Allosterically Regulated Structural Fluctuation and Microtubule-Binding Affinity of KIF1A– A Simulation Study of Coarse-Grained Model –Macoto Kikuchi, Ryo Kanada∗ and Fumiko Takagi (Cybermedia Center, Osaka University, Japan. ∗ Department of Biophysics, Kyoto University, Japan)

Model

I′II′

III′IV′V′0′

MK ⇋ MKT ⇋ MK∗ T → MKDP → MK∗ DP
MKD ⇋ MK↿⇂↿⇂↿⇂↿⇂↿⇂↿⇂↿⇂K ⇋ KT ⇋ K∗ T → KDP → K∗ DP → KD ⇋ K0I

IIIII

IVV0Figure: KIF1A-MT hydrolysis cycle
K:KIF1A, M:MT, T:ATP, D:ADP, DP:ADP·Pi0’, I’, II’,III’:Strong-binding mode, IV’,V’:Weak-binding mode

MethodA coarse-grained Cα Go-like model for each nucleotide state.
2 Nucleotides are also represented by a few beads
3 Langevin dynamics simulations for investigating the structuralfluctuations when MT is absent.
1

Reference states
IIIIIIIVV

A

Total energy
Vtot = Vp(protein) + Vn(nucleotide) + Vpn(interaction) + Vnn(for ADP·Pi)

2

Intra-molecule energy()2()2∑∑(0)(0)
Vp,n(Γ, Γ(0)) =Kr bi − bi+Kθ θi − θiResults without nucleotide
A 0.25∆Θ0Q switch II[simulation]
Burst frequency∆ΘQ switch II

100

30

[experiment]
Kd [nM]

Frequency

1Pi-2

C40

Pi-1-2-1

C20

10WT
0001

3
6steps ( 10 )24

5Figure: Dynamics of helix α4 for (A)ATP-1 (B) ADP·Pi-2. ∆Θ:Relative angleof α4 to the main body. QswitchII:Contact
fraction of the native contacts between
the switch II and the main body. (C) The
histogram of the duration time steps forburst (blue: on-burst, and red: off-burst)
in the ATP-1 state.

0.05

0.1

0.15

ADP4Pi - 2

3steps ( 10 )Figure: Temperature dependence of theburst frequency without nucleotide.

Figure: Burst frequency with and without
nucleotide

10ADP

60ADP

2WT

0.1

ADP

10.2

ATP0100.

ADP

0.5

10

Figure: Superposition of the five reference structures. Highlighted are the switch II cluster
regions.

0.3(T = 1.3)

Pi-1

11[experiment]
K d [nM]-80

0.8

0.4 0.6Qswitch II0ADPB00.2010 )80

004ATPADP.Pi-2 (1VFZ)
ADP (1I5S)

3B

Table: Nucleotide states and the corresponding analog states. PDB IDs indicate of X-raystructures used as the reference structures of Gō-like models.
ATP-1 (1I6I)
ATP-2 (1VFV)
ADP.Pi-1 (1VFX)

6explicit nucleotide

-2

1I5S

2steps (0.1

ATP

1VFZ

1.

0.2

-1

1VFX

00.05.

T = 1.3

ATP

1VFV

00.1

ADP

1I6I

0.5

0Pi-2

AMP-PCP AMP-PNP ADP-AlFx ADP-Vi ADP

1T = 1.25

ADP

ADP·Pi-1 ADP·Pi-2 ADP

T = 1.3

0.2

Pi-1

PDB ID

ATP-20.15

0.4

ADPanalog

ATP-1

ATP-1
ATP-2
ADP Pi-1
ADP Pi-2
ADP

0.2

T = 1.35-2
ATPweakP( Q switch II )80-80

nucleotide state

1-1ATPstrong

L12

α4The helix α4 at the MT binding
α4site intermittently exhibits
L11bonds
angles[() 1()]
abrupt large structural
∑
(0)
(0)
B+
Kϕ 1 − cos(ϕi − ϕi ) +1 − cos 3(ϕi − ϕi )fluctuations (Burst).
2dihedrals 2 Frequency of the Burst varies
12
10

( )12
nativenon-native(0)(0)
systematically according to thecontacts
contactsrr∑∑  ij 
Cij
+. nucleotide states andknc 5 knnc
 − 6
 +rijrij
rij
correlates strongly with thei<j−3i<j−3experimentally observed
binding affinity to MT.
Γ(0) : referencestructure,rij : distance between the ith and jth Cα,bi = ri i+1
Figure: Snapshots of ATP-1 state. (A) off
3 Interaction energy
burst: QswitchII ∼ 0.75. (B) on burst: 1210 non-native◦
native|∆Θ|∼80and QswitchII ∼ 0.15. blue:α4)((0)(0)contacts
contacts12
∑
∑
rik
C
β8, res: α4, green: L12, orange: L11  rik nnc
−6,Vpn({ri,k }) =kpn
kpn 5
+rik
rik
rik
i,ki,kEffect of temperature and nucleotide(1)
0.8
Similar for Vnn
0.3
0.6

A

binding mode

β8

Burst frequency

0′

1

Burst frequency

Kinesin switches its microtuble (MT) binding affinity between strong
binding mode and weak binding mode depending on nucleotide states.
For understanding this allosteric regulation mechanism, we studied thestructural fluctuations of the motor head of the single-headed kinesin
KIF1A in different nucleotide states using computer simulations ofcoarse-grained Go-like model.

Bust: A large structural fluctuation. .

Motivation

0.2

Burst frequency[simulation]

Figure: (A) Probability distribution QswitchII(B) Nucleotide state dependence of theburst frequency at T = 1.3 and theequilibrium dissociation constants Kd
obtained experimentally(R.Nitta et al.,Science 305(2004) 678) (C) Burst
frequency and the equilibrium
dissociation constants.

ConclusionWe propose the following mechanism for the MT affinity regulation:
1 The flexibility of the MT binding site (helix α4 in particular) is
allosterically controlled according to the nucleotide states during theATP hydrolysis process.
2 The affinity to MT is regulated through the flexibility of the MT bindingsite.
3 The more flexible the MT biniding site is, the stronger the binding
affinity becomes.
R.Kanada, F.Takagi and M.Kikuchi, Proteins 83 (2015) 809.