SmgGDSによる低分子量G蛋白質の認識と機能bioxtal.spring8.or.jp/ja/spruc/SSB_12_6.pdfSmgGDSによる低分子量G蛋白質の認識と機能...

SmgGDSによる低分子量G蛋白質の認識と機能

奈良先端科学技術大学院大学藤間　祥子

大阪大学蛋白質研究所セミナー　第42回SPring-8先端利用技術ワークショップSep.9th(Mon)

1

2

GDP�

GDI�

GEF�

GDP/GTPexchange�

GTPhydrolysis

GTP�effector�

Plasmamembrane�

GDP�

GAP�

Extraction

Release

RhoorRab

Cytoplasm�

H-Ras�1� 189�

CAAX�

(hypervariableregion)HVR

PBR�

166�

G-domain�

G1�G2� G3� G4� G5�

P-loop�SwitchI�

SwitchII�

H-RasGDP(PDBid:4q21)�

H-RasGTP(PDBid:5p21)�

lipid�

SmallGTPases　

614smallGTPasestructuresaredepositedintothePDB (byInterPRoasof2016)

3

�� RALGDS

��

��RalGDS-like2

��Epac2

��

Histone RhoGEF PH RasGEFN RasGEF

RasGEFN RasGEF RA

RasGEFN RasGEF RA

cNMP DEP cNMP RasGEFN RasGEF

RasGEFN RasGEF C1EFh EFh

� ��

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DrrA_P4M

ZnF_A20 VPS9

uDENN DENN dDENN

Rabaptin RAb5-bind

Mss4

Sec2p

VPS9

Sec2p

Mon1

SH3BP5

uDENN DENN dDENN

��

��

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Sec7 PH

Sec7 PH

Sec7 PH

RCC1 RCC1 RCC1_2 RCC1RCC1RCC1

RasGEF

RabGEF

ArfGEF

RanGEF

Sec_N� Sec7�

Sec7� PH�

Sec7�DCB� Sec7_N� DUF_1981�

Sec7�DCB� Sec7_N� DUF_1981�

Lowcomplexity

Coiledcoil

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RhoGEF PHSEC14 SPEC SH3

PDZ RGS-like RhoGEF PH

PDZ RGS RhoGEF PH

RhoGEF PH

RhoGEF PH

CH C1 SH3 SH3SH2

RhoGEF PH PDZ PDZDEP

��

SH3 RhoGEF PH

IpaB_EvcA

ARM ARM ARMARM ARM

RGS-like RhoGEF PH

RII_binding_1� RhoGEF� PH�C1�


RhoGEF� PH�C1�

RhoGEF� PH�

PH� RhoGEF� PH�PDZ�RBD�

SEC14� SPEC� SPEC� SPEC� SPEC� SPEC� SPEC� RhoGEF� PH� SH3� RhoGEF� PH� SH3� IGc2� S_Tkc�


SH3� DOCK_N� DOCK_C2� DHR-2�


SEC14� SPEC� SPEC� SPEC� SPEC� SPEC� SPEC� SPEC� RhoGEF� PH� RhoGEF� PH� IGc2� FN3� S_Tkc�


EH� EFh� SH3� SH3� SH3� SH3� SH3� RhoGEF� PH� C2�


DUF3398� PH� DOCK-C2� DHR-2�


DUF3398� DOCK-C2� DHR-2�




RhoGEF

RhoGEF PH

(Toma-Fukai&Shimizu,inrevision,Molecules)

DomainarchitecturesofGEFs　

Over300GFEstructures(133complexstructures)aredepositedintothePDB(bymanuallyasof2019)

DomainarchitecturesofGEFs(determinedinComplexstructure)

低分子量G蛋白質とは

GEF GDP GTP

細胞増殖, 細胞分化, 細胞成長

GAP 低分子量 G蛋白質

(Ras, Rho...)

細胞膜

Raf PI3K Rho kinase

GEF Guanine-nucleotide Exchange Factor ： GAP GTPase activating protein ：

P-loop Switch I Switch II HVR

GDP結合型RhoA (PDB ID: 1FTN)

P-loop Switch I Switch II

CaaX

HVR G-domain

PBR Poly-basic region ： HVR Hypervariable region ：

Mg2+

GDP

ドメイン構成

脂質修飾 1 193

PBR

GEF�

GDP/GTPexchange�

GTPhydrolysis

GTP�effector�

Plasmamembrane�

GDP�

GAP�

Cytoplasm�

Raf� PI3K� Rhokinase�

Cellproliferation,Celldifferentiation,Cellgrowth�

RhoA�1� 193�

CAAX�

HVR(hypervariableregion)

PBR�

166�

G-domain�

G1�G2� G3� G4� G5�

P-loop�SwitchI�

SwitchII�

lipid�

GDP-boundRhoAPDBID:1FTN

4

RhoA(SmallGTPase)　

5

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RhoGEF PHSEC14 SPEC SH3

PDZ RGS-like RhoGEF PH

PDZ RGS RhoGEF PH

RhoGEF PH

RhoGEF PH

CH C1 SH3 SH3SH2

RhoGEF PH PDZ PDZDEP

��

SH3 RhoGEF PH

IpaB_EvcA

ARM ARM ARMARM ARM

RGS-like RhoGEF PH



RhoGEF� PH�C1�

RhoGEF� PH�

PH� RhoGEF� PH�PDZ�RBD�















RhoGEF

RhoGEF PH Dbs:RhoAPDBid:1lb1

DblfamilyRhoGEFs　

DOCK7:Cdc42PDBid:6AJ4

Dockfamily

SmgGDS

?(Peifer,etal.,1994,Cell)

SmgGDSとは

SmgGDS

GDP GTP

RhoA/C 170 122

A

SmgGDS-558

SmgGDS-607

1 558

1 607

ARM

A B D E F G H I J K L M

B C D E F G H I J K L M

・RhoA, RhoCに対するGEFである (Hamel et al, JBC., 2010) ・Armadillo-repeat motif (ARM)から成り，既知のGEF活性ドメインを有しない・ARM数の異なる2つのアイソフォームを持つ・２つのアイソフォームは低分子量G蛋白質の脂質修飾の有無を識別する (Schuld et al, JBC., 2014 , Berg et al, JBC., 2010)

