Kinesin-1 is an ATP-driven, processive motor that transports cargo along microtubules in a tightly regulated stepping cycle. Efficient gating mechanisms ensure that the sequence of kinetic events proceeds in proper order, generating a large number of successive reaction cycles. To study gating, we created two mutant constructs with extended neck-linkers and measured their properties using single-molecule optical trapping and ensemble fluorescence techniques. Due to a reduction in the inter-head tension, the constructs access an otherwise rarely populated conformational state where both motor heads remain bound to the microtubule. ATP-dependent, processive backstepping and futile hydrolysis were observed under moderate hindering loads. Based on measurements, we formulated a comprehensive model for kinesin motion that incorporates reaction pathways for both forward and backward stepping. In addition to inter-head tension, we find that neck-linker orientation is also responsible for ensuring gating in kinesin.

Kinesin-1 is a dimeric motor that transports cargo along microtubules, taking 8.2-nm steps in a hand-over-hand fashion. The ATP hydrolysis cycles of its two heads are maintained out of phase by a series of gating mechanisms, which lead to processive runs averaging ∼1 μm. A key structural element for inter-head coordination is the neck linker (NL), which connects the heads to the stalk. To examine the role of the NL in regulating stepping, we investigated NL mutants of various lengths using single-molecule optical trapping and bulk fluorescence approaches in the context of a general framework for gating. Our results show that, although inter-head tension enhances motor velocity, it is crucial neither for inter-head coordination nor for rapid rear-head release. Furthermore, cysteine-light mutants do not produce wild-type motility under load. We conclude that kinesin-1 is primarily front-head gated, and that NL length is tuned to enhance unidirectional processivity and velocity.DOI: http://dx.doi.org/10.7554/eLife.07403.001

Kinesin-1 is a dimeric motor protein, central to intracellular transport, that steps hand-over-hand toward the microtubule (MT) plus-end, hydrolyzing one ATP molecule per step. Its remarkable processivity is critical for ferrying cargo within the cell: over 100 successive steps are taken, on average, before dissociation from the MT. Despite considerable work, it is not understood which features coordinate, or "gate," the mechanochemical cycles of the two motor heads. Here, we show that kinesin dissociation occurs subsequent to, or concomitant with, phosphate (P i ) release following ATP hydrolysis. In optical trapping experiments, we found that increasing the steady-state population of the posthydrolysis ADP·P i state (by adding free P i ) nearly doubled the kinesin run length, whereas reducing either the ATP binding rate or hydrolysis rate had no effect. The data suggest that, during processive movement, tethered-head binding occurs subsequent to hydrolysis, rather than immediately after ATP binding, as commonly suggested. The structural change driving motility, thought to be neck linker docking, is therefore completed only upon hydrolysis, and not ATP binding. Our results offer additional insights into gating mechanisms and suggest revisions to prevailing models of the kinesin reaction cycle.single molecule | mechanochemistry | optical tweezers | molecular motors S ince its discovery nearly 30 years ago (1), kinesin-1-the founding member of the kinesin protein superfamily-has emerged as an important model system for studying biological motors (2, 3). During "hand-over-hand" stepping, kinesin dimers alternate between a two-heads-bound (2-HB) state, with both heads attached to the microtubule (MT), and a one-head-bound (1-HB) state, where a single head, termed the tethered head, remains free of the MT (4, 5). The catalytic cycles of the two heads are maintained out of phase by a series of gating mechanisms, thereby enabling the dimer to complete, on average, over 100 steps before dissociating from the . A key structural element for this coordination is the neck linker (NL), a ∼14-aa segment that connects each catalytic head to a common stalk (9). In the 1-HB state, nucleotide binding is thought to induce a structural reconfiguration of the NL, immobilizing it against the MT-bound catalytic domain (2,3,(10)(11)(12)(13)(14)(15)(16)(17). This transition, called "NL docking," is believed to promote unidirectional motility by biasing the position of the tethered head toward the next MT binding site (2,3,(10)(11)(12)(13)(14)(15)(16)(17). The completion of an 8.2-nm step (18) entails the binding of this tethered head to the MT, ATP hydrolysis, and detachment of the trailing head, thereby returning the motor to the ATP-waiting state (2,3,(10)(11)(12)(13)(14)(15)(16)(17). Prevailing models of the kinesin mechanochemical cycle (2,3,10,14,15,17), which invoke NL docking upon ATP binding, explain the highly directional nature of kinesin motility and offer a compelling outline of the sequence of events following ATP binding....

