Kinesin-2 KIF3AC and KIF3AB Can Drive Long-Range Transport along Microtubules

AbstractMammalian KIF3AC is classified as a heterotrimeric kinesin-2 that is best known for organelle transport in neurons, yet in vitro studies to characterize its single molecule behavior are lacking. The results presented show that a KIF3AC motor that includes the native helix α7 sequence for coiled-coil formation is highly processive with run lengths of ∼1.23 μm and matching those exhibited by conventional kinesin-1. This result was unexpected because KIF3AC exhibits the canonical kinesin-2 neck-linker sequence that has been reported to be responsible for shorter run lengths observed for another heterotrimeric kinesin-2, KIF3AB. However, KIF3AB with its native neck linker and helix α7 is also highly processive with run lengths of ∼1.62 μm and exceeding those of KIF3AC and kinesin-1. Loop L11, a component of the microtubule-motor interface and implicated in activating ADP release upon microtubule collision, is significantly extended in KIF3C as compared with other kinesins. A KIF3AC encoding a truncation in KIF3C loop L11 (KIF3ACΔL11) exhibited longer run lengths at ∼1.55 μm than wild-type KIF3AC and were more similar to KIF3AB run lengths, suggesting that L11 also contributes to tuning motor processivity. The steady-state ATPase results show that shortening L11 does not alter kcat, consistent with the observation that single molecule velocities are not affected by this truncation. However, shortening loop L11 of KIF3C significantly increases the microtubule affinity of KIF3ACΔL11, revealing another structural and mechanistic property that can modulate processivity. The results presented provide new, to our knowledge, insights to understand structure-function relationships governing processivity and a better understanding of the potential of KIF3AC for long-distance transport in neurons

Kar3Vik1 Uses a Minus-End Directed Powerstroke for Movement along Microtubules

<div><p>We have used cryo-electron microscopy (cryo-EM) and helical averaging to examine the 3-D structure of the heterodimeric kinesin-14 Kar3Vik1 complexed to microtubules at a resolution of 2.5 nm. 3-D maps were obtained at key points in Kar3Vik1’s nucleotide hydrolysis cycle to gain insight into the mechanism that this motor uses for retrograde motility. In all states where Kar3Vik1 maintained a strong interaction with the microtubule, we found, as observed by cryo-EM, that the motor bound with one head domain while the second head extended outwards. 3-D reconstructions of Kar3Vik1-microtubule complexes revealed that in the nucleotide-free state, the motor’s coiled-coil stalk points toward the plus-end of the microtubule. In the ATP-state, the outer head is shown to undergo a large rotation that reorients the stalk ∼75° to point toward the microtubule minus-end. To determine which of the two heads binds to tubulin in each nucleotide state, we employed specific Nanogold®-labeling of Vik1. The resulting maps confirmed that in the nucleotide-free, ATP and ADP+Pi states, Kar3 maintains contact with the microtubule surface, while Vik1 extends away from the microtubule and tracks with the coiled-coil as it rotates towards the microtubule minus-end. While many previous investigations have focused on the mechanisms of homodimeric kinesins, this work presents the first comprehensive study of the powerstroke of a heterodimeric kinesin. The stalk rotation shown here for Kar3Vik1 is highly reminiscent of that reported for the homodimeric kinesin-14 Ncd, emphasizing the conservation of a mechanism for minus-end directed motility.</p> </div

Nanogold®-labeling shows Kar3 is in contact with the MT in the nucleotide-free and AMPPNP states.

<p>Longitudinal views of helical averages of Nanogold®-labeled GCN4-Kar3CFVik1C536C in the nucleotide free (<b>A</b>) and AMPPNP (<b>B</b>) states. The density corresponding to the gold label is colored orange. (<b>C & D</b>) The X-ray crystal structures of tubulin (PDB accession: 1JFF; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053792#pone.0053792-Lowe1" target="_blank">[33]</a>) and Kar3Vik1 (PDB accession: 4ETP; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053792#pone.0053792-Rank1" target="_blank">[6]</a>) docked into the scaffolds of Nanogold®-labeled GCN4-Kar3CFVik1C536C helical averages in the nucleotide-free (<b>C</b>) and AMPPNP states (<b>D</b>). Vik1 C536, the residue that was labeled with Nanogold® is colored in orange showing close agreement with the location of the Nanogold® in the helical reconstructions. The ADP molecule bound in the Kar3 active site on the crystal structure is shown in blue and helix α4 of Kar3 is shown in red. Color key: tubulin - turquoise, Kar3 - gray, Vik1 - yellow, GCN4 coiled-coil (isosurface) or SHD coiled-coil (crystal structure) - pale blue, Nanogold® - orange.</p