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2018年11月7日
横浜市立大学 生命医科学研究科
池口 満徳/• 5I>H=9;DIN/ ,."• ;ACGK;DI.0–

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1N G@E7!/%

Conformational Changes of β subunit
upon ATP binding
γγ

βE

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Drug
Extrusion

H+

Drug

Binding

AccessBinding

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H+Access

Drug

H+
AccessBindingDrugExtrusion

S. Murakami, PNE, 52 (2007), 406-414

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9=<@/$
ü9?6K*#Mhttp://www.nobelprize.org/NHashido, Ikeguchi, Kidera,FEBS. Lett., 579, 5549, 2005
Hashido, Kidera, Ikeguchi,Biophys. J., 93, 373, 2007

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1969-1977     
M. Levitt, The birth of computational structural biology, Nature Struct. Biol.
8, 392, (2001)

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分子動力学シミュレーションの解析・応用

MD7"70B38QR• %<?A1C!B–

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$<93/&46'46,B.S,.S• '<)C!B• C!B– LEOI8<=+93/,.S•  (CB–DJMFP+DOGMKP+DOHNKPCB• % C!B•– 9<@-;+$=%5B<.S*85B%CB– NMR>SAXS:9<*8C#MD•   – 

MD68:<>6;=.: RMSD, RMSF
Root Mean Square Deviation (RMSD)ü %1.*34ü .
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7RMSD from crystal structure)

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Root Mean Square Deviation (RMSD)

RMSD =

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ü MD&  ,*&%(ü /1-5!#*8&RMSD' $*/1-5RMSD'

domain A Time (ns)domain B

RCAOroguchi, MI et al., Biophys. J., 96, 2808 (2009)

RCB

MDORTWZOUXG.$: RMSD, RMSF
Root Mean Square Deviation (RMSD)
ü '0;JGBLM-%ü 3G4
ü 65G

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G7685H#/*=?EKPSNX7685H@:RMSD % =∑( )( (%) − )-./,(21RMSF 4 =∑5 )( (%) − )67.,(81

Root Mean Square Fluctuation (RMSF)
ü 0;JGBL[ü)Y")&\G4ü J:D:K+9:+Mü ]!02HJ><@:ü VZQ2HJ><=:Conformational Changes of β subunit
upon ATP bindingγγ

βEβE

βTPβTPProtein Fluctuation (RMSF)
Empty; Open)ATP bound; Closed)
ADP bound; Closed)

ü Fluctuation of C-terminal domain: βE>βTP>βDPü Despite fairly similar conformations of βTP and βDP,
fluctuations of C-terminal domain are different.

Y. Ito & M. Ikeguchi, J. Comp. Chem. 31, 2175 (2010)

βE

βTP

MD
•

#%& !"$ #– "'• –

$ # #'MD!#%"$Correlation Matrix &ü 'ü ü :, PCA
Cij =Δri ⋅ Δr j
Δri2 Δr 2j $

Cij = 1 Cij = 0 Cij = −1

Correlated Motion in Protein
Cij = 1 Cij = 0 Cij = −1

Y. Ito & M. Ikeguchi, J. Comp. Chem. 31, 2175 (2010)

MD9;=?A9>@2#: &!, PCA
Correlation Matrix B&!C
ü $&2ü 1

üCij =Δri ⋅ Δr j
Δri2 Δr 2j(-(57:<8@*6)5Principal Component Analysis (PCA)
B#Cü '$ü $71#Cij = 1 Cij = 0 Cij = −17ü 7 3,5+/.
$ 24)0%*6)5!7"∆"# ∆"$% = 1 ⋯ 3*

MD"#$&("%') PCA Principal Component Analysis (PCA)

PCA

ü PCA ! ü  !
 Computational Biochemistry and Biophysics, Becker et al. eds., 2001.

PCAF F1-ATPase β;@B?<=DE
ü Top mode 4$7*3 30 % :68
ü Top ten modes 4$7*3 70 % :68

ü 43AC>4 ATP3:,4$# 2 )1 :.($7*/580$("79-235$7'/'8(&8%Y. Ito & M. Ikeguchi, Chem. Phys. Lett. 490, 80 (2010)0'!

