William M. Behnke-Parks

Direct measurement of the full, sequence-dependent folding landscape of a nucleic acid

Nucleic acid hairpins provide a powerful model system for understanding macromolecular folding, with free-energy landscapes that can be readily manipulated by changing the hairpin sequence. The full shapes of energy landscapes for the reversible folding of DNA hairpins under controlled loads exerted by an optical force clamp were obtained by deconvolution from high-resolution, single-molecule trajectories. The locations and heights of the energy barriers for hairpin folding could be tuned by adjusting the number and location of G:C base pairs, and the presence and position of folding intermediates were controlled by introducing single-nucleotide mismatches.

A universal pathway for kinesin stepping

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 the 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. Owing to a reduction in the inter-head tension, the constructs access an otherwise rarely populated conformational state in which both motor heads remain bound to the microtubule. ATP-dependent, processive backstepping and futile hydrolysis were observed under moderate hindering loads. On the basis of 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 found that neck-linker orientation is also responsible for ensuring gating in kinesin.

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

Background: Kinesin-5 motors are important for formation and maintenance of the bipolar mitotic spindle. Results: ATP binding triggers coupled conformational changes of kinesin-5 specific structural elements in the microtubule-bound motor domain. Conclusion: Kinesin-5 mechanochemistry is tuned to its cellular functions. Significance: Subnanometer resolution structure determination of microtubule-bound kinesin-5s and kinetics experiments reveal the molecular basis of their motor properties and of drug inhibition. Kinesin-5 is required for forming the bipolar spindle during mitosis. Its motor domain, which contains nucleotide and microtubule binding sites and mechanical elements to generate force, has evolved distinct properties for its spindle-based functions. In this study, we report subnanometer resolution cryoelectron microscopy reconstructions of microtubule-bound human kinesin-5 before and after nucleotide binding and combine this information with studies of the kinetics of nucleotide-induced neck linker and cover strand movement. These studies reveal coupled, nucleotide-dependent conformational changes that explain many of this motors properties. We find that ATP binding induces a ratchet-like docking of the neck linker and simultaneous, parallel docking of the N-terminal cover strand. Loop L5, the binding site for allosteric inhibitors of kinesin-5, also undergoes a dramatic reorientation when ATP binds, suggesting that it is directly involved in controlling nucleotide binding. Our structures indicate that allosteric inhibitors of human kinesin-5, which are being developed as anti-cancer therapeutics, bind to a motor conformation that occurs in the course of normal function. However, due to evolutionarily defined sequence variations in L5, this conformation is not adopted by invertebrate kinesin-5s, explaining their resistance to drug inhibition. Together, our data reveal the precision with which the molecular mechanism of kinesin-5 motors has evolved for force generation.

Loop L5 acts as a conformational latch in the Mitotic Kinesin Eg5

All members of the kinesin superfamily of molecular motors contain an unusual structural motif consisting of an α-helix that is interrupted by a flexible loop, referred to as L5. We have examined the function of L5 in the mitotic kinesin Eg5 by combining site-directed mutagenesis of L5 with transient state kinetics, molecular dynamics simulations, and docking using cryo electron microscopy density. We find that mutation of a proline residue located at a turn within this loop profoundly slows nucleotide-induced structural changes both at the catalytic site as well as at the microtubule binding domain and the neck linker. Molecular dynamics simulations reveal that this mutation affects the dynamics not only of L5 itself but also of the switch I structural elements that sense ATP binding to the catalytic site. Our results lead us to propose that L5 regulates the rate of conformational change in key elements of the nucleotide binding site through its interactions with α3 and in so doing controls the speed of movement and force generation in kinesin motors.

The ATPase cycle of the mitotic motor CENP-E.

We have previously shown that the mitotic motor centrosome protein E (CENP-E) is capable of walking for more than 250 steps on its microtubule track without dissociating. We have examined the kinetics of this molecular motor to see if its enzymology explains this remarkable degree of processivity. We find that like the highly processive transport motor kinesin 1, the enzymatic cycle of CENP-E is characterized by rapid ATP binding, multiple enzymatic turnovers per diffusive encounter, and gating of nucleotide binding. These features endow CENP-E with a high duty cycle, a prerequisite for processivity. However, unlike kinesin 1, neck linker docking in CENP-E is slow, occurring at a rate closer to that for Eg5, a mitotic kinesin that takes only 5–10 steps per processive run. These results suggest that like kinesin 1, features outside of the catalytic domain of CENP-E may also play a role in regulating the processive behavior of this motor.

Loop L5 Assumes Three Distinct Orientations during the ATPase Cycle of the Mitotic Kinesin Eg5 A TRANSIENT AND TIME-RESOLVED FLUORESCENCE STUDY

Background: Loop L5 shapes the enzymology of kinesin motors to meet specific physiologic roles. Results: L5 is in a dynamic equilibrium of three conformations whose relative proportions shift during the ATPase cycle. Conclusion: Interactions between L5 and helix α3 modulate the kinetics of microtubule binding. Significance: Combining transient kinetics with time-resolved fluorescence reveals important insights in the structural dynamics of molecular motors. Members of the kinesin superfamily of molecular motors differ in several key structural domains, which probably allows these molecular motors to serve the different physiologies required of them. One of the most variable of these is a stem-loop motif referred to as L5. This loop is longest in the mitotic kinesin Eg5, and previous structural studies have shown that it can assume different conformations in different nucleotide states. However, enzymatic domains often consist of a mixture of conformations whose distribution shifts in response to substrate binding or product release, and this information is not available from the “static” images that structural studies provide. We have addressed this issue in the case of Eg5 by attaching a fluorescent probe to L5 and examining its fluorescence, using both steady state and time-resolved methods. This reveals that L5 assumes an equilibrium mixture of three orientations that differ in their local environment and segmental mobility. Combining these studies with transient state kinetics demonstrates that there is a major shift in this distribution during transitions that interconvert weak and strong microtubule binding states. Finally, in conjunction with previous cryo-EM reconstructions of Eg5·microtubule complexes, these fluorescence studies suggest a model in which L5 regulates both nucleotide and microtubule binding through a set of reversible interactions with helix α3. We propose that these features facilitate the production of sustained opposing force by Eg5, which underlies its role in supporting formation of a bipolar spindle in mitosis.

A Universal Pathway for Kinesin Stepping

Kinesin-1 is a processive motor that transports cargo along microtubules by hydrolyzing ATP 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 both single-molecule optical trapping and ensemble fluorescence techniques. Due to a reduction in the inter-head tension, the constructs can 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 intramolecular strain, we find that neck-linker orientation is also responsible for ensuring gating in kinesin-1.