Plasmodium falciparum Lammer (PF14_0431), kinase domain, in complex with AMP-PNP

Target ID:

PP-LAMMER

Deposition Date:

Friday 29th January 2010

Families:

Protein Kinase

Related Diseases:

Parasitic disease

Structure type:

protein-ligand

Download Coordinates:

3LLT

Authors:

Wernimont AK, Tempel W, Lin YH, Loppnau P, MacKenzie F, Sullivan H, Weadge J, Kozieradzki I, Cossar D, Sinesterra G, Vedadi M, Arrowsmith CH, Edwards AM, Bountra C, Weigelt J, Bochkarev A, Hui R, Qiu W, Hutchinson A

Site:

Toronto

3LLT

tabs

Structure Details

LAMMER kinases, named for its signature amino acid sequence motif, are Cdc2-like kinases (CLK) found in most eukarytotic genomes including humans, yeast and protozoa. They are dual-specificity kinases known to autophosphorylate at tyrosine residues but to phosphorylate only serine and threonine on substrates [1]. In Plasmodium parasites, the gene PF14_0301 has been found to encode a LAMMER kinase [2]. The signature motif, which is slightly modified to LAMMES in the parasite, is understood to affect substrate specificity. The kinase domain of the protein is found in the C-terminus, with the N-terminus domain featuring a number of RS/SR motifs known to be phosphorylation targets. Structurally, LAMMER kinases feature a unique insert between the 7 and 8 beta strands. This insert takes the form of a beta-hairpin (shown in orange).

Materials and Methods

LAMMER

PDB:3LLT

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:AAK38173.1
Entry Clone Source:
SGC Clone Accession:PP-LAMMER:D534-E875
Tag:mgsshhhhhhssgrenlyfq
Host:BL21(DE3)-V2R-pACYC LamP

Construct

Prelude:
Sequence:DDEIVHFSWKKGMLLNNAFLVIRKMGDGTFGRVLLCQHIDNKKYYAVKVVRNIKKYTRSAKIEADILKKIQNDDINNNNIVKYHGKFMYYDHMCLIFEPLGPSLYEIITRNNYNGFHIEDIKLYCIEILKALNYLRKMSLTHTDLKPENILLDDPYFEKSLITVRRVTDGKKIQIYRTKSTGIKLIDFGCATFKSDYHGSIINTRQYRAPEVILNLGWDVSSDMWSFGCVLAELYTGSLLFRTHEHMEHLAMMESIIQPIPKNMLYEATKTNGSKYVNKDELKLAWPENASSINSIKHVKKCLPLYKIIKHELFCDFLYSILQIDPTLRPSPAELLKHKFLE
Vector:pET15-MHL

Growth

Medium:TB
Antibiotics:
Procedure:PF14_0431 was expressed in E. coli BL21(DE3)-V2R-pACYC LamP cells in Terrific Broth (TB) in the presence of carbenicillin /chloramphenicol (100 microg/mL and 34 microg/mL respectively). A single colony was inoculated into 25 mL of LB with of in a 25 mL(100 microg/mL and 34 microg/mL respectively) Falcon tube and incubated with shaking at 220 rpm overnight at 37 degC. Then the culture was transfered into 1 L of TB with 100 microg/mL carbenicillin, 34 microg/mL chloramphenicol and 0.5 mL of antifoam (Sigma) in a 1 L bottle and cultured using the LEX system to an OD 600 of ~5, cooled to 15 degC, and induced with 0.5 mM isopropyl-1-thio-D-galactopyranoside (IPTG) overnight at 15 degC.

Purification

Procedure
The cleared lysate was loaded onto a column prepacked with 10 g DE52 (Whatman) anion exchange resin (previously activated with 2.5 M NaCl and equilibrated with Binding Buffer); and subsequently onto a 1.0 - 2.5 mL Ni-NTA (Qiagen) column pre-equilibrated with Binding Buffer at approximately 1 - 1.5 mL/min. After the lysate was loaded, the DE52 was further washed with 20 mL of Binding Buffer. The Ni-NTA column was then washed with 200 mL of Wash Buffer at 2 - 2.5 mL/min. After washing, the protein was eluted with 15 mL of Elution Buffer. TCEP was then added to 5 mM after approximately 15 minutes after elution.

The sample was then loaded onto a Superdex S200 26/60 column equilibrated with Gel Filtration Buffer. The fractions from the peak eluting at 232 mL corresponding to monomeric protein were pooled and concentrated using a 15 mL Amicon Ultra centrifugal filter device (Millipore). The protein sample identity and purity were evalulated by mass spectroscopy and SDS-PAGE gel. The concentrated protein was stored at 4 degC.

Extraction

Procedure
The culture was harvested by centrifugation. Pellets from 4 L of culture were resuspended to approximately 40 mL/L of cell culture in Binding Buffer with the addition of 400uL/ L protease inhibitors (1 tablet dissolved in 25ml of ddH2O, Roche complete EDTA-free). Resuspended pellets stored at -80 degC were thawed overnight at 4 degC on the day before purification. Prior to mechanical lysis, each pellet from 1 L of culture was pretreated with 0.5 % CHAPS, 100 ul of protease inhibitors(1 tablet dissolved in 25ml of ddH2O, Roche complete EDTA-free) and 500 units of benzonase for 40 minutes at room temperature. Cells were mechanically lysed with a microfluidizer (Microfluidizer Processor, M-110EH) at approximately 18000 psi; and the cell lysate was centrifuged using a Beckman JA-25.50 rotor at ~75000 x g (24000 rpms) for 20 minutes at 10 degC.
Concentration:
Ligand
MassSpec:
Crystallization:The protein was crystallized at 18 degC in 0.2M Na Acetate, 1.85 M NH4SO4, 0.1M Na Cacodylate pH5.6 with ligands 5 mM AMPPNP using the Sitting drop vapor diffusion method.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Bullock AN, Das S, Debreczeni JE, Relios P, Fedorov O, Niessen FH, Guo K, Papagrigoriou E, Amos AL, Cho S, Turk BE, Ghosh G and Knapp S (2009). Kinase Domain Insertions Define Distinct Roles of CLK Kinases in SR Protein Phosphorylation. Structure.
Li JL, Targett GAT and Baker DA (2001). Primary structure and sexual stage-speci®c expression of a LAMMER protein kinase of Plasmodium falciparum. International Journal of Parasitology.