Human ras-related C3 botulinum toxin substrate 3 (GTPase Rac3) in complex with GPPnhP

Target ID:

RAC3A

Deposition Date:

Tuesday 12th September 2006

Families:

GTPases

Related Diseases:

SignallingNeurobiologyCancer

Structure type:

apo

Download Coordinates:

2IC5

Authors:

E.UGOCHUKWU,X.YANG,Y.ZAO,J.ELKINS,C.GILEADI,N.BURGESS,S.COLEBROOK,O.GILEADI,O.FEDOROV,G.BUNKOCZI,M.SUNDSTROM,C.ARROWSMITH,J.WEIGELT,A.EDWARDS,F.VON DELFT,D.DOYLE,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

2IC5

tabs

Structure Details

Cells respond in many ways to environmental changes including altering gene expression, physically moving locations, changes to cell-cell interactions, differentiation and even altering their life spans. This is achieved through a signalling network that consists of several signalling pathways. The network receives multiple signals, interprets the signals, amplified and then transmits the signals. One of the best characterised signalling pathways involves the ras GTPase family. The importance of this family of small GTPases to human health was first recognised through the identification of the human H-Ras, N-Ras and K-Ras genes as being oncogenic.

The structure of this RAC3 crystal form has revealed the inactive-state conformation of the switch 1 loop of the bound GppNHp. Interestingly the main chain remained mostly unaltered when compared to the
stucture of RAC3 in complex with GDP. These new structural features improve our understanding of the structural changes that occur as the GTPase changes from the active to inactive state.

Rac1, Rac2 and Rac3 are a subfamily within the Rho family of small GTPases. Rac1 is expressed in most cell tissues (Moll et al, 1991), Rac2 is specifically expressed in hematopietic cells (Shirat et al, 1990) whereas Rac3 is localised mostly to the adult brain and expression in the developing nervous system (Albertinazzi et al, 1998; Haataja et al, 1997).

Rac3 has been shown to activate the JNK/stress activated kinase pathway (Haataja et al, 1997).
Up-regulation of this pathway and the p21-activated kinase
(see PAK-4 structure) by activated-GTP bound Rac3 occurs in breast cancer cells (Mira et al, 2000). Moreover, activated Rac3 is involved in the development of leukemia with the Rac3 knockout mice having a longer average survival rate (Cho et al, 2005). Hence, prevention of Rac3 activation by blocking binding of GTP would make Rac3 a potential therapeutic intervention point against certain leukemias and cancers.

Materials and Methods

Entry Clone Source: MGC

Entry Clone Accession: IMAGE:4284122

SGC Construct ID: RAC3A-c012

GenBank GI number: gi|4826962

Vector: pNIC28-Bsa4. Details [PDF]; Sequence [FASTA] or [GenBank]

Tags and additions: N-terminal hexahistidine tag with a TEV cleavage site

Sequence:
This is a mutant form of RAC 3 with the Cys178 replace by Gly. The Cys178Gly mutation is indicated in red.

mhhhhhhssgvdlgtenlyfq*smQAIKC
VVVGDGAVGKTCLLISYTTNAFPGEYIPT
VFDNYSANVMVDGKPVNLGLWDTAGQEDY
DRLRPLSYPQTDVFLICFSLVSPASFENV
RAKWYPEVRHHCPHTPILLVGTKLDLRDD
KDTIERLRDKKLAPITYPQGLAMAREIGS
VKYLECSALTQRGLKTVFDEAIRAVLG

Residues in lower case are the histidine tag followed by the TEV recognition sequence with the cleavage site marked with a *.

Host : BL-21(DE3)R3

Growth medium, induction protocol: Freshly transformed E. coli cells was used to inoculate 1 litre of TB plus 100 µg/ml ampicillin. When OD₆₀₀ reached ~0.5 the temperature was shifted down from 37°C to 25°C for 1 hour before induction with the addition of 1 mM IPTG. Protein expression was allowed to carry on for a further 4 hours before harvest.

Extraction buffer, extraction method: All extraction steps were carried out at 4°C.

Extraction buffer (EX): 50 mM Hepes pH 8.0, 150 mM NaCl, 5 % Glycerol, 10 mM Imidazole pH 8.0, 10 mM MgCl₂ . 1 tablet proteinase inhibitor in 10ml EX buffer was added to the 1L growth pellet. Total vol: 45 mls (estimate). Cell breakage: 5 passes through the Emulsiflex C5 high pressure homogeniser. Total vol: 50 mls (estimate). Centrifuge for 40 mins at 16000 rpm and 4°C to remove cell debris. Discard pellet.

