Human Dihydropyrimidinase-related protein 3

Target ID:

DPYSL3A

Deposition Date:

Friday 26th April 2013

Families:

Miscellaneous

Structure type:

apo

Download Coordinates:

4BKN

Authors:

S. Mathea, J. M. Elkins, J. Alegre-Abarrategui, L. Shrestha, N. Burgess-Brown, S. Puranik, D. Coutandin, A. Bradley, M. Vollmar, F. von Delft, C. Bountra, C. Arrowsmith, A. Edwards, S. Knapp

Site:

Oxford

4BKN

tabs

Structure Details

Dihydropyrimidinase-related protein 3 (DPYSL3, also referred to as CRMP4) is predominantly expressed in neuronal and testicular cells. Recently, a missense mutation in the human DPYSL3 gene was reported to be associated with ALS (Blasco et al., 2013).

The biological significance of DPYSL3 is highlighted by a number of model organism experiments. In cultured rat neurons, DPYSL3 inhibits axon outgrowth (Alabed et al., 2010). In zebrafish, the depletion of DPYSL3 causes abnormal cell positioning during neurulation (Tanaka et al., 2012). And the C. elegans homologue UNC 33 organises axon-dendrite sorting (Maniar et al., 2011).

DPYSL3 is capable of forming homotetramers and heterotetramers with other DPYSL proteins (Wang et al., 1997). The flexible C-terminus has been shown to be phosphorylated by GSK3, Cdk5 and DYRK2 (Cole et al., 2006). Due to its ability to bind tubulin, the 62 kDa protein has been suggested to play a role in axonal cargo transport (Khanna et al., 2012).

In the long term, further understanding of DPYSL function may help to treat neurodegenerative disorders.

Materials and Methods

Entry Clone Source: MGC
SCG Construct ID: DPYSL3A-c010
Vector: pFB-6HZB

Entry Clone DNA Sequence

ATGTCCTACCAAGGCAAGAAGAACAT
CCCGCGGATCACGAGTGACCGTCTCC
TTATCAAGGGAGGCAGAATCGTCAAT
GATGATCAGTCCTTTTATGCTGATAT
TTACATGGAAGATGGCTTAATAAAAC
AAATTGGAGACAATCTGATTGTTCCT
GGAGGAGTGAAGACCATTGAAGCCAA
TGGGAAGATGGTGATCCCTGGAGGCA
TCGATGTCCATACTCACTACCAGATG
CCATATAAGGGAATGACCACAGTAGA
TGACTTCTTCCAAGGGACAAAGGCGG
CTTTAGCAGGTGGCACCACCATGATC
ATTGACCATGTGGTGCCTGAGCCTGA
GTCCAGCCTGACTGAGGCCTATGAGA
AATGGAGAGAGTGGGCTGATGGGAAG
AGTTGCTGTGACTATGCCCTGCATGT
GGACATCACCCACTGGAATGACAGCG
TCAAGCAGGAAGTGCAGAACCTCATC
AAGGACAAAGGGGTTAACTCCTTCAT
GGTTTATATGGCTTATAAGGATTTGT
ATCAAGTATCTAACACAGAGCTCTAT
GAGATCTTCACCTGCCTGGGAGAGCT
GGGGGCCATTGCTCAAGTTCATGCTG
AGAATGGGGATATCATTGCCCAGGAG
CAAACCCGCATGTTGGAAATGGGGAT
AACTGGCCCAGAAGGCCATGTACTGA
GCAGGCCAGAAGAGCTGGAAGCTGAG
GCTGTGTTCCGTGCCATCACCATTGC
CAGCCAAACCAATTGCCCTCTCTACG
TCACAAAGGTCATGAGCAAGAGTGCA
GCTGACCTCATCTCACAAGCCAGGAA
AAAAGGAAATGTAGTCTTTGGTGAGC
CCATCACTGCCAGCCTCGGCATAGAT
GGAACCCATTATTGGAGCAAGAACTG
GGCCAAGGCGGCTGCATTTGTGACAT
CCCCACCCCTGAGCCCTGACCCAACT
ACTCCGGACTACATCAACTCCTTGCT
GGCCAGCGGGGATCTGCAGCTATCTG
GGAGTGCCCACTGCACCTTCAGCACT
GCCCAGAAAGCAATTGGGAAGGACAA
CTTCACAGCCATTCCTGAGGGCACCA
ATGGTGTGGAGGAGCGGATGTCTGTC
ATCTGGGACAAGGCTGTGGCCACAGG
GAAAATGGACGAAAACCAGTTCGTGG
CTGTGACAAGCACAAACGCTGCCAAG
ATCTTCAACCTGTATCCCCGCAAGGG
AAGAATATCTGTGGGTTCTGACAGCG
ACCTCGTCATCTGGGATCCAGATGCT
GTGAAGATCGTCTCTGCCAAGAACCA
CCAGTCTGCGGCAGAGTACAACATCT
TTGAAGGGATGGAGCTGCGCGGGGCT
CCTCTGGTTGTCATCTGCCAGGGCAA
GATCATGCTGGAAGATGGCAACCTGC
ACGTGACCCAGGGGGCTGGCCGCTTC
ATACCCTGCAGCCCGTTCTCCGACTA
TGTCTACAAGCGCATTAAAGCACGGA
GGAAGATGGCAGACCTGCATGCCGTC
CCAAGGGGCATGTACGATGGGCCTGT
GTTTGACCTGACCACCACCCCCAAAG
GTGGCACCCCCGCAGGCTCTGCTCGG
GGCTCTCCTACTCGGCCGAACCCACC
TGTGAGGAATCTTCATCAGTCGGGAT
TTAGCCTGTCAGGCACTCAAGTGGAT
GAGGGGGTTCGCTCAGCCAGCAAGCG
CATCGTGGCGCCCCCAGGCGGCCGTT
CTAATATCACATCTCTGAGTTAA

