High-resolution structures of kinesin on microtubules provide a basis for nucleotide-gated force-generation

Proteins that associate with microtubules (MTs) are crucial to generate MT arrays and establish different cellular architectures. One example is PRC1 (protein regulator of cytokinesis 1), which cross-links antiparallel MTs and is essential for the completion of mitosis and cytokinesis. Here we describe a 4-Å-resolution cryo-EM structure of monomeric PRC1 bound to MTs. Residues in the spectrin domain of PRC1 contacting the MT are highly conserved and interact with the same pocket recognized by kinesin. We additionally found that PRC1 promotes MT assembly even in the presence of the MT stabilizer taxol. Interestingly, the angle of the spectrin domain on the MT surface corresponds to the previously observed cross-bridge angle between MTs cross-linked by full-length, dimeric PRC1. This finding, together with molecular dynamic simulations describing the intrinsic flexibility of PRC1, suggests that the MT-spectrin domain interface determines the geometry of the MT arrays cross-linked by PRC1.ells rely on the microtubule (MT) cytoskeleton to help organize organelles (1), control cellular morphology (2), provide mechanical stability (3, 4), and form the spindle apparatus used to segregate chromosomes during cell division (5-8). The diverse functions of MTs are made possible through the action of motors (i.e., kinesin and dynein) and nonmotor MT-associated proteins (MAPs) that tightly regulate the MT network. Some of these proteins act by binding directly to the ends of MTs, either to the highly dynamic plus end, such as the conserved end-binding proteins (9, 10), or to the minus end (11,12). Other MT regulators, such as MAP1 and MAP2/Tau, bind along the MT lattice, stabilizing it and helping build parallel arrays, most notably in axons (13,14). Members of the MAP65 family, which includes human protein regular of cytokinesis 1 (PRC1) and its budding yeast ortholog Ase1, form antiparallel MT arrays important for setting the spindle midzone and determining the location of the cytokinetic ring (8,(15)(16)(17)(18)(19). In addition to binding selectively to antiparallel MTs, PRC1 recruits other spindle-organizing factors and therefore is an essential component of the mitotic spindle (15,20). Proper functioning of PRC1 requires cell-cycle-dependent localization and regulation. PRC1 contains two nuclear localization signals (NLSs) in its C-terminal domain (Fig. 1A) and is found almost exclusively in the nucleus during interphase (21-23). As the cell enters mitosis, PRC1 localizes to the spindle and becomes concentrated at the midzone by late anaphase (22), similar to observations for Ase1 (24). PRC1 is subject to phosphorylation by several cyclin-cyclin-dependent kinase (CDK) complexes (21) and together with kinesin-4 can regulate MT antiparallel overlap at the spindle midzone (20,23,25,26).Previous studies have identified that the dimerization domain of PRC1 is within the N-terminal domain (Fig. 1A) and that the primary MT binding region relies on a set of conserved residues within the central spectrin domain (PRC1-S) (15,22,2...

Section: Prc1's Disordered C-terminal Domain Forms Electrostatic Intementioning

confidence: 64%

Near-atomic cryo-EM structure of PRC1 bound to the microtubule

Kellogg

Howes

et al. 2016

“…This model contrasts with the conventional model of ATP binding driving full NL docking (34,39). Structural support for the conformational changes that enable NL docking upon ATP binding, either by clamshell-like closure of the "nucleotide cleft" and the associated opening of the "docking cleft" via rotation of the N-terminal subdomain (49) or by reorientation of switch I/II and P-loop subdomains (50), come from comparisons of the no-nucleotide structure to the ADP-aluminum fluoride (ADP-AlFx) structure. If the ADP·AlFx structures actually represent the ADP·P i state (19,51,52) rather than the ATP state [which is further supported by the low tethered head resolution from cryo-EM in AMP-PNP (53)], our data are in full agreement with these structural analyses.…”

Section: Discussionmentioning

confidence: 83%

Kinetics of nucleotide-dependent structural transitions in the kinesin-1 hydrolysis cycle

Mickolajczyk

Deffenbaugh

Arroyo

et al. 2015

124

222

To dissect the kinetics of structural transitions underlying the stepping cycle of kinesin-1 at physiological ATP, we used interferometric scattering microscopy to track the position of gold nanoparticles attached to individual motor domains in processively stepping dimers. Labeled heads resided stably at positions 16.4 nm apart, corresponding to a microtubule-bound state, and at a previously unseen intermediate position, corresponding to a tethered state. The chemical transitions underlying these structural transitions were identified by varying nucleotide conditions and carrying out parallel stopped-flow kinetics assays. At saturating ATP, kinesin-1 spends half of each stepping cycle with one head bound, specifying a structural state for each of two rate-limiting transitions. Analysis of stepping kinetics in varying nucleotides shows that ATP binding is required to properly enter the onehead-bound state, and hydrolysis is necessary to exit it at a physiological rate. These transitions differ from the standard model in which ATP binding drives full docking of the flexible neck linker domain of the motor. Thus, this work defines a consensus sequence of mechanochemical transitions that can be used to understand functional diversity across the kinesin superfamily.kinesin | iSCAT | microscopy | structural kinetics | structure-function K inesin-1 is a motor protein that steps processively toward microtubule plus-ends, tracking single protofilaments and hydrolyzing one ATP molecule per step (1-6).Step sizes corresponding to the tubulin dimer spacing of 8.2 nm are observed when the molecule is labeled by its C-terminal tail (7-10) and to a two-dimer spacing of 16.4 nm when a single motor domain is labeled (4,11,12), consistent with the motor walking in a handover-hand fashion. Kinesin has served as an important model system for advancing single-molecule techniques (7-10) and is clinically relevant for its role in neurodegenerative diseases (13), making dissection of its step a popular ongoing target of study.Despite decades of work, many essential components of the mechanochemical cycle remain disputed, including (i) how much time kinesin-1 spends in a one-head-bound (1HB) state when stepping at physiological ATP concentrations, (ii) whether the motor waits for ATP in a 1HB or two-heads-bound (2HB) state, and (iii) whether ATP hydrolysis occurs before or after tethered head attachment (4,11,(14)(15)(16)(17)(18)(19)(20). These questions are important because they are fundamental to the mechanism by which kinesins harness nucleotide-dependent structural changes to generate mechanical force in a manner optimized for their specific cellular tasks. Addressing these questions requires characterizing a transient 1HB state in the stepping cycle in which the unattached head is located between successive binding sites on the microtubule. This 1HB intermediate is associated with the force-generating powerstroke of the motor and underlies the detachment pathway that limits motor processivity. Optical trapping (7,19,21,22) and sin...