ドメイン構成

SmgGDS　

6

A�B�D�E�F�G�H�I� J�K�L�M�

A�B�C�D�E�F�G�H�I� J�K�L�M�

1�

1� 607�

122� 170�

SmgGDS-558�

SmgGDS-607�

ARM(armadillo-repeatmotif)�

•  SmgGDSisaGEFforRhoAandRhoC(Hamel,etal.,2010,J.B.C.)•  SmgGDShastwomajorsplicevariants(SmgGDS-558and607)whicharecomposedofARMsonly(don’thaveaknownGEFdomain)•  SmgGDScanrecognizelipidationstateofSmallGTPases(Schuld,etal.,2014,J.B.C.,Berg,etal.,2014,J.B.C.)

StructureofhSmgGDS-558　



1�

1� 607�

122� 170�

SmgGDS-558�

SmgGDS-607�


46

図 10 SmgGDSのドメイン構成と SmgGDS-558 (61-558) 単体結晶構造

SmgGDSのドメイン構成 (左) と SmgGDS-558 (61-558) 単体結晶構造リボン図 (右) 。右

図括弧内の数字は末端残基の残基番号を表す。

62

図 18 ダミーアトムモデルと SmgGDS-558 (61-558) 単体結晶構造の比較

ダミーアトムモデルは白色のダミー原子で、SmgGDS-558 (61-558) 単体結晶構造を緑色の

リボン図で表示した。RhoAと考えられる溶液構造を矢印で示した。

Crystalstructure SAXSstructure(Complex)

Dammyatom/CrystalStructure

(Shimizu,etal.,2017,J.Biol.Chem) 7

PXstructurevsSAXSstructure

299.8kDa=7.78mer=8mer

SEC-MALSò�

hPRMT8ŉ¤ �ŃĩĩĩĩĩƊą��ûŗcpĺłĲŔ�

SEC-SAXSŨŹƃŚŬźżūſ

SAXSŨŹƃŚŬźżūſ�

ĔVÁ1��ň)O%ňųŪŝƁŠ�

Sphericalcage1�

18)O/ĔVÁ1��Sphericalcage2�

Sphericalcage3�

Ó��ŃŌŒŕľSphericalCage�ûŉÓ�/Ňőœ¬Ļľ�

Trigonalform1ňÓ��û

SAXSŨŹƃŚŬźżūſ�

ĔVÁ1��ň)O%ňųŪŝƁŠ�

Sphericalcage1�

18)O/ĔVÁ1��Sphericalcage2�

Sphericalcage3�

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Trigonalform1ňÓ��û

hPRMT8Ó��

hPRMT8PRMTcore

ミリストイル化（脂質修飾） hPRMT8Ó��û�

(ç~¨�û)�

C

N

(Toma-Fukai,etal.,2016J.Mol.Biol.)

(Hasegawa,etal.,2014FEBSJ.)

PX SAXS PX SAXS

PX SAXS PX SAXS

8

SmgGDS-558の静電ポテンシャル

負領域 (ARM B-F)

正領域 (ARM H, I)

N

C

90°

D119 E151

E164

E162 E168 E188

D190 E193

E197

E204 D206

D230 D232

E234 D235 D236

E239 D254 E255

D245

K346 H330

R378 M379

R337

K372

N338 S334

R296 N293

正領域拡大図負領域拡大図

61-558

170 122 A

SmgGDS-558

SmgGDS-607

1 558

1 607

ARM

A B D E F G H I J K L M

B C D E F G H I J K L M

ElectrostaticpotentialsurfaceofSmgGDS-558



1�

1� 607�

122� 170�

SmgGDS-558�

SmgGDS-607�


Positiveregion(ARMH&I)

Negativeregion(ARMB-F)

EnlargedviewatPositiveregion EnlargedviewatNegativeregion

9

hSmgGDS-558activateslipidmodifiedRhoA

【Nucleotideexchangereaction】

0

1

2

3

4

5

6

7

8

Control WT H379A R386A N387A K421A R427A M428A

Rel

ativ

e di

ssoc

iatio

n ra

tio

0

1

2

3

4

5

6

7

8

Control WT H330A R337A N338A K372A R378A M379A

Rel

ativ

e di

ssoc

iatio

n ra

tio

*** ***

***

***

***

***

RhoA vs SmgGDS-558 RhoA vs SmgGDS-607

Non-farnesylated Farnesylated

Non-farnesylated Farnesylated

FTaseRhoAL193A

FPP(基質)

30℃,1h Onice,1h Superdex75

N.Kuhlmannet.al.,2015,J.B.C.

【invitrolipidation】

hSmgGDS-558prefersfarnesylatedhRhoA,　　　　　　　　　　　　hSmgGDS-607prefersunmodifiedhRhoA　BothvariantschangetheirpreferencesdependingonmaturationstateofRhoA

10(Shimizu,etal.,2017,J.Biol.Chem)

hSmgGDS-558/ŵřſŰŢſ/RhoAí4�PX-SAXSò�Ġ

100µm�

-7

-6

-5

-4

-3

-2

-1

00 0.05 0.1 0.15 0.2 0.25

experimentaldata

crystalstructure

q (Å-1)�

SmgGDS-558/RhoA�

90°�

Dummy atom model�

Inte

nsity

(log

sca

le)�

<Ó��û>� <SAXS�û>�

(ç~¨�û)�11

CrsytalstructureofhSmgGDS/hRhoAfarnesylated

<CrystalStructure> <SAXSStructure>

<Helicalassemblyincrystal>

90°

ARMB

ARMD

ARME

ARMF

ARMG

ARMH

ARMI

ARMJ

ARMK

ARMLARMMSwitchIIbinding

region(=positiveregion)

