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The C-terminal region of the motor protein MCAK controls its structure and activity through a conformational switch.

<div class='caption-title'>(A) Top: schematic diagram showing the different functionaldomains of full-length MCAK. Bottom: table representing the constructs used andgiven names. (B) Coomassie-stained gel showing a resin-basedbinding assay for purified His-M, His-NM, and CT domains to either glutathioneagarose beads containing GST (as a control) or the GST-CT domain. The starrepresents residual GST. (C) Top, gel filtration elution profileof MCAK motor alone (M, red) and MCAK motor bound to the CT domain (M +CT, cyan). Bottom, coomassie-stained gel showing the size-exclusionchromatography profile of M and M + CT.</div>
<div class='caption-title'>(A) Size-exclusion chromatography elution profiles of motordomain alone (red) and motor domain-CT domain complex (cyan). The horizontalred and cyan lines correspond to SEC-MALS calculated masses for motor domainalone and motor domain-CT domain complex, respectively. (B)Calibration curve for estimation of Stokes radii of motor domain alone (red)and motor domain-CT domain complex (cyan). (C) Table to showthe calculated apparent masses and stoke radii of the motor domain-CT domaincomplex and motor domain alone. The motor domain is drawn in orange and theCT domain in red, to represent the formation of the possible complexes andtheir predicted size. (D) Steady state intrinsic tryptophanfluorescence emission spectra profile for the titration of the CT domain(CT) ranging from 0 to 15.6 μM, into 1 μM of motor domainafter excitation at 295 nm. (E) Effect of the CT domaintitration on tryptophan (magenta) and aromatic residue (green) fluorescencequenching of the motor domain. The extent of fluorescence quenching of themotor domain is represented as a percentage of fluorescence change measuredfor aromatic residues (280 nm) and tryptophan (295 nm) with increasingconcentration of wild type CT domain. Relative change in fluorescence afterbackground correction is shown as a function of CT domain concentration.Error bars represent the standard deviation.</div>
<div class='caption-title'>(A) Kinesin motor domain dimers (cyan and green) bound tothe CT domain (yellow, spacefill) of MCAK. ADP is in red.(B) Motor-CT domain interface showing the electron densitymap (2Fobs − Fcalc), contoured at σ= 1.00 for the CT domain of MCAK. (C) Interactionswithin the motor-CT domain complex of less than 4 Å arerepresented by dotted lines. Oxygen and nitrogen atoms are colored redand blue. (D) Overlay of the human motor domain and Cterminus structure (blue) with the structure of murine MCAK (pink, PDB:1V8J). The respective neck regions containing the α0 and necklinker are in royal blue and magenta, respectively. The CT domain of MCAKis drawn in yellow as a sphere model with oxygen and nitrogen atoms inblue and red.</div>
Talapatra SK, Harker B, Welburn JP
OpenAccess logo Elife 4 (2015)
Cited: 27 times
EuropePMC logo PMID: 25915621

Abstract

The precise regulation of microtubule dynamics is essential during cell division. The kinesin-13 motor protein MCAK is a potent microtubule depolymerase. The divergent non-motor regions flanking the ATPase domain are critical in regulating its targeting and activity. However, the molecular basis for the function of the non-motor regions within the context of full-length MCAK is unknown. Here, we determine the structure of MCAK motor domain bound to its regulatory C-terminus. Our analysis reveals that the MCAK C-terminus binds to two motor domains in solution and is displaced allosterically upon microtubule binding, which allows its robust accumulation at microtubule ends. These results demonstrate that MCAK undergoes long-range conformational changes involving its C-terminus during the soluble to microtubule-bound transition and that the C-terminus-motor interaction represents a structural intermediate in the MCAK catalytic cycle. Together, our work reveals intrinsic molecular mechanisms underlying the regulation of kinesin-13 activity.

Reviews - 4ubf mentioned but not cited (1)

  1. Parts list for a microtubule depolymerising kinesin. Friel CT, Welburn JP. Biochem Soc Trans 46 1665-1672 (2018)

Articles - 4ubf mentioned but not cited (6)

  1. A structural model for microtubule minus-end recognition and protection by CAMSAP proteins. Atherton J, Jiang K, Stangier MM, Luo Y, Hua S, Houben K, van Hooff JJE, Joseph AP, Scarabelli G, Grant BJ, Roberts AJ, Topf M, Steinmetz MO, Baldus M, Moores CA, Akhmanova A. Nat Struct Mol Biol 24 931-943 (2017)
  2. Insight into microtubule disassembly by kinesin-13s from the structure of Kif2C bound to tubulin. Wang W, Cantos-Fernandes S, Lv Y, Kuerban H, Ahmad S, Wang C, Gigant B. Nat Commun 8 70 (2017)
  3. Cryo-EM reveals the structural basis of microtubule depolymerization by kinesin-13s. Benoit MPMH, Asenjo AB, Sosa H. Nat Commun 9 1662 (2018)
  4. The C-terminal region of the motor protein MCAK controls its structure and activity through a conformational switch. Talapatra SK, Harker B, Welburn JP. Elife 4 (2015)
  5. The depolymerase activity of MCAK shows a graded response to Aurora B kinase phosphorylation through allosteric regulation. McHugh T, Zou J, Volkov VA, Bertin A, Talapatra SK, Rappsilber J, Dogterom M, Welburn JPI. J Cell Sci 132 jcs228353 (2019)
  6. Interplay between the Kinesin and Tubulin Mechanochemical Cycles Underlies Microtubule Tip Tracking by the Non-motile Ciliary Kinesin Kif7. Jiang S, Mani N, Wilson-Kubalek EM, Ku PI, Milligan RA, Subramanian R. Dev Cell 49 711-730.e8 (2019)


