Intramolecular Strain coordinates Kinesin Stepping Behavior along Microtubules

Ahmet Yildiz1, Michio Tomishige, Arne Gennerich,1 and Ronald D. Vale1,2

1.Department of Cellular and Molecular Pharmacology and the Howard Hughes Medical Institute, University of California, 2.Department of Applied Physics, University of Tokyo,

INSPIRATION
An engineer's perspective: Nanomachines Interest in class generated when motor proteins were discussed Tremendous amount of research is STILL going on in this field

INTRODUCTION
Kinesins: Motor proteins that move along microtubule filaments. Movement is powered by ATP hydrolysis Contains two domains, two neck linker (mechanical elements), neck coiled coil Kinesins advance 8 nm along the microtubule per ATP hydrolysed

INTRODUCTION
GATING MECHANISM

CHEMICAL GATING The ATP binding to the nucleotide-free front head is inhibited until the rear head detaches.
MECHANICAL GATING After detachment, the rear end will not attach to the tubulin filament until ATP hydrolysis occurs. POWER STROKE Detachment of the rear end is triggered by a tension generated from a power stroke in the front head.

HYPOTHESIS
Whether intramolecular tension, generated by 'neck linkers' coordinates the movement of two kinesin motor domains Support of the POWER stroke Gating mechanism

OVERVIEW
Does tension have any role to play in Kinesin stepping? Decrease tension Increase tension If tension does have a role, how is it affecting the movement? Coupling ATP hydrolysis How does the coordination happen? Motor domain sensing Neck linker's role

MODELING
Cys light kinesin gene used as a template for mutagenesis Between neck linker and neck coiled coil, inserts of varying lengths of proline and glycine serine residues were inserted Inserts were flanked by two N- terminal lysines and a C-terminal glycine Motility Assay Sea urchin axonemes were immobilized. Biotinylated Kinesin motor was then Perfused in BRB12 casein buffer

TECHNIQUES
PCR Cloning insertion of proline residues or 7 repeats of glycine - serine residues Total Internal Reflection Fluorescence Microscopy measurement of speed and run lengths of Kinesin Quantum dot measuring the step size and sideways movement of a single kinesin molecule. Optical trap provides external force to single GFP tagged kinesin motor coupled to 1 micron beads via an anti-GFP antibody.

Result 1 MOTILITY PROPERTIES OF KINESIN WITH EXTENDED NECK LINKERS

Processivity remains for at least 100 steps

Velocity decreases with increase in inserts

Turnover number of ATP is nearly same

Coupling of ATP hydrolysis with stepping is IMPAIRED

CHEMICAL CROSSLINKING OF KINESIN NECK LINKER

Neck linkers cross linked using by insertion of Cysteine using a crosslinking agent

Speed histogram showing the peak at 116 nm/s of Kinesin with 13P extended linker

Speed histogram showing the two peaks after cross linking

Result 2 STUDY OF STEPPING BEHAVIOUR OF KINESIN WITH EXTENDED LINKERS

Highly Variable steps are taken by Kinesin mutants(at constant speed), proving that the Mechanical properties of neck linkers do matter in stepping behaviour

Result 3 EFFECT OF TENSION ON CHEMOMECHANICAL COUPLING

External Load on the attached beads (using anti-GFP) was applied using Optical trap method

Individual traces of WT, 13P, 14GS,and 26P under 3 pN (blue), 6 pN (green) and 9 pN (red) forward load at 1 mM ATP show stepwise movement (inserts).

Histograms of center-of-mass steps at a 9 pN assisting load show a larger average step size for 26P.

CHEMO-MECHANICAL COUPLING WAS RESTORED

On calculating, stepping rates for WT (56.2 s-1: 449 nm/s velocity divided by a 7.98 nm step) and 26P kinesin (54.73 s-1: 630 nm/s velocity divided by an 11.51 nm step)

Result 4: ATP-INDEPENDENT KINESIN MOVEMENT UNDER LOAD

Has ATP got any role to play in coordination and movement of Kinesin?

Stepping movement was seen under two dimensional forces, ruling out the possibility of slipping on microtubules. This proves the fact that tension is what mediates the co-ordination in motor heads.

Summarizing, force required for forward movement of Kinesin is dependent on state of the rear head.

Result 5: ROLE OF NECK LINKERS IN COORDINATION AND MOVEMENT OF KINESIN MOTOR HEADS
Neck linkers in WT Kinesin were replaced by 19 Proline construct

It shows that a kinesin lacking its mechanical element and ATP, can undergo 8 nm unidirectional stepping if external tension is applied.
Motor domains sense only TENSION and acts accordingly !

CONCLUSION
The results prove that intramolecular tension does play a role in stepping Suggest a POWER stroke model for communication between kinesin heads (i) Intramolecular strain promotes rear head detachment (ii) The neck linker has to dock to pull the detached head Sensor for tension-induced attachment is within the microtubule interface of the kinesin motor domain itself

DISCUSSION

STRENGTHS OF THE PAPER

Direct evidence that intramolecular tension facilitates kinesin stepping External load (an increase in tension) can cause kinesin movement even without ATP binding or neck linkers Proposed a POWER stroke model for communication between the motor domains

DISCUSSION

WEAKNESSES OF THE PAPER

This model does not account for ATP gating which surely must also exist Does strain dependent release operate at very low ATP concentrations? (One head-bound state by Mori et al)

Drawbacks
Modeling:
Cys light kinesin shows different kinetic parameters1.

Introduction of Lysine
Increase in electrostatic interactions in linkers, may have reason for increase in processivity(which was contradictory to previous results).

Tubulin Gating Mechanism

Also stands as possibility was not even considered, two head binding configuration being strained2 is basis of this mechanism.

References: 1.Stepping and stretching. How kinesin uses internal strain to walk processively,J. Biol. Chem., 278 (2003), pp. 1855018556 by Rosefeld et al. 2. Direct observation of the binding state of the kinesin head to the microtubule by Nicholas R. Guydosh et al.

Thank You