+$

Principal Component Analysis: Mode 1-4üRelaxation from interactions withadjacent subunits

Mode 1: magenta
Mode 2: yellow
Mode 3: cyan

Mode 4: green
βE→βDP: red
Y. Ito & M. Ikeguchi, Chem. Phys. Lett. 490, 80 (2010)üIntrinsic flexibility is well correlatedwith structural transition

MD!!("./•  $&')(–  $# '#0• $ )(–  $#  (0• )(– ,*-+"$  %#0Interaction between ligand and protein

Interaction between CYP1A2 and EPA

Isobe, YI, MI, Arita, Sci. Rep. 8: 7951 (2018)

MD*

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1

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2)3, − 2)38:4; ∆" = −%& ' ( ln exp −Free Energy Perturbation%& ')),62  Thermodynamic Integration=497
5 67 8"+*,Bennett Acceptance Ratio (BAR))(
ΔGbindΔGcomplex=ΔGligand=ΔGcomplex

ΔGligand

MDFEP, TI, BAR

ΔGbind = ΔGcomplex − ΔGligand

MD   Fujitani et al., JCP, 2005

ΔΔGFEP+ 
relative binding free energy  
Wang et al. JACS, 2015

816(MDMD816(MDTemperature;;;'/7)<9<(73.24-5,1&"':-90<!;;; ;;;'/7)<High

Low 816( MD #$
+:16:*%

TemperatureHighLow

Replica 3

Replica 2

Replica 2

Replica 2

Replica 3

Replica 0

Replica 1

Replica 0

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Replica 0

Replica 1

Replica 1



…
…
…
…

.)-%MD(REMD)!"



Torsion angle [θ]MDTorsion angle []: Oseltamivir &*+.1&,0#
REMD

# .)-%MD !"   
$0'/(1

Time [ns]ΔSClausius [cal/mol/K]

Time [ns]

18.4

MD: 
REMD: 1817.617.2 0

Hikiri, MI, et al. J Chem Theory Comput. 2016 12: 5990.

50

100150Time [ns]200

38:<>+Folding/Unfolding+=9<1

MD(REMD)
UnfoldFold

38:<>(10)

ΔSc

REMD
(255K-400K)<>*"$.

16000

Fold

Unfold

12000
8000
4000

Simulation time [ns]00 1 2 3 4 5 6 7#-+RMSD (Å)

Fold/Unfold

100
PFold(T) [%]Unfold :Fold :#-+RMSD (Å)

7;4027<?6?5(T=305K)

8060

Tm(REMD)

40Tm()200 250 280 310 340 370 400

Temperature [K]• REMD(38:<>*&!' +Folding/Unfolding+/• Folding/Unfolding+/ .%)* • REMD(+(Tm=305K),(315K)*!



Folding  ΔSc(T)Unfold

Fold

2.0
ΔGFoldUnfold(T) [kcal/mol]

ΔSc

-10

Fold
ΔSUnfold

:

ΔS

:

0.5
0.0-0.5

Temperature [K]0.0-20-30-40
285

1.0-1.0 250 280 310 340 370 400

ΔSFoldUnfold(T) [cal/mol/K]

ΔSc(T) [cal/mol/K]

01.5295305315

Temperature [K]Hikiri, MI, et al. J Chem Theory Comput. 2016 12: 5990.

325335-5.0

-10.0
-15.0
-20.0
-25.0

-30.0 250 280 310 340 370 400

Temperature [K]MD..'7*/FG• 246(87– 20*&6%)"0*& #%H• 2!87– 20*&+-+-#7%H• 87– A:D>/23"0*& #%H• 

8 7– 9?B;E"9D<B@E"9D=C@E87•  87– 025$1"3 ,72%H

Hierarchy of Protein Dynamics
10-15
femto
bondvibration

10-12
pico

10-9
nano

methylrotation

10-6
microloop
motion10-3
milli100
seconddomainmotion103
hourslow dynamics

protein folding

side chain rotamers

106daydrug binding
Conventional MD Simulations
Enhanced Sampling, Ensemble Simulations, etc.

Method: umbrella sampling simulations

Umbrella Sampling MDµs - mseq. MDeq. MDFree energy

Basin aroundopen structure

Basin aroundclosed structure
Conformations

Method: umbrella sampling simulations
0.5-13)
,5/25+((%MD#%!
$
4/2* MD"%&
Replica Exchange Umbrella
Sampling (REUS)Free energy)'

6Reaction Coordinate7
Conformations

Method: umbrella sampling simulations

/-2,01) $&(#% ))$!$*.1+3) 'Free energy)"'

4Reaction Coordinate5ConformationsFree Energy Simulations
1. Initial Path
Nudged Elastic Bandopenclose

2. Umbrella Sampling Simulations
For each intermediates along path, simulations
with restraint,

w j = K rmsd (ΔDrmsd − ΔDmin ) 2

ΔDrmsd = rmsd(X, X open ) − rmsd(X, X closed )was carried out.
Restraint potential is applied to both main chains
and side chains.
B. Roux JACS (2005) C.L. Brooks PNAS (2007)

3. WHAM
Weighted Histogram
Analysis Method was used
for removing restraints andcalculating free energy
profiles along structuraltransition between open
and closed conformations.