Column 1 : Low pressure chromatography using Bio-Rad Econo column (2.5 cm x 13 cm).

Buffers: Wash buffer I (WB1): 50 mM Tris pH 8.0, 150 mM NaCl, 5% Glycerol, 10mM MgCl₂ , 10mM Imidazole pH 8.0; Wash Buffer II (WBII): 50 mM Tris pH 8.0, 150mM NaCl, 5% Glycerol, 10mM MgCl₂, 30mM Imidazole pH 8.0; Elution buffer (EB): 50 mM Tris pH 8.0, 150 mM NaCl, 5% Glycerol, 10mM MgCl₂, 250mM Imidazole pH 8.0.

Procedure: Total volume of Ni-NTA added to BioRad drip column: 4 mls (50%).

Resin washed with 12.5 ml of WB1. The supernatant was applied to a column using 5 ml pipette and allowed to pass over the resin. The flow through was collected in a 50 ml falcon tube and applied once more to the column. Two wash steps followed. Wash with 12.5 ml of WBI. Wash with 12.5 ml column vols of WBII. Elute with 14 mls of EB into 7x2 ml fractions.

Nucleotide Exchange and TEV cleavage: After elution the protein was concentrated to a final volume of 5 mls for nucleotide exchange and the protein concetration measured. To this was added a 25-fold excess of Gpp(NH)p, 5 mM EDTA, 1 mM DTT 100 microlitres of TEV protease and 14 microlitres of calf alkaline phosphatase (10,000U/ml), mixed and left overnight at 4°C.

The next day MgCl₂ was added to a final concetration of 10 mM before gel filtration.

GF buffer: 50 mM Tris pH 8.0, 500 mM NaCl, 0.5mM TCEP

Any remaining TEV protease and uncleaved RAC3 was removed by placing 200 µl of 50 % Ni-NTA agarose in a 1.5 ml eppendorf tubes, add 1ml of GF buffer mix, spin down and remove buffer. Repeat this resin wash step once.

Add the TEV treated protein sample to the resin and mix for 30 min. Finally spin down resin and collect the supernatant which contains the cleaved RAC3A.

Concentration : RAC3A was concentrated to 22 mg/ml before aliquoting into 50 microl volumes and freezing in the -80°C freezer.

Mass spec characterization :
Native Mass Spec for detection of non-covalently bound complexes indicated:

8+ charge state	exp mass	9+ charge state	exp mass	abs diff*	rel diff
A 2558.2	20 458	A 2274.0	20 457	+545
B 2560.8	20 478	B 2276.4	20 479		+Na⁺
C 2563.4	20 499	C 2278.4	20 497		+2Na⁺

*rel. to 19 912
Peaks A/B/C correspond to RAC3A plus Na-GppNHp or Na-GTP (+545) with one or two additional Na ions.

Crystallisation: Crystals grew from a 1:2 ratio mix of RAC3A(GppNHp)-to-reservoir (0.1M BIS-TRIS pH 5.5; 25 % PEG₃₃₅₀).

Data Collection: Resolution: 1.9 Å; X-ray source: Rotating anode, Rigaku FR-E superbright

References

Albertinazzi C, Gilardelli D, Paris S, Longhi R, de Curtis I. (1998). Overexpression of a neural-specific rho family GTPase, cRac1B, selectively induces enhanced neuritogenesis and neurite branching in primary neurons. J. Cell Biol. 142: 815-825.

Cho YJ, Zhang B, Kaartinen V, Haataja L, de Curtis I, Groffen J, Heisterkamp N. (2005). Generation of rac3 mutant mice: role of Rac3 in Bcr/Abl-caused lymphoblastic leukemia. Mol. Cell. Biol. 25: 5777-5785.

Haataja L, Groffen J, Heisterkamp N. (1997). Characterization of RAC 3, a novel member of the Rho family. J. Biol. Chem. 272: 20384-20388.

Mira JP, Benard V, Groffen J, Sanders LC, Knaus UG. (2000). Endogenous, hyperactive Rac3 controls proliferation of breast cancer cells by a p21-activated kinase-dependent pathway. Proc. Natl. Acad. Sci. U S A. 97: 185-189.

Moll J, Sansig G, Fattori E, van der Putten H. (1991). The murine rac1 gene: cDNA cloning, tissue distribution and regulated expression of rac1 mRNA by disassembly of actin microfilaments. Oncogene 6: 863-866.