1. Interhead tension determines processivity across diverse N-terminal kinesins
M.Yahara

2. Interhead tension determines processivity
across diverse N-terminal kinesins
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA vol.108 p SEP
Shankar Shastry and William O. Hancock
Department of Bioengineering, Pennsylvania State University
William O. Hancock
This is the second introduction of the paper for me.
Today, I introduced the paper from PNAS on September the 27th in 2011.
Title is “Interhead tension determines processivity across diverse N-terminal kinesins”
written by Shankar Shastry and William O. Hancock.

3. X-ray structure of Kinesin 1
Light Blue
Yellow
This is the X-ray Structure of the conventional kinesin.
The left one is the whole figure.
What the colors showed are written below the figures.
As you know, Kinesin 1 has the tetrameric structure.
The two α-Helixs in the center of the picture are called coiled-coil.
Two globular part is called kinesin’s head, including motor domain like ATP and Microtubule binding site.
Left one is the enlarged view of motor domain.
Please be careful about the changed direction.
And , what is connecting these two parts is called Neck Linker.
This is the principal factor of what I talked today.
Heavy Chain
Blue
Green
Light　Chain
Red
… ADP
… Neck Linker(NL)
Wikipedia キネシン

4. Neck Linker
When ATP bind to the kinesin, Neck Linker docks the core motor domain.
Neck Linker domain limits the entropic spring properties
In general, Neck Linker Domain has three important roles.
First , when ATP bind to the kinesin, Neck Linker docks the core motor domain.
This process is thought to be the key structural changes in kinesin’s step.
Second, Neck Linker domain limits the entropic spring properties
for the kinesin motor domain to bind the next binding site.
Third, when both heads are simultaneously bound to the microtubule,
through Neck Linker domains, interhead mechanical forces are transmitted, and causes asymmetric movement of heads.
Interhead mechanical forces are transmitted.

5. Processivity The Degree of Processivity
is defined as the number of steps a motor takes
per interaction with a microtubule.
Differences of processivity in diverse N-terminal kinesins
The Degree of Processivity is defined as the number of steps a motor takes per interaction with a microtubule.
This degree of processivity is expressed as a distance from one kinesin’s attachment to detachment. , ｔhat is Run Length ,in short.
As you know, diverse kinesins are existed ,
and it was reported that each diverse kinesin ,that is N-terminal kinesin, showed different processivity.
In general, like differences in motor speed, it was thought to result from differences in the biochemical properties of their catalytic motor domain.
In another words, it was thought these differences depend on the differences in the rates of inherent kinetic cycle.
For instance, a slow rate of hydrolysis or slow rate of detachment from the microtubule should enhance processivity.
whereas fast detachment in the ADP or ADP-Pi state should reduce processivity.
Differences in the rate of ATP hydrolysis cycle?

6. Neck Linker Domain Constructs
Head
Neck Linker Domain(amino acids)
Kinesin-1 14
kinesin-2 17
Kinesin-3
Kinesin-5 18
Kinesin-7
However, the neck linker domain ,which also have the important role to kinetics of kinesin’s stepping,
Neck linker varies between kinesin families, ranging from 14 to 18 amino acids.
This figure shows the amino acids sequences of the neck linker domain.
Focusing this point, They did experiments to investigate the role of neck linker length.

7. Processivity of Kinesin-1
kinesin-2+GFP
kinesin-1+GFP
Head kinesin-2
Rod kinesin-1 +GFP
First, they investigated the influence of neck linker length on kinsin-1 processivity.
They modified kinesin-1 C-terminal of the neck linker sequences,
and examined single motor runs.

8. Processivity of Kinesin-1
kinesin-1+GFP
Head kinesin-2
Rod kinesin-1 +GFP
When the Neck Linker domain was extended, corresponding to the last one or three residues of the kinesin-2 neck linker, the run-length was dropped.
And , shortening the one length abolished the processivity.
These results demonstrate that kinesin-1 processivity is significantly reduced when the neck linker domain is changed by even one amino acid.