Tag: N-terminal His ₆-Z-TEV

Construct Protein Sequence

mghhhhhhssgvdnkfnkerrrarre
irhlpnlnreqrrafirslrddpsqs
anllaeakklndaqpkgtenlyfq*s
MSYQGKKNIPRITSDRLLIKGGRIVN
DDQSFYADIYMEDGLIKQIGDNLIVP
GGVKTIEANGKMVIPGGIDVHTHYQM
PYKGMTTVDDFFQGTKAALAGGTTMI
IDHVVPEPESSLTEAYEKWREWADGK
SCCDYALHVDITHWNDSVKQEVQNLI
KDKGVNSFMVYMAYKDLYQVSNTELY
EIFTCLGELGAIAQVHAENGDIIAQE
QTRMLEMGITGPEGHVLSRPEELEAE
AVFRAITIASQTNCPLYVTKVMSKSA
ADLISQARKKGNVVFGEPITASLGID
GTHYWSKNWAKAAAFVTSPPLSPDPT
TPDYINSLLASGDLQLSGSAHCTFST
AQKAIGKDNFTAIPEGTNGVEERMSV
IWDKAVATGKMDENQFVAVTSTNAAK
IFNLYPRKGRISVGSDSDLVIWDPDA
VKIVSAKNHQSAAEYNIFEGMELRGA
PLVVICQGKIMLEDGNLHVTQGAGRF
IPCSPFSDYVYKRIKARRKMADLHAV
PRGMYDGPVFDLTTTPKGGTPAGSAR
GSPTRPNPPVRNLHQSGFSLSGTQVD
EGVRSASKRIVAPPGGRSNITSLS

residues in lower case are cleaved by TEV protease

Host: Insect cells (Sf9)

Expression & Harvest

Exponentially growing Sf9 cells (2xÂ 10 ⁶Â cells/mL) were infected with high titre baculovirus stock (1:100) and incubated in shaker flasks (50 hours, 92 rpm, 27Â°C). Following this, the cell suspensions were centrifuged (15 minutes, 800x g, 4Â°C), and the cell pellets resuspended in PBS. After another centrifugation (15Â minutes, 800xÂ g, 4Â°C), the cell pellets were stored at -80Â°C.

Purification

A 6Â g cell pellet was thawed, resuspended in 40Â mL lysis buffer (50Â mM HEPES/NaOH pHÂ 7.4, 500Â mM NaCl, 20Â mM imidazole, 0.5Â mM TCEP) and sonicated (2 minutes, amplitude 35%, on ice). The cell lysate was cleared by centrifugation (45Â minutes, 100,000xÂ g, 4Â°C). The supernatant was combined with 5Â mL Ni Sepharose and loaded onto a gravity flow column. The resin was rinsed thrice with lysis buffer. Proteins bound to the resin were eluted with 5x 10Â mL elution buffer (50Â mM HEPES/NaOH pHÂ 7.4, 500Â mM NaCl, 300Â mM imidazole, 0.5Â mM TCEP) and diluted with 50Â mL no salt buffer (20Â mM HEPES/NaOH pHÂ 7.4, 0.5Â mM TCEP).