Lipidbindingpocket(onnegativeregion) RhoA�

1� 193�13� 28� 61� 181�20� 38� 78� 189�

P-loop�SwitchI� SwitchII�

PBR � CAAX �HVR�

GTP,GDP,Mg2+認識


SwitchIIBindingregion

Lipidbindingpocket

12(Shimizu,etal.,2018,P.N.A.S)



1�

1� 607�

122� 170�

SmgGDS-558�

SmgGDS-607�


複合体中のRhoA 重ね合わせ

SmgGDS-558凹面とRhoAの相互作用

D65

N338 Y66

D254 L252

R337 K372

D67

R68 L69

R70

E204

L72

S73 N293

Y74

D298

D245

RhoA SmgGDS-558

R5 K6

Y66 D67 R68 L69 R70 S73 K98 E102

E162 E164 E204 D245 L252 D254 N293 D298 R337 N338 K372 D409 H410

Switch II

ARM D

ARM E

ARM F

ARM G

ARM H

ARM I

ARM J 水素結合塩橋

A3

D65

HVR

Switch I Switch II

P-loop

Switch II (Partially disordered)

P-loop (Disordered)

Switch I (Disordered)

相互作用残基

GDP

Mg2+

GDP結合型RhoA (PDB ID: 1FTN)

RhoArecognitionmechanismonswitchIIbindingregion

13

Interactionresidues

-Saltbridge--Hydrogenbond

GDP-boundRhoAPDBID:1FTN RhoAinComplex Overlay

構造に基づく変異体解析

Pulldown

GEF活性測定

Y66A R68E

L69A R70E L72A

D67R

S73A

WT

ファルネシル化RhoA 未修飾RhoA

Y66A R68E

L69A R70E L72A

D67R

S73A

WT

SmgGDS-607 SmgGDS-558

25

75 kDa

25

75 kDa

GST-SmgGDS

His6-RhoA (未修飾)

