Theory and simulations of condensin mediated loop extrusion in DNA

Condensation of hundreds of mega-base-pair-long human chromosomes in a small nuclear volume is a spectacular biological phenomenon. This process is driven by the formation of chromosome loops. The ATP consuming motor, condensin, interacts with chromatin segments to actively extrude loops. Motivated by real-time imaging of loop extrusion (LE), we created an analytically solvable model, predicting the LE velocity and step size distribution as a function of external load. The theory fits the available experimental data quantitatively, and suggests that condensin must undergo a large conformational change, induced by ATP binding, bringing distant parts of the motor to proximity. Simulations using a simple model confirm that the motor transitions between an open and a closed state in order to extrude loops by a scrunching mechanism, similar to that proposed in DNA bubble formation during bacterial transcription. Changes in the orientation of the motor domains are transmitted over ~50 nm, connecting the motor head and the hinge, thus providing an allosteric basis for LE.

Kinesin-binding protein remodels the kinesin motor to prevent microtubule-binding

Kinesins are tightly regulated in space and time to control their activation in the absence of cargo-binding. Kinesin-binding protein (KIFBP) was recently discovered to bind the catalytic motor heads of 8 of the 45 known kinesin superfamily members and inhibit binding to microtubules. In humans, mutation of KIFBP gives rise to Goldberg-Shprintzen syndrome (GOSHS), but the kinesin(s) that is misregulated to produce clinical features of the disease is not known. Understanding the structural mechanism by which KIFBP selects its kinesin binding partners will be key to unlocking this knowledge. Using a combination of cryo-electron microscopy and crosslinking mass spectrometry, we determined structures of KIFBP alone and in complex with two mitotic kinesins, revealing regions of KIFBP that participate in complex formation. KIFBP adopts an alpha-helical solenoid structure composed of TPR repeats. We find that KIFBP uses a 2-pronged mechanism to remodel kinesin motors and block microtubule-binding. First, KIFBP engages the microtubule-binding interface and sterically blocks interaction with microtubules. Second, KIFBP induces allosteric conformational changes to the kinesin motor head that displace a key structural element in the kinesin motor head (α-helix 4) required for microtubule binding. We identified two regions of KIFBP necessary for in vitro kinesin-binding as well as cellular regulation during mitosis. Taken together, this work establishes the mechanism of kinesin inhibition by KIFBP and provides the first example of motor domain remodeling as a means to abrogate kinesin activity.

On the theory of condensin mediated loop extrusion in genomes

The condensation of several mega base pair human chromosomes in a small cell volume is a spectacular phenomenon in biology. This process, involving the formation of loops in chromosomes, is facilitated by ATP consuming motors (condensin and cohesin), that interact with chromatin segments thereby actively extruding loops. Motivated by real time videos of loop extrusion (LE), we created an analytically solvable model, which yields the LE velocity as a function of external load acting on condensin. The theory fits the experimental data quantitatively, and suggests that condensin must undergo a large conformational change, triggered by ATP binding and hydrolysis, that brings distant parts of the motor to proximity. Simulations using a simple model confirm that a transition between an open and closed states is necessary for LE. Changes in the orientation of the motor domain are transmitted over ~ 50 nm, connecting the motor head and the hinge, thus providing a plausible mechanism for LE. The theory and simulations are applicable to loop extrusion in other structural maintenance complexes.

Parsing the roles of neck-linker docking and tethered head diffusion in the stepping dynamics of kinesin

Kinesin walks processively on microtubules (MTs) in an asymmetric hand-over-hand manner consuming one ATP molecule per 16-nm step. The individual contributions due to docking of the approximately 13-residue neck linker to the leading head (deemed to be the power stroke) and diffusion of the trailing head (TH) that contributes in propelling the motor by 16 nm have not been quantified. We use molecular simulations by creating a coarse-grained model of the MT–kinesin complex, which reproduces the measured stall force as well as the force required to dislodge the motor head from the MT, to show that nearly three-quarters of the step occurs by bidirectional stochastic motion of the TH. However, docking of the neck linker to the leading head constrains the extent of diffusion and minimizes the probability that kinesin takes side steps, implying that both the events are necessary in the motility of kinesin and for the maintenance of processivity. Surprisingly, we find that during a single step, the TH stochastically hops multiple times between the geometrically accessible neighboring sites on the MT before forming a stable interaction with the target binding site with correct orientation between the motor head and the α/β tubulin dimer.

Parsing the roles of neck-linker docking and tethered head diffusion in the stepping dynamics of kinesin

Kinesin walks processively on microtubules (MTs) in an asymmetric hand-over-hand manner consuming one ATP molecule per 16 nm step. The contributions due to docking of the approximately thirteen residue neck linker to the leading head (deemed to be the power stroke), and diffusion of the trailing head contribute in propelling the motor by 16 nm have not been quantified. We use molecular simulations by creating a new coarse-grained model of the microtubule-kinesin complex, which reproduces the measured stall force as well as the force required to dislodge the motor head from the MT, to show that nearly three quarters of the step occurs by bidirectional stochastic motion of the TH. However, docking of the neck linker to the leading head constrains the extent of diffusion and minimizes the probability that kinesin takes side steps implying that both the events are necessary in the motility of kinesin, and for the maintenance of processivity. Surprisingly, we find that during a single step the trailing head stochastically hops multiple times between the geometrically accessible neighboring sites on the MT prior to forming a stable interaction with the target binding site with correct orientation between the motor head and the α/ß tubulin dimer.Significance StatementLike all motors, the stepping of the two headed conventional Kinesin on the microtubule is facilitated by conformational changes in the motor domain upon ATP binding and hydrolysis. Numerous experiments have revealed that docking of the thirteen residue neck linker (NL) to the motor domain of the leading plays a critical role in propelling the trailing head towards the plus end of the microtubule by nearly 16 nm in a single step. Surprisingly our molecular simulations reveal that nearly three quarters of the step occurs by stochastic diffusion of the trailing head. Docking of the NL restricts the extent of diffusion, thus forcing the motor to walk with overwhelming probability on a single protofilament of the MT.