The next purification step was performed using an AKTAprime system combined with a 5Â ml SP Sepharose column. After equilibrating the column with no salt buffer, the sample was loaded. The column was rinsed with 10% high salt buffer (20Â mM HEPES/NaOH pHÂ 7.4, 2.5Â M NaCl, 0.5Â mM TCEP). The protein was eluted by a linear gradient of 10-100% high salt buffer in 150Â mL.

Fractions containing protein were pooled, combined with recombinant TEV protease (mass ratio 1:25) and incubated with rotation (over night, 4Â°C). Following this, 5Â mL Ni Sepharose was added, and the suspension was loaded onto a gravity flow column. The flowthrough was collected and concentrated to 5.2Â mL.

Finally, the protein was purified by gel filtration using an AKTAxpress system with an S200 16/600 column and GF buffer (20Â mM HEPES/NaOH pHÂ 7.4, 500Â mM NaCl, 0.5Â mM TCEP). Fractions containing protein were pooled, concentrated and stored at -80Â°C.

Crystallisation

Crystals grew from a 1:2 ratio of protein solution (12.6Â mg/mL) and precipitant solution (20% PEG 3.3K, 0.1Â M Bis-Tris-propane pHÂ 8.5, 10% Ethylene Glycol, 0.2Â M KSCN), using the vapour diffusion method.

Data Collection

Crystals were cryo-protected by equilibration into precipitant solution containing 25% ethylene glycol and flash frozen in liquid nitrogen. Data was collected at Diamond, beamline I02.

References

Alabed YZ, Pool M, Ong Tone S, Sutherland C, Fournier AE. GSK3 beta regulates myelin-dependent axon outgrowth inhibition through CRMP4. J Neurosci. 2010 Apr 21;30(16):5635-43.
Blasco H, Bernard-Marissal N, Vourc'h P, Guettard YO, Sunyach C, Augereau O, Khederchah J, Mouzat K, Antar C, Gordon PH, Veyrat-Durebex C, Besson G, Andersen PM, Salachas F, Meininger V, Camu W, Pettmann B, Andres CR, Corcia P; French ALS study group. A rare Motor Neuron Deleterious Missense Mutation in the DPYSL3 (CRMP4) Gene is Associated with ALS. Hum Mutat. 2013 Apr 8. doi: 10.1002/humu.22329.
Cole AR, Causeret F, Yadirgi G, Hastie CJ, McLauchlan H, McManus EJ, Hernández F, Eickholt BJ, Nikolic M, Sutherland C. Distinct priming kinases contribute to differential regulation of collapsin response mediator proteins by glycogen synthase kinase-3 in vivo. J Biol Chem. 2006 Jun 16;281(24):16591-8.
Khanna R, Wilson SM, Brittain JM, Weimer J, Sultana R, Butterfield A, Hensley K. Opening Pandora's jar: a primer on the putative roles of CRMP2 in a panoply of neurodegenerative, sensory and motor neuron, and central disorders. Future Neurol. 2012 Nov 1;7(6):749-771.
Maniar TA, Kaplan M, Wang GJ, Shen K, Wei L, Shaw JE, Koushika SP, Bargmann CI. UNC-33 (CRMP) and ankyrin organize microtubules and localize kinesin to polarize axon-dendrite sorting. Nat Neurosci. 2011 Nov 20;15(1):48-56.
Tanaka H, Morimura R, Ohshima T. Dpysl2 (CRMP2) and Dpysl3 (CRMP4) phosphorylation by Cdk5 and DYRK2 is required for proper positioning of Rohon-Beard neurons and neural crest cells during neurulation in zebrafish. Dev Biol. 2012 Oct 15;370(2):223-36.
Wang LH, Strittmatter SM. Brain CRMP forms heterotetramers similar to liver dihydropyrimidinase. J Neurochem. 1997 Dec;69(6):2261-9.