1. Volume 24, Issue 8, Pages 1322-1334 (August 2016)
Structural Basis of Backwards Motion in Kinesin-1-Kinesin-14 Chimera: Implication for Kinesin-14 Motility 
Masahiko Yamagishi, Hideki Shigematsu, Takeshi Yokoyama, Masahide Kikkawa, Mitsuhiro Sugawa, Mari Aoki, Mikako Shirouzu, Junichiro Yajima, Ryo Nitta 
Structure 
Volume 24, Issue 8, Pages (August 2016)
DOI: /j.str
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2. Structure 2016 24, 1322-1334DOI: (10.1016/j.str.2016.05.021)
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3. Figure 1 Structures of Ncd and Kinesin-1
(A) The constructs of kinesin-14 Ncd and kinesin-1.
(B) The amino acid sequences of the catalytic core of Ncd and kinesin-1 have high homology. The N-terminal neck helix (neck-motor junction) of Ncd and the cover strand of kinesin-1 have little homology. The C-terminal neck mimic of Ncd and the neck linker of kinesin-1 have slight homology. Five residues at the base of Ncd neck mimic (AASVN) and kinesin-1 neck linker (GQRAK) are shown in red and blue, respectively.
(C) The 3D structures of Ncd (PDB: 3U06, chain B) and kinesin-1 (PDB: 4HNA). Both catalytic cores, including the switch II cluster, show high structural homology. The C-terminal neck mimic of Ncd and the neck linker of kinesin-1 have slight structural homology (Heuston et al., 2010). The N-terminal neck helix of Ncd and the cover strand of kinesin-1 have little structural homology. Five residues at the base of the Ncd neck mimic (AASVN) and the kinesin-1 neck linker (GQRAK) are shown in red and blue, respectively. The neck mimic is closely located in the switch II cluster and the neck helix in 3D structure.
Structure  , DOI: ( /j.str )
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4. Figure 2 Ncd-Kinesin-1 Chimera Constructs and their Motilities
(A) Avi-tag fused monomeric Ncd, kinesin-1, and chimera constructs. The avi-tag was fused to the N terminus of Ncd (residues 325–700), chimeras, and the C terminus of kinesin-1 (1–340).
(B) Scheme of the in vitro polarity-marked microtubule sliding assay. Single-headed kinesins with a biotinylated tag sequence (avi-tag) are anchored to biotinylated BSA via biotin-streptavidin linkage. Polarity-marked microtubules slide on single-headed kinesins.
(C) Fluorescence image of a polarity-marked microtubule. The minus-end segment of the microtubule is brighter than the plus-end segment. Scale bar, 5 μm.
(D) Monomeric kinesin's motility. Direction and velocity (mean ± SEM) were determined. Typical kymographs of the polarity-marked microtubules sliding driven by Ncd, chimeras, and kinesin-1 are shown. These kymographs show that avi-Ncd and avi-nKn664 are sliding microtubules with their plus ends leading, whereas avi-nKn669 and kinesin-1 are sliding with their minus ends leading. ATPase kcat is given as mean ± SEM.
Structure  , DOI: ( /j.str )
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5. Figure 3
Cryo-EM Reconstructions of Two nKn Chimeras in Two Different Nucleotide States
nKn664 reconstructions (A, C, E, and G); nKn669 reconstructions (B, D, F, and H). Two nKn chimeras in the nucleotide-free state are shown from the right side (A, B) and from the surface (E, F) of the microtubule. Two nKn chimeras in the ATP state are shown from the right side (C, D) and from the surface (G, H) of the microtubule. The additional density seen in the nKn664-free is shown as the dark blue segment (A). The additional density seen in the nKn664-ATP is shown as the pink segment (C).
Structure  , DOI: ( /j.str )
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6. Figure 4
Cryo-EM Reconstructions of Two nKn Chimeras with Fitted Atomic Models
(A) The nKn664-free structure. The kinesin-1-free model (PDB: 4LNU) (Cao et al., 2014) was perfectly fitted except for the small protrusions at the N terminus (yellow arrowheads) and the C terminus (magenta arrowheads). The C-terminal tail of β-tubulin (E hook) was also assigned to the additional density at the intra-tubulin-dimer interface.
(B) The nKn664-ATP structure. The kinesin-1-ATP model (PDB: 4HNA) (Gigant et al., 2013) was fitted well except for the N-terminal neck-helix-neck-motor junction (yellow), the switch II cluster (green), and the C-terminal α6-neck mimic (red and magenta) of the catalytic core, which were further refined using MDFF and Modeller.