※青字はSwitch IIの残基

Structure-basedmutationanalysis


<Pulldownassay>

<GEFassay>

ResiduesonSwitchIIbindingregion

RhoA Farnesylated-RhoA

SwitchI&IIstructuresofallsmallGTPasesinComplexwithGEF

15

22

Table GEF:Small GTPase structures 630 631

No PDB ID Year GEF SmallG GEF Reso Ligand*1 Disorder*2

(p,sw1,sw2) HVR*3

1 1LFD 1998 RALGDS H-Ras RasGEF 2.1 GNP no, no ,no no

2 1BKD 1998 SOS1 H-Ras RasGEF 2.8 � no, no ,no no

3 1NVU 2000 SOS1 H-Ras RasGEF 2.2 � no, no ,no no

4 1NVV 2003 SOS1 H-Ras RasGEF 2.2 GNP no, no ,no no

5 1NVW 2003 SOS1 H-Ras RasGEF 2.7 GNP no, no ,no no

6 1NVX 2003 SOS1 H-Ras RasGEF 3.2 GTP no, no ,no no

7 1XD2 2004 SOS1 H-Ras RasGEF 2.7 GDP no, no ,no no

8 3CF6 2008 Epac2 Rap1B RapGEF 2.2 SO4 no, no ,no no

9 4NYJ 2014 SOS1 H-Ras RasGEF 2.9 GNP no, no ,no no

10 4NYM 2014 SOS1 H-Ras RasGEF 3.6 � no, no ,no no

11 4MGI 2014 Epac Rap1b RapGEF 2.8 SO4 no, no ,no no

12 4MGK 2014 Epac Rap1b RapGEF 2.7 SO4 no, no ,no no

13 4MGY 2014 Epac Rap1b RapGEF 2.6 SO4 no, no ,no no

14 4MGZ 2014 Epac Rap1b RapGEF 3.0 SO4 no, no ,no no

15 4MH0 2014 Epac Rap1b RapGEF 2.4 SO4 no, no ,no no

16 4URU 2015 SOS1 H-Ras RasGEF 2.8 � no, no ,no no

17 4URV 2015 SOS1 H-Ras RasGEF 2.6 � no, no ,no no

18 4URW 2015 SOS1 H-Ras RasGEF 2.8 � no, no ,no no

19 4URX 2015 SOS1 H-Ras RasGEF 2.5 � no, no ,no no

20 4URY 2015 SOS1 H-Ras RasGEF 2.5 � no, no ,no no

21 4URZ 2015 SOS1 H-Ras RasGEF 2.2 � no, no ,no no

22 4US0 2015 SOS1 H-Ras RasGEF 2.2 � no, no ,no no

23 4US1 2015 SOS1 H-Ras RasGEF 2.7 L71 no, no ,no no


25 5CM8 2015 Rgl2 (Rlf) RalA RalGEF 2.6 � no, no ,no no

26 6AXG 2017 RasGRP4 H-Ras RasGEF 3.3 � no, yes, no no

27 6AXF 2017 RasGRP Rap1b RapGEF 3.1 � no, no ,no no

28 6D55 2018 SOS1 HRas RasGEF 1.7 Na, FMT, GOL, Mg,

GNP, FWA no, no ,no no

29 6D5W 2018 SOS1 HRas RasGEF 2.5 FVV, Mg, GNP no, no ,no no

30 6D56 2018 SOS1 HRas RasGEF 1.7 Na, FMT, GOL, Mg, GNP, FVN

no, no ,no no

31 6D59 2018 SOS1 HRas RasGEF 1.7 Na, FMT, GOL, Mg, GNP, FVJ

no, no ,no no

32 6D5E 2018 SOS1 HRas RasGEF 1.8

Na, FMT, GOL, Mg,

GNP, FVG, CL

no, no ,no no

23

33 6D5G 2018 SOS1 HRas RasGEF 1.9

FMT, GOL, Mg, GNP,

FVD, Cl,BME

no, no ,no no

34 6D5H 2018 SOS1 HRas RasGEF 1.8 FMT, GOL, MG, GNP,

FV7, Cl no, no ,no no

35 6D5J 2018 SOS1 HRas RasGEF 1.8 Na, FMT, GOL, MG, GNP, FV4

no, no ,no no

36 6D5L 2018 SOS1 HRas RasGEF 1.7 Na, FMT, GOL, MG, GNP, FW7

no, no ,no no

37 6D5V 2018 SOS1 HRas RasGEF 2.0 Mg, GNP, FVY no, no ,no no

38 6D5M 2018 SOS1 HRas RasGEF 2.1 Mg, GNP, FW4 no, no ,no no

39 5WFO 2018 SOS1 HRas RasGEF 2.0 Mg, GNP, 5UU no, no ,no no

40 5WFP 2018 SOS1 HRas RasGEF 2.1 Mg, GNP, 5UX no, no ,no no

41 5WFQ 2018 SOS1 HRas RasGEF 2.3 Mg, GNP, 5UV no, no ,no no

42 5WFR 2018 SOS1 HRas RasGEF 2.5 Mg, GNP, 5UW no, no ,no no

43 6BVI 2018 SOS1 HRas RasGEF 1.8 NA, FMT, EC4, GOL, Mg, GNP

no, no ,no no

44 6BVJ 2018 SOS1 HRas RasGEF 1.7 NA, FMT, EAS, GOL, Mg, GNP

no, no ,no no

45 6BVK 2018 SOS1 HRas RasGEF 1.8 NA, FMT,

EAV, GOL, Mg, GNP

no, no ,no no

46 6BVL 2018 SOS1 HRas RasGEF 1.7 NA, FBY,

EAV, GOL, Mg, GNP

no, no ,no no

47 6BVM 2018 SOS1 HRas RasGEF 1.8 NA, FBY,

EBV, GOL, Mg, GNP

no, no ,no no

48 6EPL 2019 SOS1 KRas RasGEF 2.6 GOL no, no ,no no

49 6EPM 2019 SOS1 KRas RasGEF 2.5 BQ5, GOL no, no ,no no

50 6EPN 2019 SOS1 KRas RasGEF 2.5 BQ2, DMS, GOL no, no ,no no

51 6EPO 2019 SOS1 KRas RasGEF 2.4 GOL, BPW no, no ,no no

52 6EPP 2019 SOS1 KRas RasGEF 2.4 GOL, BOQ no, no ,no no

53 1LB1 2002 Mcf21(Dbs) RhoA RhoGEF 2.8 � no, no ,no no

54 1X86 2004 ARHGEF12 RhoA RhoGEF 3.2 PO4 no, no ,no no

55 1XCG 2004 ARHGEF11 (PDZ-rhoGEF) RhoA RhoGEF 2.5 � no, no ,no no

56 2RGN 2007 ARHGEF25 (p63RhoGEF) RhoA RhoGEF 3.5 � no, no ,no no

57 3KZ1 2010 ARHGEF11 (PDZ-rhoGEF) RhoA RhoGEF 2.7 GSP no, no ,no no

58 3LW8 2010 IpgB2 RhoA RhoGEF 1.9 GDP no, no ,no no

59 3LWN 2010 IpgB2 RhoA RhoGEF 2.3 GDP no, no ,no no

60 3LXR 2010 IpgB2 RhoA RhoGEF 1.7 GDP no, no ,no no

61 3T06 2011 ARHGEF11 (PDZ-rhoGEF) RhoA RhoGEF 2.8 � no, no ,no no

62 4D0N 2014 AKAP13 RhoA RhoGEF 2.1 GDP no, no ,no no

63 4XH9 2015 NET1 RhoA RhoGEF 2.0 � no, yes, no no

24

(ARHGEF8)

