1. Volume 20, Issue 11, Pages 2626-2638 (September 2017)
Mechanism of Catalytic Microtubule Depolymerization via KIF2-Tubulin Transitional Conformation 
Tadayuki Ogawa, Shinya Saijo, Nobutaka Shimizu, Xuguang Jiang, Nobutaka Hirokawa 
Cell Reports 
Volume 20, Issue 11, Pages (September 2017)
DOI: /j.celrep
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2. Cell Reports 2017 20, 2626-2638DOI: (10.1016/j.celrep.2017.08.067)
Copyright © 2017 The Author(s) Terms and Conditions

3. Figure 1
Catalytic MT Depolymerization by KIF2 Requires the Presence of ATP
(A) Microtubules (MTs) exist as free tubulin dimers, tubulin protofilament rings (pf-rings), and polymerized MTs.
(B) Domain structure diagrams of MmKIF2A/C minimal core constructs (KIF2core). KIF2core consists of the conserved motor domain and KIF2-specific neck.
(C) AFM images of the pf-ring after incubation with KIF2Acore at high concentration (KIF2Acore:tubulin dimer = 1:100) or low concentration (1:2,000) in the presence of ATP analogs. Catalytic depolymerization by a small amount of KIF2Acore requires the completion of ATP hydrolysis. Scale bar, 100 nm.
(D) AFM images of catalytic depolymerization of the pf-ring via KIF2Acore. KIF2core (magenta arrowhead) binds the inside the pf-ring, and a crack (white arrowhead) appears near the KIF2core binding site (cyan arrowhead). The large KIF2core-tubulin complex was dissociated from the pf-ring (green arrowhead).
See also Figure S1 and Movie S1.
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4. Figure 2
Size Distribution of the KIF2-Tubulin Complex during ATP Hydrolysis in Solution
(A) High-resolution size-exclusion chromatography (HiRes SEC) shows the size distribution of the KIF2-tubulin complex. Traces are shown as follows: tubulin alone, dotted black; KIF2Acore alone, gray; reaction mixture of KIF2Acore and tubulin with ATP-γS, blue; AMP-PCP, dotted orange; ADP-BeFx, magenta; AMP-PNP, dotted light blue; ADP-Vi, purple; and ADP-AlFx, dotted green. Colloidal blue staining of each fraction is also shown.
(B) Size distribution of the KIF2-tubulin complex. Quantification of each population was achieved based on the peak area of the chromatogram. The 1st peak is indicated in magenta, and the 2nd peak is in light blue.
(C) Relative molar ratio of KIF2Acore and tubulin dimers in each peak fraction on HiRes SEC.
(D) Size distribution of the KIF2Acore-tubulin dimer complex in the presence of ADP-BeFx, as analyzed by HiRes SEC-RI (refractive index)-MALS (multi-angle light scattering). Traces are colored as follows: MALS, red; RI, blue; and UV, green.
See also Figure S2.
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5. Figure 3
Size Determination of the KIF2-Tubulin Complex during ATP Hydrolysis
(A) Size distribution of the KIF2Acore-tubulin complex with ADP-BeFx, as analyzed by AUC (analytical ultracentrifugation). Raw absorbance distributions, the best-fit model (upper), and the sedimentation coefficient distribution [c(s)] (lower) were calculated in the SEDFIT program.
(B) MALDI-TOF spectrum of the KIF2Acore-tubulin complex. In the 1st peak in the ADP-BeFx state (upper), the large complex size (257 kDa) corresponds to the sum of its components: KIF2Acore (51 kDa) + tubulin dimer (103 kDa) × 2. In the 2nd peak in ADP-AlFx, the small complex (154 kDa) consists of one KIF2Acore (51 kDa) and one tubulin dimer (103 kDa).
(C) Summary of size distribution of the KIF2Acore-tubulin complex in nucleotide states.
See also Figure S3.
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6. Figure 4
SAXS Analysis of the Transitional KIF2-Tubulin Complex in Its Pre-hydrolysis State
(A) ABS280, Rg, and I(0)/ABS280 versus number of images from SEC-SAXS experiments of the KIF2Ccore-tubulin complex with ADP-BeFx.
