Origin of the surprising mechanical stability of kinesin’s neck coiled coil

JI Qing

河北工业大学 生物物理研究所

June 2014, Beijing

Institute of Biophysics

Hebei University of Technology

1. Macromolecular dynamics: structure, function and life

R.Vale & R.Milligan, Science, 2000.

ATP hydrolysis:

55 /G kJ mol

5 8

24 /

W pN nm

kJ mol

Work in one step:

molecular motor

ATPase

Kinesin

One motor head:350 amino acids

Driving motility in life.

Tight coupling

2. Neck coiled coil: FunctionKinesin’s mechanochemical cycling needs exquisite inter-head communication and cooperation and gating between the mechanical steps and chemical steps.

Inter-head tension is crucial.

8 nm

Neck linker (4 nm)

The neck coiled coil should keep zippered under inter-head tension.

High efficiency — Tight coupling — No futile hydrolysis.

Experimantal facts: (1) Tomishige & Vale (JCB, 2000)Cross-linking coiled coil had relatively little effect on motor activity.

(3) Bornschlögl, Woehlke and Rief (PNAS, 2009)

The unconventional N-terminal parts of the neck coiled coil exhibit a surprising mechanical stability.The opening of only 2 N-terminal coiled-coil turns will destabilize the complete neck and lead to unfolding.

Substitution of Trp 340, the first hydrophobic core d position residue of the coiled-coil, with an Ala residue resulted in a greater than expected decrease in stability and helicity of the coiled-coil structure.

(2) Tripet and Hodges (JSB, 2002)

3. Neck coiled coil: Structure

Heptad :

The first two turns

The first heptad

abcdefg

A E E W K K K Y E K E K E K ----- W339 346 370

a b c d e f g a b c d e f g d

The neck domain (residues 339–370, 2KIN)

A B

A

B

Structure of conventional coiled coilCohen and Parry (Proteins, 1990)

The key features of coiled coil are as follows:

Fig. 2

Fig. 1

Thormählen, Marx, Sack, and Mandelkow (JSC,1998)Fig. 3

The sequence of neck coiled coil

4. Neck coiled coil: Structure-function relationship

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Dis

tanc

e (Å

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Time (ps)

ALA339:CA-ALA114:CA TRP342:CA-TRP117:CA TYR346:CA-TYR121:CA

50pN

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Time (ps)

ALA339:CA-ALA114:CA TRP342:CA-TRP117:CA TYR346:CA-TYR121:CA

150pN

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tanc

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Time (ps)

ALA339:CA-ALA114:CA TRP342:CA-TRP117:CA TYR346:CA-TRY121:CA

100pN

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10

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Dis

tanc

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Time (ps)

ALA339:CA-ALA114:CA TRP342:CA-TRP117:CA TYR346:CA-TYR121:CA

170pN

4.1 Mechanical response to stretching forces

50 pN:

150 pN:

Partially open and then stable

Stable

100 pN:

Partially open and then stable

170 pN:

Totally open

4.2 Details of the opening process

第一个台阶： Fig. 3

4 ns 5.5 ns 7.5 ns

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4

6

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d 339-3

39 (Å)

Time (ps)

100pN

4 ns

5.5 ns

7.5 nsThe first platform:

Backbone hydrogen bonds breaking

The second platform:

Peptide planes rotation

The unzippering process is stopped at Y346.

Y346

Y346

4.3 Tyrosine buckle and its mechanical function

The buckle structure

O’Shea et al. (Science,1991)

Fig 7. Schematic drawing (not to scale) showing differences betweenpositions a and d.

vdW Radii

H 1.2 Å

O 1.52 Å

N 1.55 Å

C 1.7 Å

Bondi (JPC,1964)

aa’

d’d

4.4 The buckle mechanics

In equilibrium:

0 c sF F

0c x F N

(A + B)

(A)

? 0y N

Constraint force Stretching force

Contact force

Ny is balanced by the special hydrophobic pressure.

A E E W K K K Y E K E K E K 339 342 346

a b c d e f g a b c d e f g

The first two heptads in the neck domain

Lyotropic liquid crystal

N

N’

Nx

Ny

N’x

N’y

Nx = Fc = Fs

y

x

N

N

y x sN N F N’y = N’x = F’s

Micelle

Hydrophobic pressure

phph Forces in equilibrium

0y N P

For (A), define

hdP p

In equilibrium:

P must have an upper limit and so does Fs.

Conclusion

The surprising mechanical stability of the neck coiled coil arises from the cooperation between the special hydrophobic pressure and the steric hindrance of the tyrosine buckle.

5. Special hydrophobic pressure?

The average distance between the Cs of the two Y346s is 4.678Å (the shortest).

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Time (ps)

ALA339:CA-ALA114:CA TRP342:CA-TRP117:CA TYR346:CA-TYR121:CA GLU349:CA-GLU124:CA ASN353:CA-ASN128:CA LEU356:CA-LEU131:CA ILE360:CA-ILE135:CA

100pN

Average

The mechanical function of the Y-buckle is sensitive to the C-C distance.

'y yN N

'x xN N

The special arrangement of the neck residues results in the special hydrophobic pressure.

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r 3 (Å)

Time (ps)

TRP342:CE2-LYS118:HD1100 pN

r3 3.777 Å

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Dis

tance

(Å)

Time (ps)

LYS348:NZ-LYS345:NZ LYS344:NZ-LYS348:NZ LYS343:NZ-LYS344:NZ

100 pN

l1 (blue) 10.382 Å

l2 (red) 11.264 Å

l3 (black) 10.379 Å

Average

Average

A E E W K K K Y E K E K E K 339 342 346

a b c d e f g a b c d e f g

(1) W342(A) vs K343(B)

(2) KKK

(3) ……

Conclusion

Each detail has a reason.

My students in this work

Acknowledgement

刘书霞 (Shu-Xia Liu)

吕 刚 (Gang Lü)

柳睿殊 (Rui-Shu Liu)

耿轶钊 (Yi-Zhao Geng)

张 辉 (Hui Zhang)

The National Natural Science Foundation of China ， No. 90403007

The National Natural Science Foundation of China ， No. 10975044

卓益忠（ Yin-Zhong Zhuo)

包景东（ Jing-Dong Bao)

张红雨（ Hong-Yu Zhang)

郝柏林（ Bo-Lin Hao)

欧阳钟灿（ Zhong-Can Ouyang)

陈润生（ Run-Sheng Chen)