9. Processivity of Kinesin-2
kinesin-1+GFP
Head kinesin-2
Rod kinesin-1 +GFP
Next, Kinesin-2 has a 17 amino acid neck linker and this is three residues longer than Kinsin-1.
And this kinsin-2 is 4-fold less processive than kinesin-1.
So,they assumed that if the longer neck domain reduced the processivity,
shortening the kinsin-2 neck linker domain should enhance processivity as the figure fit the blue line.
To test this , they fused the Kinsin-2, head and shortening neck linker to the neck coil and rod
and examined the run length.

10. Processivity of Kinesin-2
kinesin-1+GFP
Head kinesin-2
Rod kinesin-1 +GFP
P→A
This red line was the results.
The figure didn’t fit the blue one.
So, they examined the structure , using with the molecular dynamics simulations.
From this simulation, the proline existing in the neck linker sequences may shorten the length of neck linker.
They changed the proline to alanine, and the experiment by same way was done.

11. Processivity of Kinesin-2
kinesin-1+GFP
Head kinesin-2
Rod kinesin-1 +GFP
This white spots with red line is the result.
This figure matched the blue one.
So, they concluded that Neck Linker length determines the degree of processivity in kinesin-1 and kinesin-2 motors.
This is the research they wrote in 2010.

12. N-terminal kinesins kinesin-3 kinesin-5 kinesin-7
In this paper I introduced to you, they extended the objects and tried to identify the degree to which the length of the neck linker controls motor properties.
They used heavy chain’s dimer of another three N-terminal kinesin ; kinesin-3,5,7.
They combined their motor domain and neck linker domain to kinesin-1’s rod domain as the same way.
kinesin-3
kinesin-5
kinesin-7

13. Processivity of Kinesin-3
Original kinesin-3 has 17 neck linker length.
They investigated the run length , shortening the neck linker sequences.
And the one Kinesin-3 head and neck linker fused to the kinesin-1 rod showed similar figure to the kinsin-1’s.

14. Processivity of Kinesin-5
Wild type Kinesin-5 is constructed as a tetramer, and has 18 neck linker length.
Kinesin-5 is usually said to be minimally processive,
although dimeric Kinesin-5 showed the motor activities in a microtubule gliding assay.
Some processive runs are observed in 17 and 18 neck linker length.
But the high fluoresence intensity of the moving spots was observed , and this suggests these were aggregates, and not single molecule events.
So, this results could not be reliable about 17 and 18.
When the neck linker length was shortened to 14 residues by deleting the last four amino acids, processiviity increased dramatically .
This result demonstrates shortening the neck linker enhance processivity, in other words, enhance head-head coordination to asymmetric movement of the heads.

15. Processivity of Kinesin-7
Until now, the relation between the neck linker length and run length is clearly demonstrated.
But about kinesin-7 the result is different from these.
Kinesin-7 has the 18 neck linker length, and was showed high processivity same as kinesin-1 which has 14 residue neck linker length.
And in contrast to the other N-terminal kinesins, kinesin-7 processivity decreased when its neck linker was shortened.

16. Processivity of Kinesin-7
Kin7_TR;T→R
They noted the difference between RAK sequences in other N-terminal kinesins and TAK sequences in kinesin-7,
and build up the hypothesis that threonine(T) in TAK sequences destabilized the α6 ,being strong relationship to another threonine structurally, and this becomes the cause of extending neck linker length.
So, they changed the residue threonine(T) to arginine(R).
This mutated kinesin7 neck linker showed the figure similar to others’.

17. Processivity depend on Neck Linker Length
Taken up together, these results showed that processivity depend so strongly on neck linker length.

18. Processivity depend on Neck Linker Length
They also plotted motor velocity as a function of run length.
This results showed , although changing neck linker length dramatically changed the run length, it has a minimal influence on motor velocity.

19. Conclusions
The N-terminal kinesin neck linker domain is the primary determinant of processivity.
The varieties of run length of diverse N-terminal kinesins results not from tuning of biochemical rate , but from differences of neck linker length.

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