64 6BC0 2017 ARHGEF28 (p190RhoGEF) RhoA RhoGEF 2.2 GSP no, no ,no no

65 5JHG 2017 ARHGEF11 RhoA RhoGEF 2.5 GOL no, no ,no no

66 6BCA 2017 ARHGEF18 (LbcRhoGEF) RhoA RhoGEF 2.0 GSP no, no ,no no

67 6BCB 2017 ARHGEF18 (p114RhoGEF) RhoA RhoGEF 1.4 GSP no, no ,no no

68 5JHH 2017 ARHGEF11 RhoA RhoGEF 2.3 RAO no, no ,no no

69 5ZHX 2018 SmgGDS RhoA RhoGEF 3.5 yes, yes,

yes yes

70 1FOE 2000 Tiam Rac1 RhoGEF 2.8 SO4 no, no ,no no

71 2NZ8 2007 Trio Rac1 RhoGEF 2.0 � no, no ,no no

72 2VRW 2008 Vav1 Rac1 RhoGEF 1.9 � no, no ,no no

73 2YIN 2011 DOCK1 Rac1 RhoGEF 2.7 � no, no ,no no

74 3B13 2012 DOCK2 Rac1 RhoGEF 3.0 � no, no ,no no

75 3BJI 2007 Vav1 Rac1 RhoGEF 2.6 � no, no ,no no

76 4YON 2015 P-Rex1 Rac1 RhoGEF 2.0 � no, no ,no no

77 5FI0 2016 P-Rex1 Rac1 RhoGEF 3.3 � no, no ,no yes

78 5O33 2017 Kalirin Rac1 RhoGEF 1.6 GDP no, no ,no no

79 6BC1 2018 ARHGEF28 (p190RhoGEF) Rac1 RhoGEF 2.9 GSP no, no ,no no

80 1KZ7 2002 Mcf2l (Dbs) Cdc42 RhoGEF 2.4 � no, no ,no no

81 1GZS 2002 SOPE Cdc42 RhoGEF 2.3 PO4 no, no ,no no

82 1KI1 2002 ITSN1 Cdc42 RhoGEF 2.3 PO4 no, no ,no no

83 1KZG 2002 Mcf2l (Dbs) Cdc42 RhoGEF 2.6 � no, no ,no no

84 2DFK 2006 ARHGEF9 (Collybistin II) Cdc42 RhoGEF 2.2 PO4 no, no ,no yes

85 2WM9 2009 DOCK9 Cdc42 RhoGEF 2.2 GOL no, no ,no no

86 2WMN 2009 DOCK9 Cdc42 RhoGEF 2.4 GDP no, no ,no no

87 2WMO 2009 DOCK9 Cdc42 RhoGEF 2.2 GDP no, no ,no no

88 3GCG 2009 map (L0028) Cdc42 RhoGEF 2.3 � no, yes, no no

89 3QBV 2012 ITSN1 Cdc42 RhoGEF 2.7 GDP no, yes, no no

90 3VHL 2012 DOCK8 Cdc42 RhoGEF 2.1 PO4 no, no ,no no

91 6AJ4 2019 DOCK7 Cdc42 RhoGEF 3.2 � no, no ,no yes

92 6AJL 2019 DOCK7 Cdc42 RhoGEF 3.2 � no, no ,no yes

93 3CX6 2008 ARHGEF11 (PDZRhoGEF) Galpha-13 RhoGEF 2.5 GDP no, no ,no no

94 3CX7 2008 ARHGEF11 (PDZRhoGEF) Galpha-13 RhoGEF 2.3 GSP no, no ,no no


96 1SHZ 2005 ARHGEF1 (p115RhoGEF) Gnai1,13 RhoGEF 2.9 GDP+ALF no, no ,no no

97 2NTY 2007 ROPGEF8 ROP4 RopGEF 3.1 GDP no, yes, no no

98 2WBL 2009 ROPGEF8 ROP7 RopGEF 2.9 � no, yes, no no

25

99 2WWX 2009 SidM/DrrA Rab1 RabGEF 1.5 � no, no ,no no

100 3L0I 2010 SidM/DrrA Rab1 RabGEF 2.9 SO4 no, no ,no no

101 3JZA 2010 DrrA/SidM Rab1b RabGEF 1.8 PO4 no, no ,no no

102 5O74 2017 DrrA/SidM Rab1b RabGEF 2.5 GDP no, yes, yes no

103 2OT3 2007 RabGEF1 (RABEX) Rab21 RabGEF 2.1 � no, yes, no no

104 3TW8 2011 DENND1B Rab35 RabGEF 2.1 � no, yes, no no

105 4Q9U 2014 RABGEF1 Rab5A RabGEF 4.6 � yes, yes, no no

106 2FU5 2006 MSS4 Rab8A RabGEF 2.0 � no, yes yes no

107 4LHX 2013 RAB3IP (Rabin8) Rab8A RabGEF 3.1 SO4 no, no ,no no

108 4LHY 2013 RAB3IP (Rabin8) Rab8A RabGEF 3.1 GDP no, no ,no no

109 4LHZ 2013 RAB3IP (Rabin8) Rab8A RabGEF 3.2 GTP no, no ,no no

110 4LI0 2013 RAB3IP (Rabin8) Rab8A RabGEF 3.3 GDP no, no ,no no

111 2EFD 2010 AtVps9a RabF2B (Ara7) RabGEF 3.0 � no, yes,no no

112 2EFE 2010 AtVps9a RabF2B (Ara7) RabGEF 2.1 GDP no, no, no no

113 2EFH 2010 AtVps9a RabF2B (Ara7) RabGEF 2.1 GDP no, yes, no no

114 2EFC 2010 AtVps9a RabF2B (Ara7) RabGEF 2.1 GDP no, no, no no

115 4G01 2013 Vps9a RabF2B (Ara7) RabGEF 2.2 GDP no, no, no no

116 2OCY 2007 Sec2p Sec4p RabGEF 3.3 GDP no, no, no no

117 2EQB 2007 Sec2p Sec4p RabGEF 2.7 � no, no, no no

118 4ZDW 2015 Sec2p Sec4p RabGEF 2.9 � no, no, no no

119 3CUE 2008 TRS23 (TRAPPI assembly)

Ypt1p RabGEF 3.7 � no, yes, no no

120 5LDD 2017 Mon1-Ccz1 Ypt7 RabGEF 2.5 SO4 no, no, no no

121 6IXV 2019 SH3BP5 Rab11 RabGEF 3.8 PO4 no no, no no

122 6EKK 2019 DENND 1A Rab35 RabGEF 1.8 GDP, SO4, EDO no no, no no

123 6DJL 2019 SH3BP5 Rab11 RabGEF 3.1 � no no, no no

124 1RE0 2003 GEA1 ARF1 ArfGEF 2.4 GDP no, no, no no

125 1R8Q 2003 CYTH2 (Arno) ARF1 ArfGEF 1.9 G3D no, no, no no

126 1R8S 2003 CYTH2 (Arno) ARF1 ArfGEF 1.5 GDP no, no, no no

127 1S9D 2003 CYTH2 (Arno) ARF1 ArfGEF 1.8 GDP no, yes, no no

128 4C0A 2013 IQSEC1 ARF1 ArfGEF 3.3 G3D no, no, no no

129 6FAE 2018 IQSEC2 ARF1 ArfGEF 2.3 � no, yes, no no

130 4KAX 2013 CYTH3 ARF6 ArfGEF 1.9 GTP no, no, no no

131 5EE5 2016 ARFGEF1 ARL1 ArfGEF 2.3 GTP no, no, no no

132 5J5C 2016 ARFGEF1 ARL1 ArfGEF 3.4 GTP no, no, no no

133 1I2M 2001 RCC1 RAN RanGEF 1.8 SO4 no, yes, no no

*1 The names of the ligands are described below. 632

22



(p,sw1,sw2) HVR*3
































no, no ,no no


no, no ,no no


Na, FMT, GOL, Mg,

GNP, FVG, CL

no, no ,no no

SwitchI&IIdisorderstructureis2/133

SmgGDS

MSS4

(Toma-Fukai&Shimizu,inrevision,Molecules)

CommonGEFmechanism

16

SOS1:Hras(PDBid:1bkd)�

SOS1

H-Ras

SOS1Cdc25(930-946)

H-RasGDP(PDBid:5p21)