“…1B for Eg5. These labeling sites were selected based on prior structural studies (2,6,7,(9)(10)(11)(12)(17)(18)(19) to detect changes in the distance between the NL or switch-1 and relatively fixed locations in β1 and β7 by using time-resolved FRET between a fluorescent donor (AEDANS) and a nonfluorescent acceptor (DDPM) (20,21).…”

Section: Resultsmentioning

confidence: 99%

“…The NL has been proposed to isomerize between two conformations: one that is flexible and termed undocked, and the other that is ordered and termed docked, where it interacts with a cleft in the motor domain formed by the twisted β-sheet and is oriented along the MT axis (5-7). NL isomerization (5,8) is hypothesized to be the force-generating transition in kinesin motors (6,7,(9)(10)(11), and its position has also been proposed to coordinate the ATPase cycles of processive kinesin dimers by regulating nucleotide binding and hydrolysis (11).…”

mentioning

confidence: 99%

“…MT binding accelerates ADP dissociation, thereby allowing ATP to bind, the NL to dock, and mechanical work to be performed. ATP hydrolysis and phosphate release are then followed by dissociation from the MT to complete the cycle (5,(7)(8)(9)(10)14). This model also argues that: (i) NL docking of the MT-attached motor domain moves the tethered, trailing head into a forward position, where it undergoes a biased diffusional search to attach to the next MT-binding site (11,14); (ii) switch-1, which coordinates the γ-phosphate of ATP, alternates between two conformations, referred to as "open" and "closed," and the NL alternates between docked and undocked (5,6,10,(13)(14)(15); and (iii) coordination between the conformations of switch-1 and the NL regulates the timing of the ATPase cycles of the two motor domains in processive kinesin dimers (11).…”

mentioning

confidence: 99%

See 1 more Smart Citation

The structural kinetics of switch-1 and the neck linker explain the functions of kinesin-1 and Eg5

Muretta

Jun

Gross

et al. 2015

Kinesins perform mechanical work to power a variety of cellular functions, from mitosis to organelle transport. Distinct functions shape distinct enzymologies, and this is illustrated by comparing kinesin-1, a highly processive transport motor that can work alone, to Eg5, a minimally processive mitotic motor that works in large ensembles. Although crystallographic models for both motors reveal similar structures for the domains involved in mechanochemical transduction-including switch-1 and the neck linker-how movement of these two domains is coordinated through the ATPase cycle remains unknown. We have addressed this issue by using a novel combination of transient kinetics and time-resolved fluorescence, which we refer to as "structural kinetics," to map the timing of structural changes in the switch-1 loop and neck linker. We find that differences between the structural kinetics of Eg5 and kinesin-1 yield insights into how these two motors adapt their enzymologies for their distinct functions. here are more than 42 kinesin genes in the human genome, representing 14 distinct classes (1). All are members of the P-loop NTPase superfamily of nucleotide triphosphate hydrolases (2-4). Like other NTPases, kinesins share a conserved Walker motif nucleotide-binding fold (2, 4) that consists of a central twisted β-sheet and three nucleotide-binding loops, which are termed switch-1, switch-2, and the P-loop. Kinesins also share a common microtubule (MT) binding interface, which isomerizes between states that either bind MTs weakly or strongly, and a mechanical element, termed the neck linker (NL). The NL has been proposed to isomerize between two conformations: one that is flexible and termed undocked, and the other that is ordered and termed docked, where it interacts with a cleft in the motor domain formed by the twisted β-sheet and is oriented along the MT axis (5-7). NL isomerization (5, 8) is hypothesized to be the force-generating transition in kinesin motors (6, 7, 9-11), and its position has also been proposed to coordinate the ATPase cycles of processive kinesin dimers by regulating nucleotide binding and hydrolysis (11).Spectroscopic and structural studies have led to a model to explain how kinesins generate force (5-7, 9, 10, 12-15) (summarized in SI Appendix, Fig. S1), which proposes that the conformations of the nucleotide binding site, the MT-binding interface, and the NL are all determined by the state of the catalytic site. It predicts that when unbound to the MT, the motor contains ADP in its catalytic site and its NL is undocked. MT binding accelerates ADP dissociation, thereby allowing ATP to bind, the NL to dock, and mechanical work to be performed. ATP hydrolysis and phosphate release are then followed by dissociation from the MT to complete the cycle (5,(7)(8)(9)(10)14). This model also argues that: (i) NL docking of the MT-attached motor domain moves the tethered, trailing head into a forward position, where it undergoes a biased diffusional search to attach to the next MT-binding site (11,14); (i...