The bacterial adaptive immune system CRISPR-Cas9 has been appropriated as a versatile tool for editing genomes, controlling gene expression, and visualizing genetic loci. To analyze Cas9's ability to bind DNA rapidly and specifically, we generated multiple libraries of potential binding partners for measuring the kinetics of nucleasedead Cas9 (dCas9) interactions. Using a massively parallel method to quantify protein-DNA interactions on a high-throughput sequencing flow cell, we comprehensively assess the effects of combinatorial mismatches between guide RNA (gRNA) and target nucleotides, both in the seed and in more distal nucleotides, plus disruption of the protospacer adjacent motif (PAM). We report two consequences of PAM-distal mismatches: reversal of dCas9 binding at long time scales, and synergistic changes in association kinetics when other gRNA-target mismatches are present. Together, these observations support a model for Cas9 specificity wherein gRNA-DNA mismatches at PAM-distal bases modulate different biophysical parameters that determine association and dissociation rates. The methods we present decouple aspects of kinetic and thermodynamic properties of the Cas9-DNA interaction and broaden the toolkit for investigating off-target binding behavior.DNA | molecular biophysics | kinetics | sequencing | CRISPR C RISPR-associated protein 9 (Cas9) is programmed to bind its target DNA by a guide RNA (gRNA) that, once loaded, forms a ribonucleoprotein (RNP) complex. The Streptococcus pyogenes CRISPR system, the most extensively studied and applied system to date, targets a 23-bp DNA sequence containing (i) an "NGG" protospacer adjacent motif (PAM) element downstream of the single-guide RNA (sgRNA) target DNA (1) and (ii) a 20-bp sequence upstream of the PAM bearing complementarity to the gRNA (2). Genome engineering applications leverage the nuclease activity of the Cas9 RNP, but Cas9 engineered to lack the residues required for cleavage [dCas9 (nuclease-dead Cas9)] has proven valuable by enabling the creation of customizable and programmable DNA binding elements that can activate and repress gene expression with high precision (CRISPRa and CRISPRi) (3).The biophysical underpinnings of the Cas9 target search have been investigated both by directed biochemical assays (4, 5) and through measurements of off-target Cas9 activity (6-11). These studies have led to a model for binding wherein Cas9 proceeds through a series of steps starting with PAM recognition, followed by DNA melting, RNA strand invasion, and heteroduplex formation dependent on complementarity with a 5-10-bp seed. Structural data have further suggested that conformational changes in the HNH domain reposition catalytic residues and permit allosteric regulation of the RuvC domain. This conformational gating ensures that cleavage occurs only in the context of substantial homology between gRNA and target (12, 13).The specificity of Cas9 DNA binding is crucial for all potential applications of Cas9's RNA-programmable targeting. Localization of dCas9...

Summary The response of motor proteins to external loads underlies their ability to work in teams and determines the net speed and directionality of cargo transport. The mammalian kinesin-2, KIF3A/B, is a heterotrimeric motor involved in intraflagellar transport and vesicle motility in neurons. Bidirectional cargo transport is known to result from the opposing activities of KIF3A/B and dynein bound to the same cargo, but the load-dependent properties of kinesin-2 are poorly understood. We used a feedback-controlled optical trap to probe the velocity, run length and unbinding kinetics of mouse KIF3A/B under various loads and nucleotide conditions. The kinesin-2 motor velocity is less sensitive than kinesin-1 to external forces, but its processivity diminishes steeply with load, and the motor was observed occasionally to slip and reattach. Each motor domain was characterized by studying homodimeric constructs, and a global fit to the data resulted in a comprehensive pathway that quantifies the principal force-dependent kinetic transitions. The properties of the KIF3A/B heterodimer are intermediate between the two homodimers, and the distinct load-dependent behavior is attributable to the properties of the motor domains, and not to the neck-linkers or the coiled-coil stalk. We conclude that the force-dependent movement of KIF3A/B differs significantly from conventional kinesin-1. Against opposing dynein forces, KIF3A/B motors are predicted to rapidly unbind and rebind, resulting in qualitatively different transport behavior from kinesin-1.