Free Energy Profiles for Open-Close Transition
blue: open
yellow: closed
green: ATP

open

close

open

close

ü Meta-stable state is found:ATP-bound open conformation.
encounter complex

Ito, Oroguchi, Ikeguchi, JACS, 133, 3372 (2011)  transition: H-bond in P-loop

Open

H-bond partner of Asp256 is changed
from Lys162 to Thr163 (P-loop).
Mutation of Asp256, Lys162, or Thr163 results inremaining open conformation even with ATP bound.

ClosedMD7"70B38QR• %<?A1C!B–

$<93/A.2+93/–

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– 9<@-;+

$=%5B<.S*85B%CB– NMR>SAXS:9<*8

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MJMDK

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MD

MDKMD;';*/Ø ! "MD;)#'7/'@,Ø O! " +;!(LMD;% LØ O/ @ : 63>2812

MD%78 )('#% PhoB4 5%DNA/0*2
%!
CNS6NMR,312$"

%!MARBLE6NMRCHARMM27/CMAP,312&+-.$"
4PME5300K→800K→0K

Yamane, Okamura, Ikeguchi, Nishimura, Kidera, Proteins, 71, 1970 (2008)

Water-mediated interactions between PhoB and DNA
the same water
position in
NMR and crystalstructures
Glu159red lines: NMR structures
green lines: crystal structurescontrol DNA
binding activityMD7/;.
MJMDK@?5)#MD/Ø - ;4=;BIFØ O/ @-:Ø O&$9! "ACDGIAEH6<90NJ!

MD

MDKMD;';*/Ø ! "MD;)#'7/'@,Ø O! " +;!(LMD;% LØ O/ @ : 63>2812

NMR: Order Parameter
NMR provides information on
dynamics of both backbone
and side chains.

backboneHH

Order parameters (S2)represent the amplitude offluctuation of bond vectors.
MD simulations without any
restraints were conducted for
10 ns x 20 structures.
Calculated S2NH and S2axis
were compared withexperimental data.

side chain

NS2NH
F ij =HCrCHrS2axis

ri rjr2
2
31
22
S = tr F - ( tr F )22i , j = x, y , z
Best, Clarke, Karplus, J. Mol. Biol. 349, 189, (2005)

Model-free order parameter S2NH
-backbone dynamics-« Agreement with experiments

R=0.91« Small changes between
free and complex
except for β6-β7 loop
« Flexible regions:ütransactivation loop(α2-α3 loop)
üArg172 in β5

R=0.88üβ3-β4 loop

Yamane, Okamura, Kidera, Nishimura, Ikeguchi, J. Am. Chem. Soc.132, 12653, 2010

Principle of MD-SAXS
Simulation timeSAXSMD Simulation
ProteinSolution……
Initial
Structure2q

Calculations of I(q) at t = tn
I(q)

t=t1

t=t2qqt=tn……

Structure ensembleof proteinq

Average of I(q): I(q)MD-SAXS
I(q)CompareqI(q)I(q)EXq

.VPY\D[J\S"SZI\@"*VDR@)!G/*?
]HX@"*A<1=D3?8E4CDF=1:0

5F=2D9/B[QLO`a?;1=&4610]HNTOR/H\PNTOR%@"*A
$@*?+14>10

5F=1E4/VDR@)!

@(-HNTOR
1,25D3 /rVDR-LBD
H12HXCoactivator
Helix 12^HNTOR_Helix 12^H\PNTOR_HNTOR%^PDB id: 2ZLC_
H\PNTOR%^PDB id: 2ZXM_b

*@

A>1b

H\PNTOR

JB/rVDR-LBD
H12W[QLO124
7#?
6=1E
"@UQK\M@,>@3H\PNTORG'8E*A
DF=1>1zt|Dnx9x}mb/.+F"8Y. Anami, N. Shimizu, T. Ekimoto, D. Egawa, T. Itoh, M. Ikeguchi, and K. Yamamoto; J. Med. Chem. 59, 7888 (2016).

9) aehpstyuoqbKS`QN2 aEQ/.^/.+F@*j=\ZOl{NAntagonist>b&1!b&vtjZSNYifb/.+Fb@IacN
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Antagonist>
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vudaipD l]pc( cj\pQ 4&tMD-SAXS$64-Q,

8kblp_>SAXS7(O+HK 4kbm
q[hQ(

9MDQ,:"1)Y.%H7O3100 ns MDN?
TχQB4QgoeZ\mr3#sχ=0.8

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JZ D8ZORT ;.:>HB?@:>9まとめ• 生体分子の動きが機能に重要• 分子動力学シミュレーションでは、様々な力場が提案され、今も継続して改良中である• 分子動力学シミュレーションの解析は、その機能ごとに様々である‒ 例：RMSD, RMSF, 相関行列, PCA等

• 分子動力学シミュレーションは、様々に応用されている。
‒ 実験との連携も重要な点‒ 超並列計算への展開：レプリカ系の計算