(C) The nKn669-free structure. The kinesin-1-free model (PDB: 4LNU) was perfectly fitted. A small protrusion was found at its N terminus (arrowheads).
(D) The nKn669-ATP structure. The kinesin-1-ATP model (PDB: 4HNA) was fitted well except for the switch II cluster (green) and the C terminus (blue and magenta) of the catalytic core. Left and right panels represent the reconstructions seen from the right side and from the microtubule surface, respectively. Yellow and magenta arrowheads indicate the N-terminal and C-terminal ends of the catalytic core.
See also Movies S5, S6, S7, and S8.
Structure  , DOI: ( /j.str )
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7. Figure 5
Nucleotide-Binding Pocket and Nucleotide-Sensitive Switches I and II in Two nKn Chimeras
(A–D) (A) nKn664-free, (B) nKn669-free, (C) nKn664-ATP, and (D) nKn669-ATP are shown from the left side of the microtubules. Arrowheads indicate the L7-α4 interaction (latch) in nKn664-ATP (C) and the corresponding region in nKn669-ATP (D).
(E–H) Zoom-in views of the nucleotide-binding pockets in nKn664-free (E), nKn669-free (F), nKn664-ATP (G), and nKn669-ATP (H) are shown. Dotted circles indicate the pocket for the nucleotide binding (E, G).
Structure  , DOI: ( /j.str )
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8. Figure 6
Conformations of the Neck Helix, Neck-Motor Junction, Switch II, and Neck Mimic in Two nKn Chimeras
nKn664-free (A, E), nKn669-free (B, G), nKn664-ATP (C, F, I), and nKn669-ATP (D, H) reconstructions are shown with the fitted atomic models. (A–D) are shown from the front-right side of the microtubules. (E–H) The magnified view around the switch II cluster from the right side of the microtubules. Orange dotted lines indicate the interactions among the neck-motor junction, the switch II cluster, and the α6-neck mimic, which are distinct between nKn664 and nKn669 or between the nucleotide-free state and the ATP state. (I) the kinesin-14 bundle formed between the neck-motor junction and neck mimic in nKn664-ATP seen from the bottom front of the microtubule. The right panel represents the magnified view of the dotted square in the left panel, shown with the cryo-EM densities of two different contour levels. It illustrates the pathway of the conformational change to form the kinesin-14 bundle; ATP binding influences the conformations of P loop and switch II loop L11, then helix α6, and finally the neck-mimic conformation.
Structure  , DOI: ( /j.str )
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9. Figure 7 Crystal Structure of nKn664-ADP
(A) Overall conformation of nKn664-ADP seen from the microtubule-binding side.
(B) Superposition of nKn664-ADP solved here (cyan) and kinesin-1-ADP (gray, PDB: 1BG2).
(C) Superposition of nKn664-ADP and Ncd-ADP (brown, PDB: 3U06).
(D) Close-up view of the neck helix in nKn664-ADP.
(E) Close-up view of the neck helix in Ncd-ADP.
Structure  , DOI: ( /j.str )
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10. Figure 8
Conformational Change of Kinesin-1, nKn664, and nKn669 during the ADP/ATP Exchange
(A) Conformational change of kinesin-1 during ADP release.
(B) Conformational change of kinesin-1 during ATP binding.
(C) Conformational change of nKn664 during ADP release.
(D) Conformational change of nKn664 during ATP binding.
(E) Conformational change of nKn669 during ATP binding.
Structure  , DOI: ( /j.str )
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11. Figure 9 Conformational Change of nKn664 during ADP/ATP Exchange
(A–C) Schema of the conformational change of nKn664 on the microtubule are represented with its plus end toward the right.
(D–F) Conformational changes of nKn664 are shown with ribbon models from the microtubule-binding side.
(A, D) The nKn664-ADP model depicts the neck helix pointing toward the plus end of the microtubule (dotted arrow) with the neck mimic being flexible. (B, E) The nKn664-free model depicts both the neck helix and the neck mimic being flexible. (C, F) The nKn664-ATP model depicts the neck helix pointing toward the minus end of the microtubule (dotted arrow) stabilized by the kinesin-14 bundle. Dotted arrows indicate the axes of the neck helix (thick lines) and helix α6 (thin lines).
Structure  , DOI: ( /j.str )
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