DTT pH 7.2 with protein sub spectra (b, c). Positions of sub spectra (b, c) in the overall spectrum concentrations between 200 and 500 µM. Backbone assign- (a) are indicated by dashed boxes. Red peaks indicate peaks with negative intensity caused by aliasing of the ment of the CTD was performed using HNCA, HNCACB, N resonance frequency. They are the resonances of the arginine Hε/Nε groups HN(CO)CACB, HN(CO)CA, H(CCCO)NH, (H)C(CCO)NH 1 3 Solution NMR assignment of the C-terminal domain of human chTOG 223 and HNCO experiments recorded at 700, 800 and 950 MHz Assignments and data deposition on Bruker Avance spectrometers at 25 °C. Side chain reso- nance assignment was initialized using the backbone-side- The chTOG CTD expressed well in standard E. coli BL21 chain TOCSY experiments H(CCCO)NH and (H)C(CCO) cells with good yields of about 10 mg in 1 L of LB. The NH and completed using a combination of HCCH TOCSY domain gives good spectra (see Fig. 1) even though there and C NOESY-HSQC experiments. Spectra were pro- appears to be a substantial variation in peak intensity and cessed with Topspin 3.1 (Bruker) and all assignments were distribution: numerous peaks of very high intensity cluster performed with CCPN analysis 2.4 (Vranken et al. 2005). Fig. 2 Secondary chemi- cal shifts and chemical shift index (CSI) (Wishart and Sykes 1994). All values were calculated using CCPN analysis version 2.4 and figures were generated in apple numbers. a Cα, b Cβ, c C′, d Hα, e CSI. Positions of secondary structure elements based on the chemical shift analysis are indicated as bars 1 3 224 E. Rostkova et al. distribution, and reproduction in any medium, provided you give appro- around the random coil region while a slightly larger number priate credit to the original author(s) and the source, provide a link to are more widely distributed which suggests a considerable the Creative Commons license, and indicate if changes were made. amount of at least partially disordered protein in the domain. 13 1 This is confirmed by the analysis of C and H secondary chemical shifts where we can clearly confirm the existence References of the predicted four helices from our previous work on the MSPS homologue of the domain (Fig. 2). In addition, there Al-Bassam J, Chang F (2011) Regulation of microtubule dynamics is evidence for a fifth helix at the C-terminus even though by TOG-domain proteins XMAP215/Dis1 and CLASP. Trends Cell Biol 21:604–614. https ://doi.org/10.1016/j.tcb.2011.06.007 the secondary chemical shifts are slightly weaker than those Burgess SG, Bayliss R, Pfuhl M (2015) Solution NMR assignment of for the other four helices. In between we see very weak sec- the cryptic sixth TOG domain of mini spindles. Biomol NMR ondary chemical shifts suggestive of a largely disordered, Assign 9:1–3. https ://doi.org/10.1007/s1210 4-015-9620-4 flexible protein in good agreement with the appearance of Currie JD, Stewman S, Schimizzi G, Slep KC, Ma A, Rogers SL 1 15 (2011) The microtubule lattice and plus-end association of Dros- the H– N HSQC experiment. Finally, the presence of at ophila mini spindles is spatially regulated to fine-tune microtubule least 6 sidechain arginine He/Ne peaks (shown in red in dynamics. Mol Biol Cell 22:4343–4361. https ://doi.org/10.1091/ Fig. 1a)—out of a total of nine possible ones—should be mbc.E11-06-0520 noted given the relatively high pH value of 7.2. These sug- Gard DL, Becker BE, Romney SJ (2004) MAPping the eukaryotic tree of life: structure, function, and evolution of the MAP215/ gest the presence of a quite high proportion of arginines Dis1 family of microtubule-associated proteins. Int Rev Cytol in protective interactions such as salt bridges or hydrogen 239:179–272. https ://doi.org/10.1016/S0074 -7696(04)39004 -2 bonds. Gutiérrez-Caballero C, Burgess SG, Bayliss R, Royle SJ (2015) The CTD domain construct comprises 141 amino acids TACC3-ch-TOG track the growing tips of microtubules indepen- dently of clathrin and aurora-A phosphorylation. Biology Open (residues 1817–1957). It was possible to find assignments 4:170–179. https ://doi.org/10.1242/bio.20141 0843 for all of these even though backbone amide peaks were Harrison RG (2000) Expression of soluble heterologous proteins via missing for three of them (F1875, V1912, R1945). Out of fusion with NusA protein. Innovations 11:4–7 a total of 141 backbone nitrogens, 141 α carbons, 131 β Hood FE, Williams SJ, Burgess SG, Richards MW, Roth D, Straube A, Pfuhl M, Bayliss R, Royle SJ (2013) Coordination of adjacent carbons, 126 γ carbons, 86 δ carbons, 26 ε carbons and 141 domains mediates TACC3-ch-TOG-clathrin assembly and mitotic backbone carbonyls a total of 132 (94%), 141 (100%), 131 spindle binding. J Cell Biol 202:463–478. https://doi.or g/10.1083/ (100%), 102 (81%), 65 (76%), 25 (96%) and 132 (94%), jcb.20121 1127 respectively, could be assigned. 405 out of a total of 423 Ohkura H, Garcia MA, Toda T (2001) Dis1/TOG universal microtubule adaptors: one MAP for all? J Cell Sci 114:3805–3812. https://doi. backbone resonances (96%) and 543 out of a total of 647 org/10.1016/0012-1606(84)90117 -9 sidechain proton resonances (84%) were assigned. In terms Thakur HC, Singh M, Nagel-Steger L, Kremer J, Prumbaum D, Fansa of sidechain nitrogen containing groups, all six asparagine EK, Ezzahoini H, Nouri K, Gremer L, Abts A, Schmitt L, Raun- δ NH groups, three of five glutamine ε NH groups and ser S, Ahmadian MR, Piekorz RP (2014) The centrosomal adap- 2 2 tor TACC3 and the microtubule polymerase chTOG interact via two out of nine arginine ε groups could be assigned. The defined C-terminal subdomains in an aurora-A kinase-independent assignment has been deposited with the BMRB, accession manner. J Biol Chem 289:74–88. https ://doi.or g/10.1074/jbc. code 27235. M113.53233 3 van Breugel M, Drechsel D, Hyman A (2003) Stu2p, the budding yeast Acknowledgements This project is funded by a CRUK programme member of the conserved Dis1/XMAP215 family of microtubule- Grant C24461/A12772 to RB and a BBSRC project Grant BB/ associated proteins is a plus end–binding microtubule destabilizer. L023113/1 to RB and MP. NMR spectra were recorded in the Cen- J Cell Biol 161:359–369. https ://doi.org/10.1083/jcb.20021 1097 tre for Biomolecular Spectroscopy at King’s College London and at Vranken WF, Boucher W, Stevens TJ, Fogh RH, Pajon A, Llinas M, the Francis Crick Institute. We would like to thank Andrew Atkinson, Ulrich EL, Markley JL, Ionides J, Laue ED (2005) The CCPN Geoff Kelly, Tom Frankiel and Alain Oregioni for their help with data data model for NMR spectroscopy: development of a software collection. pipeline. Proteins 59:687–696. https://doi.or g/10.1002/prot.20449 Wishart DS, Sykes BD (1994) The C-13 chemical-shift index: a simple method for the identification of protein secondary structure using Open Access This article is distributed under the terms of the Crea- C-13 chemical-shift data. J Biomol NMR 4:171–180 tive Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, 1 3

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Biomolecular NMR AssignmentsSpringer Journals

Published: Mar 26, 2018

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