Reviews citing this publication (3)

  1. Control of microtubule organization and dynamics: two ends in the limelight. Akhmanova A, Steinmetz MO. Nat Rev Mol Cell Biol 16 711-726 (2015)
  2. The kinetochore-microtubule interface at a glance. Monda JK, Cheeseman IM. J Cell Sci 131 jcs214577 (2018)
  3. Molecular insight into the regulation and function of MCAK. Ritter A, Kreis NN, Louwen F, Wordeman L, Yuan J. Crit Rev Biochem Mol Biol 51 228-245 (2015)

Articles citing this publication (17)

  1. Microtubule end tethering of a processive kinesin-8 motor Kif18b is required for spindle positioning. McHugh T, Gluszek AA, Welburn JPI. J Cell Biol 217 2403-2416 (2018)
  2. A small-molecule activator of kinesin-1 drives remodeling of the microtubule network. Randall TS, Yip YY, Wallock-Richards DJ, Pfisterer K, Sanger A, Ficek W, Steiner RA, Beavil AJ, Parsons M, Dodding MP. Proc Natl Acad Sci U S A 114 13738-13743 (2017)
  3. MYC Dysregulates Mitosis, Revealing Cancer Vulnerabilities. Rohrberg J, Van de Mark D, Amouzgar M, Lee JV, Taileb M, Corella A, Kilinc S, Williams J, Jokisch ML, Camarda R, Balakrishnan S, Shankar R, Zhou A, Chang AN, Chen B, Rugo HS, Dumont S, Goga A. Cell Rep 30 3368-3382.e7 (2020)
  4. Insights into Kinesin-1 Activation from the Crystal Structure of KLC2 Bound to JIP3. Cockburn JJB, Hesketh SJ, Mulhair P, Thomsen M, O'Connell MJ, Way M. Structure 26 1486-1498.e6 (2018)
  5. GTSE1 tunes microtubule stability for chromosome alignment and segregation by inhibiting the microtubule depolymerase MCAK. Bendre S, Rondelet A, Hall C, Schmidt N, Lin YC, Brouhard GJ, Bird AW. J Cell Biol 215 631-647 (2016)
  6. NuSAP modulates the dynamics of kinetochore microtubules by attenuating MCAK depolymerisation activity. Li C, Zhang Y, Yang Q, Ye F, Sun SY, Chen ES, Liou YC. Sci Rep 6 18773 (2016)
  7. Molecular architecture of the Dam1 complex-microtubule interaction. Legal T, Zou J, Sochaj A, Rappsilber J, Welburn JP. Open Biol 6 150237 (2016)
  8. Coordination of Synthesis and Assembly of a Modular Membrane-Associated [NiFe]-Hydrogenase Is Determined by Cleavage of the C-Terminal Peptide. Thomas C, Muhr E, Sawers RG. J Bacteriol 197 2989-2998 (2015)
  9. Tension promotes kinetochore-microtubule release by Aurora B kinase. Chen GY, Renda F, Zhang H, Gokden A, Wu DZ, Chenoweth DM, Khodjakov A, Lampson MA. J Cell Biol 220 e202007030 (2021)
  10. Spindle rotation in human cells is reliant on a MARK2-mediated equatorial spindle-centering mechanism. Zulkipli I, Clark J, Hart M, Shrestha RL, Shrestha RL, Gul P, Dang D, Kasichiwin T, Kujawiak I, Sastry N, Draviam VM. J Cell Biol 217 3057-3070 (2018)
  11. The far C-terminus of MCAK regulates its conformation and spindle pole focusing. Zong H, Carnes SK, Moe C, Walczak CE, Ems-McClung SC. Mol Biol Cell 27 1451-1464 (2016)
  12. The family-specific α4-helix of the kinesin-13, MCAK, is critical to microtubule end recognition. Patel JT, Belsham HR, Rathbone AJ, Wickstead B, Gell C, Friel CT. Open Biol 6 160223 (2016)
  13. A rapid computational approach identifies SPICE1 as an Aurora kinase substrate. Deretic J, Kerr A, Welburn JPI. Mol Biol Cell 30 312-323 (2019)
  14. In Vitro FRET- and Fluorescence-Based Assays to Study Protein Conformation and Protein-Protein Interactions in Mitosis. Ems-McClung SC, Walczak CE. Methods Mol Biol 2101 93-122 (2020)
  15. A Cdk1 phosphomimic mutant of MCAK impairs microtubule end recognition. Belsham HR, Friel CT. PeerJ 5 e4034 (2017)
  16. A synthetic ancestral kinesin-13 depolymerizes microtubules faster than any natural depolymerizing kinesin. Belsham HR, Alghamdi HM, Dave N, Rathbone AJ, Wickstead B, Friel CT. Open Biol 12 220133 (2022)
  17. KIF2C Facilitates Tumor Growth and Metastasis in Pancreatic Ductal Adenocarcinoma. Huang X, Zhao F, Wu Q, Wang Z, Ren H, Zhang Q, Wang Z, Xu J. Cancers (Basel) 15 1502 (2023)


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