(B) Experimental SAXS profile of the KIF2Ccore-tubulin complex with ADP-BeFx and theoretical SAXS profiles of the model structure by CRYSOL (χ2 = 3.669).
(C) Guinier plot of the KIF2Ccore-tubulin complex. The least-squares fits of the Guinier equation to the q range that satisfy the criteria for the Guinier approximation qRg < 1.3 are shown as a solid line.
(D) Pair distribution function P(r) of the KIF2Ccore-tubulin complex.
(E) Superposition of the SAXS model of the KIF2Ccore-tubulin complex. The dummy atom model (gray surface) by DAMMIN and the model structure (blue, tubulin dimer α1β1; green, tubulin dimer α2β2; and gray, KIF2Ccore).
See also Table S1.
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7. Figure 5
Conformational Change of KIF2Ccore in Its Pre-hydrolysis State
(A) KIF2-specific structural elements of the KIF2Ccore:ADP-BeFx: positively charged neck (light green), Loop2 KVD finger (red), α4 (yellow), Loop8 (blue), and Loop12 (light blue) (Figure 4A). Bottom view from tubulin (asterisk in yellow) is presented to the right.
(B) Superposition of KIF2Ccore structures: KIF2Ccore:ADP-BeFx (colored) in the middle of pre-hydrolysis and KIF2Ccore:ADP (silver-white). Loop8 (blue), Loop10 (light blue), and α2a (purple) shifted upward.
(C) Superposition of KIF2Ccore structures between the nucleotide binding pocket and the MT binding surface: KIF2Ccore:ADP-BeFx (colored) in the middle of pre-hydrolysis and KIF2Ccore:ADP (silver-white).
(D) Conformational changes of the central axis α4 (yellow), Loop11 (purple), and Loop12 (cyan). KIF2Ccore:ADP in ADP form, KIF2Ccore:ADP-BeFx in the middle of pre-hydrolysis, and KIF2Ccore:ADP-AlFx in the ADP-Pi state.
(E) Electron density map around the nucleotide binding pocket of KIF2Ccore:ADP-BeFx.
See also Table S2.
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8. Figure 6
KIF2-Specific Structural Features Contributing to Sustaining the Large 1:2 Complex
(A) Crosslinking between KIF2Ccore Loop12 and α-tubulin C terminus. K530 on Loop12 and K401 of α-tubulin were crosslinked with DSSO.
(B) Position of the interaction of KIF2Ccore and α-tubulin in the 1:2 complex.
(C) Loss-of-function mutants do not display the large 1:2 complex (1st peak) with tubulin in the HiRes SEC-based binding assay. Loss-of-function mutants: Loop2 KVD/AAA mutant (blue dotted line), neck-less mutant (gray), neck-less Loop2 KVD/AAA mutant (orange dotted line), PAK-specific phospho-mimetic mutant (light blue), and MLK3-specific phospho-mimetic mutant (pink dotted line).
See also Figure S4.
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9. Figure 7 Model of Catalytic Microtubule Depolymerization by KIF2
(A) KIF2core targets the curled protofilament of MT ends and the pf-ring.
(B) KIF2core binds to the tubulin end of the protofilament in initial pre-hydrolysis states (ATP-γS and AMP-PCP).
(C) One KIF2core strongly binds to two tubulin dimers in the middle pre-hydrolysis states (ADP-BeFx). Using the KIF2-specific warped surface and structural features, such as the neck helix (dotted green), KVD finger (red), Loop8 (blue), and Loop11 and Loop12 (light blue), KIF2core can stabilize the curved conformation of two tubulin dimers. The strong binding strains the link between the ultimate and the penultimate tubulin dimers (straining effect). Because of the extension of the neck helix on the tubulin surface and the force propagation within the protofilament, additional binding of KIF2core is suppressed (exclusive binding).
(D) One tubulin dimer was already released from the large 1:2 complex in the ADP-Pi states (ADP-AlFx).
(E) KIF2core dissociates from the tubulin heterodimer by completion of ATP hydrolysis.
Cell Reports  , DOI: ( /j.celrep )
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