Human Dihydropyrmidinease-like 2

Target ID:

DRP2A

Aliases:

CRMP2DHPRP2DPYSL2DRP-2DRP2

Deposition Date:

Wednesday 26th April 2006

Families:

Oxidoreductases

Related Diseases:

Neurobiology

Structure type:

apo

Download Coordinates:

2GSE

Authors:

D.OGG,P.STENMARK,C.ARROWSMITH,H.BERGLUND,R.COLLINS,A.EDWARDS,M.EHN,S.FLODIN,A.FLORES,S.GRASLUND,B.M.HALLBERG,M.HAMMARSTROM,T.KOTENYOVA,P.KURSULA,P.NILSSON-EHLE,T.NYMAN,C.PERSSON,J.SAGEMARK,M.SUNDSTROM,L.HOLMBERG-SCHIAVONE,A.GTHORSELL,J.UPPENBERG,S.VAN DEN BERG,J.WEIGELT,P.NORDLUND,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Stockholm

ICM Datapack:

ICM Datapack

2GSE

tabs

Structure Details

Dihydropyrimidinase-Related Protein 2 (DPYSL2) is mostly called CRMP2 in the literature.
Five isoforms of CRMPs exists in man, namely CRMP1 to CRMP5;
they are approximately 70% identical to each other.
We have solved the structure of CRMP2 which is the most extensively studied member of the family.
Dihydropyrimidinase-Related Protein (CRMP2) is, as the name implies, related to dihydropyrimidinases.
CRMP2 is 58% identical to the human dihydropyrimidinase which catalyse the second step in pyrimidine degradation.
The CRMP2 family does not have any known catalytic activity but is involved in neuronal differentiation, axonal guidance and neuronal polarity. The protein is highly expressed in the developing nervous system.
Over-expression of CRMP2 induces the growth of numerous axons; it is also involved in the maturation of neurites and pre-existing dendrites to axons (Yoshimura T. et al.).
CRMP2 binds to tubulin heterodimers promoting microtubule assembly (Fukata Y et al.). GSK3-beta phosphorylation in the C-terminal part of the CRMP2 (Thr514) inactivates it.

The overall structure of the CRMP2 tetramer is similar to the CRMP1 structure that has been solved from mouse (Deo RC et al.).
The protein is active as a tetramer. The C-terminus of the CRMPs has been shown to be cleaved off by partial proteolysis (Deo et al.) and is therefore likely to be disordered, for this reason it was not included in the construct that was used to obtain the structure. One calcium ion is bound to each of the monomers; it is coordinated between the carbonyl oxygen of residue Thr349 and the side chain of Glu353. Glu353 is not conserved thorough the different isoforms of the CRMPs and calcium is one of the components of the crystallisation condition; therefore it is unlikely that the calcium binding is physiologically relevant.

The CRMPs are one example of enzymes that have lost there catalytic function and gained a regulatory role instead.
We have previously solved the structure of PRPSAP1 which is another example of an enzyme from nucleotide metabolism that has gained a regulatory function after losing its catalytic function.

CRMP2 is the first of the human CRMPs to be structurally characterised, it will provide valuable information of variability of the binding surfaces of the CRMP protein family.
Hyper-phosphorylated CRMP2 is found in Alzheimer disease tissue (Cole AR et al.).
It is not presently known if this is a consequence or a cause of the development of Alzheimer. CRMP2 is also a candidate gene for Schizophrenia and a number of SNPs have been observed (Fallin MD et al.). The structure will aid in interpreting mutations if/when linked to the disease.

Materials and Methods

DPYSL2

PDB:2GSE

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC067109
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal hexahistidine tag with integrated TEV protease cleavage site: mhhhhhhssgvdlgtenlyfq*s(m).
Host:Rosetta II pLysS

Construct

Prelude:
Sequence:MHHHHHHSSGVDLGTENLYFQSMTSDRLLIKGGKIVNDDQSFYADIYMEDGLIKQIGENLIVPGGVKTIEAHSRMVIPGGIDVHTRFQMPDQGMTSADDFFQGTKAALAGGTTMIIDHVVPEPGTSLLAAFDQWREWADSKSCCDYSLHVDISEWHKGIQEEMEALVKDHGVNSFLVYMAFKDRFQLTDCQIYEVLSVIRDIGAIAQVHAENGDIIAEEQQRILDLGITGPEGHVLSRPEEVEAEAVNRAITIANQTNCPLYITKVMSKSSAEVIAQARKKGTVVYGEPITASLGTDGSHYWSKNWAKAAAFVTSPPLSPDPTTPDFLNSLLSCGDLQVTGSAHCTFNTAQKAVGKDNFTLIPEGTNGTEERMSVIWDKAVVTGKMDENQFVAVTSTNAAKVFNLYPRKGRIAVGSDADLVIWDPDSVKTISAKTHNSSLEYNIFEGMECRGSPLVVISQGKIVLEDGTLHVTEGSGRYIPRKPFPDFVYKRIKARSRLAE
Vector:pNIC-Bsa4