SwitchISwitchII

P-loop

E62 A59

GDP

Mg2+K16

DOCK9

DOCK9:Cdc42(PDBid:2wm9)�

DOCK9:Cdc42-GTP-Mg2+(PDBid:2wmo)

DOCK9DHR2(345-350)

V347

GTP

Mg2+SwitchI

SwitchII

P-loop

Cdc42

Localproteinunfoldingandrefoldingmechanism

17

MSS4

Rab8

RhoA(incomplexwithGDP)

SmgGDS

RhoA(incomplexwithSmgGDS)PDBid:5zhx

RhoA(incomplexwithGDP)PDBid:1ftn

RhoA

Overlay

Overlay(Toma-Fukai&Shimizu,inrevision,Molecules)

Bi-functionalprotein:chaperonandGEF

(Itzen,eta.l,2006,EMBOJ)

Rab8(incomplexwithGDP)PDBid:4lhv

Rab8(incomplexwithMSS4)PDBid:2fu5

90°

ARMB

ARMD

ARME

ARMF

ARMG

ARMH

ARMI

ARMJ

ARMK

ARMLARMMSwitchIIbinding

region(=positiveregion)

Lipidbindingpocket(onnegativeregion) RhoA�

1� 193�13� 28� 61� 181�20� 38� 78� 189�

P-loop�SwitchI� SwitchII�

PBR � CAAX �HVR�

GTP,GDP,Mg2+recognition


SwitchIIBindingregion

Lipidbindingpocket




1�

1� 607�

122� 170�

SmgGDS-558�

SmgGDS-607�


ApoComplex

ARMB(H2)

ARMD(H1) ARMB(H3)

H1

H2H3

Complex Apo

Y118

L127L136

L149

C153N159

A156

Q124

M152

L113

V155

L139

Fo-Fcomitmap

Crypticpocket:sitethatonlybecomesapparentwhendrugsbind.(foundserendipitously)

Lipidbindingpocketisacrypticpocket

19

SmgGDS-558の隠されたポケット

ARM B

ARM D

ファルネシル化システイン

隠されたポケット

HVR

Y118

L113 L136

L149

C153 N159

A156

Q124

M152

L160

V155

L139

G114 G110

ポケットの表面残基

0

2

4

6

8

control WT G110R G114R A156R

Relat

ive

GEF a

ctivit

y

ゲラニルゲラニル化システイン

ファルネシル化システイン

GEF活性試験

ドッキングシミュレーション

TheCrypticpocketofSmgGDS-558

20

Farnesylated-Cys

Crypticpocket

Aminoacidresiduesonthepocket Dockingsimulation

GEFassay

Farnesylated-Cys

Geranylgeranylated-Cys

........

KAMOanalysistoidentifythelipidbindingpocket

SelectedClusterNo.54

126datasetswereused(resolutionofeachdatais~4Å)

Total121clusters

RawimagesweredepositedintoZenododatarepository(https://doi.org/10.5281/zenodo.1134209)21

molA molB molC molD

densitymapoflipid(Fo-Fcomitmap)

ClusterNo.118ClusterNo.121 ClusterNo.119

22

SmgGDS-558/farnesylated RhoA

Space group P212121 Wavelength (Å) 0.90000 No. of crystals 2 Unit cell (a, b, c) 93.3, 181.8, 205.3 X-ray source SPring-8 BL44XU Resolution (Å) (outer shell) 136.1-3.5 (3.71-3.50)

a No. of obs. ref. 1967740 No. of unique. ref. 44892 Completeness (%) 99.9 (100.0) Rmeas

b 0.565 (4.100) I/σ(I) 10.4 (1.2) CC1/2 0.997 (0.623) < Refinement >

Resolution (Å) 136.1-3.5 Rwork (%)

c 24.4 Rfree (%)

d 29.8 RMSD

Bond length (Å) 0.007 Bond angles (˚) 1.314 Number of atoms per asymmetric unit Protein 18760 Ligand 0 Ramachandran plots

Favored 2316 (93%) Allowed 160 (6%) Outlier 4 (0%) a The numbers in parentheses represent statistics in the highest resolution shell.

b R

meas =�(n/n-1)

1/2�

j|<I(h)> -I (h)

j|/��

j|<I(h)>, where <I(h)> is the mean intensity of symmetry-equuivalent reflections.

c R

work=�||F

o|- |F

c|/�|F

o|, where F

o and F

c are the observed and calculated structure factors for data used for

refinement, respectively. d R

free=�||F

o|- |F

c|/�|F

o| for 5% of the data not used at any stage of structural refinement.

Mergedwithtwocrystals(samesampleLot)andcollectedatBL44XU(SPring-8)

Statisticsoffinaldata3.5Åstructurecouldbedetermined!

