Structure-based discovery of an inhibitor of Arf activation by Sec7 domains through targeting of protein-protein complexes -- Viaud et al. 104 (25): 10370 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Viaud et al. 10.1073/pnas.0700773104.

Supporting Information

Files in this Data Supplement:

SI Figure 7
SI Table 1
SI Table 2
SI Figure 8
SI Movie 1
SI Figure 9

SI Figure 7
Fig. 7. Inhibitory mechanism of LM11.

All nucleotide exchange rates are measured by fluorescence kinetics. (A) Effect of LM11 on spontaneous nucleotide exchange. (B) Effect of LM11 on the dissociation of the nucleotide-free Arf1-ARNO complex by mGTP. (C) Inhibitory effect of LM11 on the apparent activation rate constant (kapp) of 1 mM [Δ17]Arf1 by 50 nM ARNO^4M after addition of 10 mM mGTP.

SI Figure 8
Fig. 8.
Arf-GTP pull-down assay.

The VHS-GAP domain of GGA3 fused to GST (GST-GGA3^VHS-GAT) was expressed in BL21 Gold E. coli and purified on a Glutathione sepharose 4FF column followed by a gel filtration on a Superdex S75 10/330 GL column (Amersham). Glutathione sepharose 4FF beads were incubated with GST-GGA3^VHS-GAT at 4°C for 1 hour with slow rotation, then washed 3 times with 10 volumes of PBS 1X. Binding of GST-GGA3^VHS-GAT was verified by SDS/PAGE. HeLa cells were treated with either 100 M of LM11, 100 mM BFA or DMSO during 2 hours at 37°C in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, GlutaMAX and an antibiotic/antimycotic mixture in 10 cm treated dishes (Nunc). Cells were cleared by one volume of PBS 1X, lysed by 1 ml of 50 mM Tris, pH 7.5/100 mM NaCl/2 mM Mgcl₂/0.1% SDS/0.5% sodium desoxycholate/1% Triton X-100/10% glycerol and a protease inhibitors cocktail during 10 min at 4°C with slow rotation, and finally centrifugated at 13,000 ´ g for 10 min at 4°C. The clear lysates were incubated with 50 µl of GST-GGA3^VHS-GAT-coated beads for 1 hour at 4°C under slow rotation. The beads were washed 3 times with 10 volumes of washing buffer (50 mM Tris, pH 7.5/100 mM NaCl/2 mM MgCl₂/1% NP-40/10% glycerol with protease inhibitors), then resuspended in 30 µl of buffer at 95°C for 5 min before centrifugation at 13,000 ´ g and analysis by SDS/PAGE. Western blot detection of Arf proteins was done with the 1D9 primary antibody (Abcam) and revealed using the SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology). Treatment of HeLa cells with 100 mM LM11 (right lanes) resulted in the inhibition of endogeneous Arf proteins to an extent similar to that seen with BFA (left lane). It is likely, given the abundance of Arf1 relative to other Arf members in cells, that this mostly reflects Arf1 inhibition.

SI Figure 9
Fig. 9. SiRNA silencing of ARNO in MDCK cells.

The targeting sequence of ARNO or a control nonsilencing siRNA was used. MDCK cells were transfected with 150 pmol of ARNO or control siRNA oligonucleotides using Lipofectamine 2000. At 2 days after transfection, MDCK cells monolayers were wounded and the level of ARNO (cytohesin 2) or cytohesin 1 (negative control) was measured at the end of wound healing experiment by immunoblot after immunoprecipitation of the cell lysates using anti-ARNO antibodies.

SI Movie 1a
SI Movie 1b

Movie 1. LM11 inhibits ARNO-dependent migration of MDCK cells. Stably transfected MDCK cells expressing exogenous wt-ARNO were treated with either 0.5% DMSO carrier solvent (a) or 100 µM of LM11 (b). Cell sheet migration after scratch wounding was analyzed by time-lapse videomicroscopy as described in the Materials and Methods.