Growth

Medium:
Antibiotics:
Procedure:20 mL TB media was inoculated with Rosetta II pLysS cells, and the culture was grown overnight at 30 ºC in 100 mL shake flasks at 175 rpm. 1.5 L TB media supplemented with 8 g/L glycerol, 50 µg/mL kanamycin and approximately 20 µL BREOX (anti-foam solution) was inoculated with 20 mL of the over night culture. The large scale cultivations were grown in 2 L bottles in the LEX system water bath at 37 °C. The temperature of the bath is regulated by connection via a circulation (pumping) system to another tempered water bath, set to 41°C. Room tempered air is circulated and transfered to the bottles by an airline tubing and a sparger inside the bottle. The bottle was transferred to a neighbouring water bath of 18 °C when the OD600 reached 2.5. The protein expression was induced by adding IPTG to a final concentration of 0.5 mM when the OD600 reached 3.5. The protein expression was continued over night.

Purification

Procedure
Columns: HiTrap Chelating and HiLoad 16/60 Superdex 200

Purification was conducted automatically on an ÄKTA xpress system operated by UNICORN software. Prior to purification columns were equilibrated with IMAC Bind/Wash1 Buffer (HiTrap Chelating) and Gel filtration buffer (Superdex 200). The protein sample was loaded on the HiTrap Chelating column that was washed with IMAC Bind/Wash1 Buffer followed by IMAC Wash2 Buffer. Bound protein was eluted from the IMAC columns with IMAC Elution Buffer and loaded onto the Gelfiltration column. The chromatogram from Gelfiltration showed one major protein peak that consisted of highly pure DRP2A-c011 confirmed by SDS-PAGE analysis. TCEP was added to the sample to a final concentration of 2 mM. The protein was concentrated to 33 mg/mL and stored at -80ºC.

Extraction

Procedure
A stock solution of Complete EDTA-free (protease inhibitor) was prepared by dissolving 24 tablets in 48 mL of IMAC binding and wash1 buffer. Cells were harvested by centrifugation for 10 min at 4000 g. The cell pellet was resuspended in 80 mL of IMAC lysis buffer complemented with 4 mL of Complete EDTA-free protease inhibitor stock and then frozen at -80˚C. The frozen cell pellet was briefly thawed by warm water. 4 µL (1000 U) benzonase was added to the sample prior to high-pressure homogenization with the HPH. The cells were run three times through the HPH before centrifugation for 20 min at 49 000 g in the Sorvall SS-34 rotor. The soluble fraction was decanted and filtered through 0.45mm prior to loading onto the ÄKTAxpress for furher purification.
Concentration:
Ligand
MassSpec:
Crystallization:0.2 M CaCl, 0.1 M Tris pH 8.5, 17% PEG 10k. Dilute the sample 1+1 with GF- buffer to 16.7 mg/ml. Set up 2 µl (protein) + 1 µl (well solution) hanging drops.
NMR Spectroscopy:
Data Collection:ESRF ID14.2 20060213
Data Processing:

References

Yoshimura T, Kawano Y, Arimura N, Kawabata S, Kikuchi A, Kaibuchi K
GSK-3beta regulates phosphorylation of CRMP-2 and neuronal polarity.
Cell. 2005 Jan 14;120(1):137-49.

Cole AR, Knebel A, Morrice NA, Robertson LA, Irving AJ, Connolly CN, Sutherland C.
GSK-3 phosphorylation of the Alzheimer epitope within collapsin response mediator proteins regulates axon elongation in primary neurons.

J Biol Chem. 2004 Nov 26;279(48):50176-80. Epub 2004 Oct 5.

Deo RC, Schmidt EF, Elhabazi A, Togashi H, Burley SK, Strittmatter SM Structural bases for CRMP function in plexin-dependent semaphorin3A signaling.
EMBO J. 2004 Jan 14;23(1):9-22.

Fallin MD, Lasseter VK, Avramopoulos D, Nicodemus KK, Wolyniec PS, McGrath JA, Steel G, Nestadt G, Liang KY, Huganir RL, Valle D, Pulver AE.
Bipolar I disorder and schizophrenia: a 440-single-nucleotide polymorphism screen of 64 candidate genes among Ashkenazi Jewish case-parent trios.
Am J Hum Genet. 2005 Dec;77(6):918-36.

Nishimura T, Fukata Y, Kato K, Yamaguchi T, Matsuura Y, Kamiguchi H, Kaibuchi K.
CRMP-2 regulates polarized Numb-mediated endocytosis for axon growth.
Nat Cell Biol. 2003 Sep;5(9):819-26. Epub 2003 Aug 24.

Fukata Y, Itoh TJ, Kimura T, Menager C, Nishimura T, Shiromizu T, Watanabe H, Inagaki N, Iwamatsu A, Hotani H, Kaibuchi K.
CRMP-2 binds to tubulin heterodimers to promote microtubule assembly.
Nat Cell Biol. 2002 Aug;4(8):583-91.