LipidationstateofallsmallGTPasesinComplexwithGEF

23

22



(p,sw1,sw2) HVR*3
































no, no ,no no


no, no ,no no


Na, FMT, GOL, Mg,

GNP, FVG, CL

no, no ,no no

23

33 6D5G 2018 SOS1 HRas RasGEF 1.9

FMT, GOL, Mg, GNP,

FVD, Cl,BME

no, no ,no no

34 6D5H 2018 SOS1 HRas RasGEF 1.8 FMT, GOL, MG, GNP,

FV7, Cl no, no ,no no

35 6D5J 2018 SOS1 HRas RasGEF 1.8 Na, FMT, GOL, MG, GNP, FV4

no, no ,no no

36 6D5L 2018 SOS1 HRas RasGEF 1.7 Na, FMT, GOL, MG, GNP, FW7

no, no ,no no

37 6D5V 2018 SOS1 HRas RasGEF 2.0 Mg, GNP, FVY no, no ,no no

38 6D5M 2018 SOS1 HRas RasGEF 2.1 Mg, GNP, FW4 no, no ,no no

39 5WFO 2018 SOS1 HRas RasGEF 2.0 Mg, GNP, 5UU no, no ,no no

40 5WFP 2018 SOS1 HRas RasGEF 2.1 Mg, GNP, 5UX no, no ,no no

41 5WFQ 2018 SOS1 HRas RasGEF 2.3 Mg, GNP, 5UV no, no ,no no

42 5WFR 2018 SOS1 HRas RasGEF 2.5 Mg, GNP, 5UW no, no ,no no

43 6BVI 2018 SOS1 HRas RasGEF 1.8 NA, FMT, EC4, GOL, Mg, GNP

no, no ,no no

44 6BVJ 2018 SOS1 HRas RasGEF 1.7 NA, FMT, EAS, GOL, Mg, GNP

no, no ,no no

45 6BVK 2018 SOS1 HRas RasGEF 1.8 NA, FMT,

EAV, GOL, Mg, GNP

no, no ,no no

46 6BVL 2018 SOS1 HRas RasGEF 1.7 NA, FBY,

EAV, GOL, Mg, GNP

no, no ,no no

47 6BVM 2018 SOS1 HRas RasGEF 1.8 NA, FBY,

EBV, GOL, Mg, GNP

no, no ,no no

48 6EPL 2019 SOS1 KRas RasGEF 2.6 GOL no, no ,no no

49 6EPM 2019 SOS1 KRas RasGEF 2.5 BQ5, GOL no, no ,no no

50 6EPN 2019 SOS1 KRas RasGEF 2.5 BQ2, DMS, GOL no, no ,no no

51 6EPO 2019 SOS1 KRas RasGEF 2.4 GOL, BPW no, no ,no no

52 6EPP 2019 SOS1 KRas RasGEF 2.4 GOL, BOQ no, no ,no no

53 1LB1 2002 Mcf21(Dbs) RhoA RhoGEF 2.8 � no, no ,no no

54 1X86 2004 ARHGEF12 RhoA RhoGEF 3.2 PO4 no, no ,no no

55 1XCG 2004 ARHGEF11 (PDZ-rhoGEF) RhoA RhoGEF 2.5 � no, no ,no no

56 2RGN 2007 ARHGEF25 (p63RhoGEF) RhoA RhoGEF 3.5 � no, no ,no no

57 3KZ1 2010 ARHGEF11 (PDZ-rhoGEF) RhoA RhoGEF 2.7 GSP no, no ,no no

58 3LW8 2010 IpgB2 RhoA RhoGEF 1.9 GDP no, no ,no no

59 3LWN 2010 IpgB2 RhoA RhoGEF 2.3 GDP no, no ,no no

60 3LXR 2010 IpgB2 RhoA RhoGEF 1.7 GDP no, no ,no no

61 3T06 2011 ARHGEF11 (PDZ-rhoGEF) RhoA RhoGEF 2.8 � no, no ,no no

62 4D0N 2014 AKAP13 RhoA RhoGEF 2.1 GDP no, no ,no no

63 4XH9 2015 NET1 RhoA RhoGEF 2.0 � no, yes, no no

24

(ARHGEF8)

