FRET measurements of kinesin neck orientation reveal a structural basis for processivity and asymmetry

Abstract

As the smallest and simplest motor enzymes, kinesins have served as the prototype for understanding the relationship between protein structure and mechanochemical function of enzymes in this class. Conventional kinesin (kinesin-1) is a motor enzyme that transports cargo toward the plus end of microtubules by a processive, asymmetric hand-over-hand mechanism. The coiled-coil neck domain, which connects the two kinesin motor domains, contributes to kinesin processivity (the ability to take many steps in a row) and is proposed to be a key determinant of the asymmetry in the kinesin mechanism. While previous studies have defined the orientation and position of microtubule-bound kinesin motor domains, the disposition of the neck coiled-coil remains uncertain. We determined the neck coiled-coil orientation using a multidonor fluorescence resonance energy transfer (FRET) technique to measure distances between microtubules and bound kinesin molecules. Microtubules were labeled with a new fluorescent taxol donor, TAMRA-X-taxol, and kinesin derivatives with an acceptor fluorophore attached at positions on the motor and neck coiled-coil domains were used to reconstruct the positions and orientations of the domains. FRET measurements to positions on the motor domain were largely consistent with the domain orientation determined in previous studies, validating the technique. Measurements to positions on the neck coiled-coil were inconsistent with a radial orientation and instead demonstrated that the neck coiled-coil is parallel to the microtubule surface. The measured orientation provides a structural explanation for how neck surface residues enhance processivity and suggests a simple hypothesis for the origin of kinesin step asymmetry and "limping."is a dimeric motor enzyme involved in microtubule-based intracellular transport (1). Kinesin utilizes ATP hydrolysis to move toward the plus ends of microtubules via a hand-over-hand mechanism in which each motor domain (or "head") alternately steps forward (2, 3). The mechanism is asymmetrical, in the sense that alternate steps are different structurally and kinetically (2, 4-7), and is processive, in that kinesin can take hundreds of steps along a microtubule before dissociating (8). Both of these mechanistic features may be important to biological function: Asymmetry allows kinesin to use energy for cargo transport rather than dissipating it in cargo rotation; processivity allows individual kinesin molecules to transport cargoes through the viscoelastic cytoplasm.Processive movement requires that kinesin maintain a tight grip on the microtubule as it moves. To this end, the catalytic cycle is gated to ensure that at least one head remains tightly bound to the microtubule at all times (9-11). However, the kinesin-1 neck, a five heptad repeat coiled-coil connected to the two heads by the short nonhelical neck linkers (Fig. 1), also appears to play a role in keeping the enzyme bound to the microtubule: Mutations on the neck surface can significantly increase or decrease processi...

Structural Correlation of the Neck Coil with the Coiled-coil (CC1)-Forkhead-associated (FHA) Tandem for Active Kinesin-3 KIF13A

“…In addition, the flexibility between the neck and stalk of kinesin-1 would endow the different orientations between them. The previous studies of kinesin-1 demonstrated that, during processive movement, the neck is parallel to the microtubule tracks, whereas the stalk seems somewhat more perpendicular to the tracks for holding cargoes (45). As for kinesin-3, NC forms the neck, whereas the CC1-FHA tandem may resemble the stalk of kinesin-1 (Fig.…”

ATP-Independent Control of Autotransporter Virulence Protein Transport via the Folding Properties of the Secreted Protein

“…Molecular driving forces are integral components of diverse cellular processes, including transport of cargo along microtubules by kinesin, the separation of double-stranded nucleic acid structures, and the unfolding of substrate proteins prior to their degradation (Zolkiewski, 2006; Enemark and Joshua-Tor, 2008; Zhang et al, 2009; Martin et al, 2010; Myong and Ha, 2010). In each of these examples, molecular motors use the chemical energy of ATP hydrolysis to produce an asymmetrical conformational change, which is used to produce a mechanical force.…”

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“…The surplus, naturally, goes into depots. Peters and Langemann, however, remained in doubt about this concept partly due to the fact that this “push” does not work invariably for all animal or human subjects (Martin et al, 2010; Cao et al, 2011). …”