The Structural Basis of Force Generation by the Mitotic Motor Kinesin-5

Abstract

Double-stranded DNA viruses, including tailed bacteriophages and mammalian herpesviruses, package their genomes into pre-formed protein capsids. The packaging process is driven by a molecular complex known as the packaging motor. This motor is a ring-shaped oligomeric ATPase that utilizes the chemical energy from ATP binding and hydrolysis to perform the mechanical work. We have recently presented a detailed mechanochemical characterization for the bacteriophage phi29 motor, a homo-pentamer that translocates DNA in cycles composed of alternating dwells and bursts (1). We now aim to investigate the behavior of the motor at different stages of packaging, as the motor needs to overcome increasing amounts of internal pressure generated by the compressed dsDNA. We find that the effect of the internal pressure on the motor dynamics is more complex than simply exerting an opposing force to packaging. Through a detailed analysis using ultra-high-resolution DNA translocation data, we find that the internal pressure affects multiple kinetic transitions in the mechanochemical cycle, including events in both the dwell phase and the burst phase. This analysis allows us to make an accurate estimation of the internal force as a function of DNA filling, which is important for the understanding of the DNA organization inside the capsid and the ejection energetics. Remarkably, the motor changes its step size and displays a new class of long-lived pauses towards the completion of packaging. Finally, we determine the structural elements in the packaging complex that are responsible for the signal transduction from the capsid to the motor.(1) In this work, we explored the possible existence of a wear mechanism developing under mild conditions using surface force apparatus (SFA). By selectively digesting different components (type II collagen, Hyaluronic acid (HA), glycosaminoglycans (GAGs)) of the cartilage, we also establish correlation between the structural properties of cartilage surface and the tribological properties. We observed stick-slip friction in articular cartilage for the first time under mild conditions. To visualize load and speed regimes of stick-slip friction occurrence we introduced 'Dynamic (friction) phase diagram'. Prolonged exposure of the cartilage surfaces to stick-slip sliding results in an increase of surface roughness similar to HA and GAGs digested cartilage suggesting that stick-slip motion is able to induce severe morphological changes of cartilage superficial zone. We also found that digestion of the different components of cartilage alters the morphology of the superficial zone in very different ways. HA and GAGs digestions increased surface roughness while collagen digestion decreased the surface roughness compared to normal cartilage. Friction forces increased up to 2, 5 and 10 times after HA, collagen and GAGs digestion, respectively, indicating no direct correlation between surface roughness and friction forces. Kinesin-5s are essential for forming the bipolar spindle during mitosis in most eukary...

Near-atomic cryo-EM structure of yeast kinesin-5-microtubule complex reveals a distinct binding footprint

“…However, immediately above and protruding away from the nucleotide binding site, density corresponding to the Cut7MDspecific loop5 is clearly visible ( Figure 1B, C). As was previously seen at lower resolution (Britto et al, 2016), this Cut7MD loop5 conformation is distinct from that observed for AMPPNP-bound human kinesin-5 loop5 (Goulet et al, 2012;Parke et al, 2010). The sequences of Cut7 and human kinesin-5 in this region of the motor domain are distinct ( Figure S2), and conformational sensitivity of this loop to even small changes in other kinesin-5s has been observed ( Figure 1C; (Behnke-Parks et al, 2011;Bell et al, 2017;Bodey et al, 2009;Liu et al, 2011).…”

“…Distal to the nucleotide binding site, the Cut7MD N-and C-termini coincide to form the functionally important cover neck bundle (CNB; Figure 1D) (Goulet et al, 2012;Khalil et al, 2008). Density corresponding to the C-terminal neck linker, which connects the Cut7 motor domain to the rest of the protein, is clearly visible docking along the length of the motor domain and directed towards the MT plus end.…”

Kinesin-5 promotes microtubule nucleation and assembly by stabilizing a lattice-competent conformation of tubulin

“…This "stapling" conformation implies a model in which the two heads cross-bridge consecutive tubulin dimers and thereby promote protofilament stability. Surprisingly, isolated Eg5 head domains, which bind to a single tubulin (Goulet et al, 2012), also promote microtubule polymerization, which rules out lattice crossbridging as the primary Eg5 polymerase mechanism. Hence, we considered a model in which Eg5 promotes microtubule polymerization by modulating the curvature of tubulin.…”

Microtubule C‐Terminal Tails Can Change Characteristics of Motor Force Production

“…While kinesin-family motor domains are relatively conserved, there are still significant differences in the motors kinetic cycles and also their necks and neck-linkers (7)(8)(9)(10)(11)(12)(13)(14), and we are only starting to understand the extent to which molecular variations adapt the motors to their specific functions (see e.g. [15][16][17][18].…”