Table 1

. Experimental HSQC ¹⁵N-¹H chemical shifts for [Δ17]Arf1-GDP

Residue type	Residue number	N	HN	HA	HA2	New or corrected chemical shift
MET	18
ARG	19	124.12	9.007	4.885
ISO	20	124.47	8.67	4.801
LEU	21	127.17	8.142	5.203
MET	22	125.42	9.124	5.77
VAL	23	113.74	8.502	5.222
GLY	24	101.42	6.537	3.896	2.886
LEU	25	123.52	10.19	4.709
ASP	26	118.35	8.796	4.082
ALA	27	121.54	10.72	3.947
ALA	28	122.06	7.518	3.803
GLY	29	101.53	8.633	3.94	4.074
LYS	30	121.44	9.735	3.784
THR	31	120.52	9.12	4.309
THR	32	118.15	8.85	4.16
ISO	33	120.96	7.972	3.211		C
LEU	34	116.59	7.879	3.513		C
TYR	35	115.13	8.156	4.034
LYS	36	120.29	8.278	4.025		C
LEU	37	115.31	7.697	3.94		C
LYS	38	113.03	7.465	3.924		C
LEU	39	117.84	8.138	3.946		C
GLY	40	103.09	7.344	3.77	4.07	C
GLU	41	119.49	8.304	4.2
ISO	42	123.06	8.224	4.145
VAL	43	117.54	7.444	4.14		N
THR	44	116.07	7.964	4.717
THR	45	120.42	9.203	4.529
ISO	46	122.01	8.43	5.139
PRO	47
THR	48	107.45	7.438	4.4
ISO	49	122.74	7.888	3.691
GLY	50	112.91	8.755	4.245	4.715
PHE	51	121.81	8.258	5.141
ASN	52	120.72	7.827	4.986		C
VAL	53	123.26	8.722	4.565
GLU	54	125.26	8.815	5.158		C
THR	55	112.67	8.944	5.57		C
VAL	56	118.14	8.261	4.548		C
GLU	57	124.76	8.435	5.274
TYR	58	124.61	9.054	4.714
LYS	59	122.9	8.756	3.504
ASN	60	115.59	8.4	4.58		C
ISO	61	121.04	8.664	4.463
SER	62	119.65	8.368	5.157		N
PHE	63	124.38	9.314	5.39
THR	64	118.39	8.297	4.917
VAL	65	125.93	9.402	5.53
TRP	66	121.42	7.65	4.95
ASP	67	115.85	8.788	5.121
VAL	68	112.98	7.86	4.91
GLY	69	106.24	8.253	4.38	3.8
GLY	70	109.02	8.735	3.98	4.102
GLN	71	118.15	8.532	4.303
ASP	72	119.75	8.518
LYS	73
ISO	74
ARG	75
PRO	76
LEU	77	114.98	7.564	3.996
TRP	78	115.83	7.399	4.07
ARG	79	114.32	7.08
HIS	80	114.93	9.04	4.23
TYR	81	114.61	7.444	4.346
PHE	82	114.62	7.7	4.775
GLN	83	117.01	7.584	4.139
ASN	84	116.84	8.89	4.494		C
THR	85	115.43	8.002	4.072
GLN	86	122.94	8.525	4.55		N
GLY	87	103.45	7.676	5.03	3.26
LEU	88	124.77	9.268	5.191
ISO	89	123.71	9.573	5.441
PHE	90	128.16	9.163	5.23
VAL	91	127.28	8.414	4.614
VAL	92	122.03	8.799	4.28
ASP	93	122.9	9.137	4.712
SER	94	122.52	8.463	4.02
ASN	95	116.62	9.135	4.739
ASP	96	118.16	6.959	4.744
ARG	97	122.98	8.089	3.83
GLU	98	117.38	8.45	4.16
ARG	99	113.13	7.402	4.85		N
VAL	100	120.17	7.068	3.87
ASN	101	118.36	8.342	4.45
GLU	102	121.7	7.719	4.123
ALA	103	120.65	8.07	4.015
ARG	104	115.65	8.154	3.52
GLU	105	116.6	8.096	3.873
GLU	106	116.03	8.307	4.261
LEU	107	121.53	8.416	3.522
MET	108	114.09	8.49	4.038
ARG	109	118.21	7.986	4.036
MET	110	117.53	7.697	4.237
LEU	111	112.91	8.015	3.834
ALA	112	118.77	7.16	4.28
GLU	113	117.46	7.37	4.29
ASP	114	128.04	9.217	4.708		N
GLU	115	115.32	9.619	4.19		C
LEU	116	113.74	7.395	4.636		N
ARG	117	121.65	7.496	4.136
ASP	118	113.74	8.466	4.924
ALA	119	121.19	7.238	4.096
VAL	120	121.48	7.553	4.322
LEU	121	125.77	8.533	5.525
LEU	122	127.62	9.243	5.129
VAL	123	124.75	9.071	5.165		N
PHE	124	124.37	8.718	5.818
ALA	125	127.92	8.685	4.5
ASN	126	120.57	8.731	5.664
LYS	127	115.55	7.659	4.56
GLN	128	111.41	8.014	3.59
ASP	129	112.62	9.78	4.31
LEU	130	121.16	7.288	4.508
PRO	131
ASN	132	113.72	8.541	4.893
ALA	133	120.77	7.335	3.813
MET	134	121.47	8.244	4.188
ASN	135	117.62	8.726	4.77
ALA	136	118.21	8.89	3.708
ALA	137	122.57	8.4	4.033
GLU	138	120.28	8.343	4.16
ISO	139	118.09	8.45	3.53
THR	140	115.89	8.216	3.32
ASP	141	119.19	7.381	4.415
LYS	142	117.69	8.592	3.993
LEU	143	113.74	8.367	4.02
GLY	144	102.89	7.379	3.68	3.8
LEU	145	114.79	7.29	3.085
HIS	146	111.04	7.997	4.555		C
SER	147	111.94	7.564	4.398
LEU	148	121.38	7.295	4.272
ARG	149
HIS	150
ARG	151	117.35	7.793	4.658
ASN	152	125.76	9.238	4.96
TRP	153	120.02	7.899	6.654
TYR	154	120.35	8.135	3.848
ISO	155	122.04	7.087	5.167
GLN	156	125.15	8.878	4.439
ALA	157	132.16	9.025	4.75
THR	158	114.78	8.701	5.074
CYS	159	119.25	8.298	4.96
ALA	160	131.27	9.455	3.801
THR	161	102.05	7.1	3.662
SER	162	113.97	7.574	4.555
GLY	163	111.11	8.304	4.41	3.252
ASP	164	122.66	7.871	4.473
GLY	165	112.16	8.657	4.714	3.73	C
LEU	166	116.5	7.162	3.691		N
TYR	167	115.93	8.47	4.069		C
GLU	168	120.98	9.64	3.73		C
GLY	169	105.51	8.183	4.053	3.817
LEU	170	120.76	8.295	4.07
ASP	171	120.47	8.901	4.386		C
TRP	172	119.51	7.57	3.895		N
LEU	173	118.57	8.097	4.091
SER	174	112.31	8.759	3.854
ASN	175	117.47	7.797	4.28
GLN	176	116.08	7.564	3.662
LEU	177	115.94	7.419	4.185		C
ARG	178
ASN	179					C
GLN	180	119.1	7.863	4.292
LYS	181	126.52	7.938	4.11