64 6BC0 2017 ARHGEF28 (p190RhoGEF) RhoA RhoGEF 2.2 GSP no, no ,no no

65 5JHG 2017 ARHGEF11 RhoA RhoGEF 2.5 GOL no, no ,no no

66 6BCA 2017 ARHGEF18 (LbcRhoGEF) RhoA RhoGEF 2.0 GSP no, no ,no no

67 6BCB 2017 ARHGEF18 (p114RhoGEF) RhoA RhoGEF 1.4 GSP no, no ,no no

68 5JHH 2017 ARHGEF11 RhoA RhoGEF 2.3 RAO no, no ,no no

69 5ZHX 2018 SmgGDS RhoA RhoGEF 3.5 yes, yes,

yes yes

70 1FOE 2000 Tiam Rac1 RhoGEF 2.8 SO4 no, no ,no no

71 2NZ8 2007 Trio Rac1 RhoGEF 2.0 � no, no ,no no

72 2VRW 2008 Vav1 Rac1 RhoGEF 1.9 � no, no ,no no

73 2YIN 2011 DOCK1 Rac1 RhoGEF 2.7 � no, no ,no no

74 3B13 2012 DOCK2 Rac1 RhoGEF 3.0 � no, no ,no no

75 3BJI 2007 Vav1 Rac1 RhoGEF 2.6 � no, no ,no no

76 4YON 2015 P-Rex1 Rac1 RhoGEF 2.0 � no, no ,no no

77 5FI0 2016 P-Rex1 Rac1 RhoGEF 3.3 � no, no ,no yes

78 5O33 2017 Kalirin Rac1 RhoGEF 1.6 GDP no, no ,no no

79 6BC1 2018 ARHGEF28 (p190RhoGEF) Rac1 RhoGEF 2.9 GSP no, no ,no no

80 1KZ7 2002 Mcf2l (Dbs) Cdc42 RhoGEF 2.4 � no, no ,no no

81 1GZS 2002 SOPE Cdc42 RhoGEF 2.3 PO4 no, no ,no no

82 1KI1 2002 ITSN1 Cdc42 RhoGEF 2.3 PO4 no, no ,no no

83 1KZG 2002 Mcf2l (Dbs) Cdc42 RhoGEF 2.6 � no, no ,no no

84 2DFK 2006 ARHGEF9 (Collybistin II) Cdc42 RhoGEF 2.2 PO4 no, no ,no yes

85 2WM9 2009 DOCK9 Cdc42 RhoGEF 2.2 GOL no, no ,no no

86 2WMN 2009 DOCK9 Cdc42 RhoGEF 2.4 GDP no, no ,no no

87 2WMO 2009 DOCK9 Cdc42 RhoGEF 2.2 GDP no, no ,no no

88 3GCG 2009 map (L0028) Cdc42 RhoGEF 2.3 � no, yes, no no

89 3QBV 2012 ITSN1 Cdc42 RhoGEF 2.7 GDP no, yes, no no

90 3VHL 2012 DOCK8 Cdc42 RhoGEF 2.1 PO4 no, no ,no no

91 6AJ4 2019 DOCK7 Cdc42 RhoGEF 3.2 � no, no ,no yes

92 6AJL 2019 DOCK7 Cdc42 RhoGEF 3.2 � no, no ,no yes


94 3CX7 2008 ARHGEF11 (PDZRhoGEF) Galpha-13 RhoGEF 2.3 GSP no, no ,no no


96 1SHZ 2005 ARHGEF1 (p115RhoGEF) Gnai1,13 RhoGEF 2.9 GDP+ALF no, no ,no no

97 2NTY 2007 ROPGEF8 ROP4 RopGEF 3.1 GDP no, yes, no no

98 2WBL 2009 ROPGEF8 ROP7 RopGEF 2.9 � no, yes, no no

25

99 2WWX 2009 SidM/DrrA Rab1 RabGEF 1.5 � no, no ,no no

100 3L0I 2010 SidM/DrrA Rab1 RabGEF 2.9 SO4 no, no ,no no

101 3JZA 2010 DrrA/SidM Rab1b RabGEF 1.8 PO4 no, no ,no no

102 5O74 2017 DrrA/SidM Rab1b RabGEF 2.5 GDP no, yes, yes no

103 2OT3 2007 RabGEF1 (RABEX) Rab21 RabGEF 2.1 � no, yes, no no

104 3TW8 2011 DENND1B Rab35 RabGEF 2.1 � no, yes, no no

105 4Q9U 2014 RABGEF1 Rab5A RabGEF 4.6 � yes, yes, no no

106 2FU5 2006 MSS4 Rab8A RabGEF 2.0 � no, yes yes no

107 4LHX 2013 RAB3IP (Rabin8) Rab8A RabGEF 3.1 SO4 no, no ,no no

108 4LHY 2013 RAB3IP (Rabin8) Rab8A RabGEF 3.1 GDP no, no ,no no

109 4LHZ 2013 RAB3IP (Rabin8) Rab8A RabGEF 3.2 GTP no, no ,no no

110 4LI0 2013 RAB3IP (Rabin8) Rab8A RabGEF 3.3 GDP no, no ,no no

111 2EFD 2010 AtVps9a RabF2B (Ara7) RabGEF 3.0 � no, yes,no no

112 2EFE 2010 AtVps9a RabF2B (Ara7) RabGEF 2.1 GDP no, no, no no

113 2EFH 2010 AtVps9a RabF2B (Ara7) RabGEF 2.1 GDP no, yes, no no

114 2EFC 2010 AtVps9a RabF2B (Ara7) RabGEF 2.1 GDP no, no, no no

115 4G01 2013 Vps9a RabF2B (Ara7) RabGEF 2.2 GDP no, no, no no

116 2OCY 2007 Sec2p Sec4p RabGEF 3.3 GDP no, no, no no

117 2EQB 2007 Sec2p Sec4p RabGEF 2.7 � no, no, no no

118 4ZDW 2015 Sec2p Sec4p RabGEF 2.9 � no, no, no no

119 3CUE 2008 TRS23 (TRAPPI assembly)

Ypt1p RabGEF 3.7 � no, yes, no no

120 5LDD 2017 Mon1-Ccz1 Ypt7 RabGEF 2.5 SO4 no, no, no no

121 6IXV 2019 SH3BP5 Rab11 RabGEF 3.8 PO4 no no, no no

122 6EKK 2019 DENND 1A Rab35 RabGEF 1.8 GDP, SO4, EDO no no, no no

123 6DJL 2019 SH3BP5 Rab11 RabGEF 3.1 � no no, no no

124 1RE0 2003 GEA1 ARF1 ArfGEF 2.4 GDP no, no, no no

125 1R8Q 2003 CYTH2 (Arno) ARF1 ArfGEF 1.9 G3D no, no, no no

126 1R8S 2003 CYTH2 (Arno) ARF1 ArfGEF 1.5 GDP no, no, no no

127 1S9D 2003 CYTH2 (Arno) ARF1 ArfGEF 1.8 GDP no, yes, no no

128 4C0A 2013 IQSEC1 ARF1 ArfGEF 3.3 G3D no, no, no no

129 6FAE 2018 IQSEC2 ARF1 ArfGEF 2.3 � no, yes, no no

130 4KAX 2013 CYTH3 ARF6 ArfGEF 1.9 GTP no, no, no no

131 5EE5 2016 ARFGEF1 ARL1 ArfGEF 2.3 GTP no, no, no no

132 5J5C 2016 ARFGEF1 ARL1 ArfGEF 3.4 GTP no, no, no no

133 1I2M 2001 RCC1 RAN RanGEF 1.8 SO4 no, yes, no no

*1 The names of the ligands are described below. 632

22



(p,sw1,sw2) HVR*3
































no, no ,no no


no, no ,no no


Na, FMT, GOL, Mg,

GNP, FVG, CL

no, no ,no no

LipidatedsmallGTPasestructureis1/133

SmgGDS

Acknowledgement

GraduateSchoolofPharmaceuticalSciences,TheUniversityofTokyoHikaruShimizu*ToshiyukiShimizu　MeijiPharmaceuticalUniversity(present　address)KenjiKontaniMusashinoUniversity(presentaddress)ToshiakiKatada

<ProteinCrystallography>SPring-8KeitaroYamashita,KazuyaHasegawa(supportedbyBINDs),AkifumiHigashiura,EikiYamashitaandBL44XU,BL41XU

BL32XUstaffKEK-PFNaohiroMatsugaki,YusukeYamadaandBL1A,BL5A,NE3Astaff<Bio-SAXS>KEK-PFShinyaSaijo,NobutakaShimizuandBL10Cstaff

24

SmgGDSによる低分子量G蛋白質の認識と機能bioxtal.spring8.or.jp/ja/spruc/SSB_12_6.pdfSmgGDSによる低分子量G蛋白質の認識と機能...

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Transcript of SmgGDSによる低分子量G蛋白質の認識と機能bioxtal.spring8.or.jp/ja/spruc/SSB_12_6.pdfSmgGDSによる低分子量G蛋白質の認識と機能...