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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
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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
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SH3 RhoGEF PH
IpaB_EvcA
ARM ARM ARMARM ARM
RGS-like RhoGEF PH
RII_binding_1� RhoGEF� PH�C1�
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�
SEC14� SPEC� SPEC� SPEC� SPEC� SPEC� SPEC� RhoGEF� PH� SH3� RhoGEF� PH� SH3� IGc2� S_Tkc�
SH3� DOCK_N� DOCK_C2� DHR-2�
SH3� DOCK_N� DOCK_C2� DHR-2�
SEC14� SPEC� SPEC� SPEC� SPEC� SPEC� SPEC� SPEC� RhoGEF� PH� RhoGEF� PH� IGc2� FN3� S_Tkc�
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�
EH� EFh� SH3� SH3� SH3� SH3� SH3� RhoGEF� PH� C2�
DUF3398� PH� DOCK-C2� DHR-2�
DUF3398� PH� DOCK-C2� DHR-2�
DUF3398� DOCK-C2� DHR-2�
DUF3398� DOCK-C2� DHR-2�
DUF3398� 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
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SH3 RhoGEF PH
IpaB_EvcA
ARM ARM ARMARM ARM
RGS-like RhoGEF PH
RII_binding_1� RhoGEF� PH�C1�
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�
SEC14� SPEC� SPEC� SPEC� SPEC� SPEC� SPEC� RhoGEF� PH� SH3� RhoGEF� PH� SH3� IGc2� S_Tkc�
SH3� DOCK_N� DOCK_C2� DHR-2�
SH3� DOCK_N� DOCK_C2� DHR-2�
SEC14� SPEC� SPEC� SPEC� SPEC� SPEC� SPEC� SPEC� RhoGEF� PH� RhoGEF� PH� IGc2� FN3� S_Tkc�
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�
EH� EFh� SH3� SH3� SH3� SH3� SH3� RhoGEF� PH� C2�
DUF3398� PH� DOCK-C2� DHR-2�
DUF3398� PH� DOCK-C2� DHR-2�
DUF3398� DOCK-C2� DHR-2�
DUF3398� DOCK-C2� DHR-2�
DUF3398� DOCK-C2� DHR-2�
DUF3398� DOCK-C2� DHR-2�
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
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)�
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�
Ó��ŃŌŒŕľSphericalCage�ûʼnÓ�/Ňőœ¬Ļľ�
Trigonalform1ňÓ��û
hPRMT8��
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
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)�
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
CrsytalstructureofhSmgGDS/hRhoAfarnesylated
SwitchIIBindingregion
Lipidbindingpocket
12(Shimizu,etal.,2018,P.N.A.S)
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)�
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
14(Shimizu,etal.,2018,P.N.A.S)
<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
24 4US2 2015 SOS1 H-Ras RasGEF 2.5 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
95 3CX8 2008 ARHGEF11 (PDZRhoGEF) Galpha-13 RhoGEF 2.5 GDP 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
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
24 4US2 2015 SOS1 H-Ras RasGEF 2.5 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
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
CrsytalstructureofhSmgGDS/hRhoAfarnesylated
SwitchIIBindingregion
Lipidbindingpocket
18(Shimizu,etal.,2018,P.N.A.S)
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)�
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
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
24 4US2 2015 SOS1 H-Ras RasGEF 2.5 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
95 3CX8 2008 ARHGEF11 (PDZRhoGEF) Galpha-13 RhoGEF 2.5 GDP 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
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
24 4US2 2015 SOS1 H-Ras RasGEF 2.5 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
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
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