All chemical shifts are reported in ppm. N and C (last column) indicate that the assignment for the residue has been added (N) or corrected (C) compared to that published in ref 1.

Table 2. Experimental chemical-shift variations induced by LM11 for [Δ17]Arf1-GDP.

Residue	H1	N1	H2	N2	D ¹H	D ¹⁵N
L21	8.174	127.467	8.187	127.423	0.013	0.044
M22	9.147	125.616	9.145	125.533	-	0.083
L34	7.876	116.575	7.9	116.628	0.024	0.053
Y35	8.145	115.122	8.159	115.106	0.014	-
L37	7.691	115.455	7.685	115.338	0.006	0.117
K38	7.462	113.177	7.441	112.876	0.021	0.301
L39	8.154	117.959	8.158	117.796	-	0.163
I42	8.243	123.011	8.256	123.117	0.013	0.106
V43	7.505	117.425	7.524	117.383	0.019	0.042
V53	8.747	123.33	8.768	123.464	0.021	0.134
T55	8.955	112.496	8.954	112.638	-	0.142
V56	8.261	117.843	8.293	118.07	0.032	0.227
E57	8.415	124.632	8.423	124.706	0.008	0.074
Y58	9.024	124.537	9.009	124.559	0.015	-
I61	8.558	120.618	8.574	120.666	0.016	0.048
F63	9.287	124.039	9.273	124.082	0.014	0.043
Q83	7.656	117.066	7.646	117.062	0.01	-
N84	8.885	116.57	8.902	116.633	0.017	0.063
G87	7.747	103.671	7.728	103.64	0.019	0.031
L88	9.261	124.765	9.264	124.923	-	0.158
I89	9.562	123.703	9.569	123.871	0.007	0.168
F90	9.127	128.109	9.139	128.119	0.012	-
L166	7.137	116.458	7.146	116.532	0.009	0.074
Y167	8.481	115.893	8.508	115.946	0.027	0.053
E168	9.704	121.275	9.692	121.092	0.012	0.183
D171	8.862	120.349	8.869	120.443	0.007	0.094
W172	7.588	119.506	7.565	119.411	0.023	0.095
L173	8.095	118.587	8.086	118.613	0.009	-
S174	8.756	112.384	8.741	112.311	0.015	0.073
N175	7.803	117.436	7.788	117.31	0.015	0.126
Q176	7.551	116.057	7.545	116.003	-	0.054

Chemical shifts are given in the absence (H1, N1) or in the presence (H2, N2) of LM11. All chemical shifts are reported in ppm. Resolution is 0.006 ppm and 0.03 ppm for ¹H and¹⁵N chemical shifts, respectively.

Seidel RD, 3rd, Amor JC, Kahn RA, Prestegard JH (2004) J Biol Chem 279:48307-48318.