Leishmania major N-myristoyltransferase in complex with myristoyl-CoA

Target ID:

PP-NMT

Deposition Date:

Wednesday 22nd April 2009

Families:

Transferases

Related Diseases:

Parasitic disease

Structure type:

protein-ligand

Download Coordinates:

3H5Z

Authors:

Qiu W, Hutchinson A, Lin Y-H, Wernimont A, Mackenzie F, Ravichandran M, Cossar D, Zhao Y, Schapira M, Arrowsmith CH, Bountra C, Weigelt J, Edwards AM, Ferguson MAJ, Fairlamb AH, Hui R

Site:

Toronto

3H5Z

tabs

Structure Details

Myristoylation is an irreversible, co-translational (during translation) protein modification found in animals, plants, fungi and viruses. It is an acylation process absolutely specific to the N-terminal amino acid glycine in proteins. A myristoyl group (a 14 carbon saturated fatty acid) is covalently attached to the alpha-amino group of an N-terminal amino acid of a protein and thus increases the lipophilicity of the N-terminal end of the protein[1]. This co-translational modification plays a vital role in membrane targeting, signal transduction, vesicular and protein trafficking.

N-myristoyltransferase(NMT) belongs to the GCN5-realted N-acetyltransferase superfamily and it has been well characterized in a broad range of eukaryotic species, including parasitic protozoa Leishmania major and Trypanosoma brucei. Gene targeting and RNAi results demonstrated that NMT is essential for viability in Leishmania major and Trypanosoma brucei. Sequence alignment and biochemical studies showed that LmNMT and TbNMT have divergent peptide binding specificity compared to the human NMT thus they could be valid chemo-therapeutics targets for developing anti-kinetoplastid drugs[2].

We have determined the X-ray structure of LmNMT in complex with its natural cofactor, myristoyl-CoA to a resolution of 1.5A. Similar to other known NMT structures, the overall LmNMT fold consists of a saddle-shaped mixed beta-sheet in the middle surrounding by several alpha helixes[3,4]. It has a pseudo- two fold symmetry that separates the structure into two parts: the N-terminal half of the NMT is contribute to the binding of myristoyl-CoA The acyl chain from myristoyl-CoA is buried deeply in a narrow pocket within the protein core. The peptide binding groove lies on the C-terminal part of LmNMT with connection to the myristoyl-CoA binding pocket. Since there is no peptide present in the structure, its binding site is found in an open conformation with a series of residues (e.g. Tyr217, His219, Ser221 and Tyr253) having dual conformation.

Materials and Methods

LmjF32.0080

PDB:3H5Z

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:Q4Q5S8_LEIMA
Entry Clone Source:
SGC Clone Accession:LmjF32.0080:MAC040-B01:C203581
Tag:N-terminal tag: mgsshhhhhhssgrenlyfqg
Host:BL21-(DE3)-V2R-pRARE2

Construct

Prelude:
Sequence:PSNSDAAHAFWSTQPVPQTEDETEKIVFAGPMDEPKTVADIPEEPYPIASTFEWWTPNMEAADDIHAIYELLRDNYVEDDDSMFRFNYSEEFLQWALCPPNYIPDWHVAVRRKADKKLLAFIAGVPVTLRMGTPKYMKVKAQEKGEGEEAAKYDEPRHICEINFLCVHKQLREKRLAPILIKEATRRVNRTNVWQAVYTAGVLLPTPYASGQYFHRSLNPEKLVEIRFSGIPAQYQKFQNPMAMLKRNYQLPSAPKNSGLREMKPSDVPQVRRILMNYLDSFDVGPVFSDAEISHYLLPRDGVVFTYVVENDKKVTDFFSFYRIPSTVIGNSNYNLLNAAYVHYYAATSIPLHQLILDLLIVAHSRGFDVCNMVEILDNRSFVEQLKFGAGDGHLRYYFYNWAYPKIKPSQVALVML
Vector:pET15-MHL

Growth

Medium:TB
Antibiotics:
Procedure:LmNMT was expressed in E. coli BL21-(DE3)-V2R-pRARE2 resistant strain in Terrific Broth (TB) in the presence of ampicillin/chloramphenicol (100 μg/mL and 34 μg/mL respectively). A single colony was inoculated into 100mL of LB with of ampicillin/chloramphenicol (100 μg/mL and 34 μg/mL respectively) in a 250 mL baffled flask and incubated with shaking at 250 rpm overnight at 37 °C. The culture was transferred into 1.0 L of TB with ampicillin/chloramphenicol (100 μg/mL and 34 μg/mL respectively) and 0.15 mL of antifoam (Sigma) in a 1 L bottle and cultured using the LEX system to an OD600 of 5-6, cooled to 15 °C, and induced with 0.5 mM isopropyl-1-thio-D-galactopyranoside (IPTG) overnight at 15 °C.

Purification

Procedure
The cleared lysate was loaded onto a 1.0-2.5 mL Ni-NTA (Qiagen) open column (pre-equilibrated with Binding Buffer) at approximately 1.5-2.0 mL/min. The Ni-NTA column was then washed with 150 mL of Wash Buffer at 2-2.5 mL/min. After washing, the protein was eluted with Elution Buffer. The eluted sample was applied to a Sephadex S200 26/60 gel filtration column pre-equilibrated with Gelfiltration Buffer on a AKTA explorer system. The fractions corresponding to the eluted protein peak were pooled and its identity and purity was evalulated by mass spectroscopy and SDS-PAGE gel. It was then concentrated using a 15 mL Amicon Ultra centrifugal filter device (Millipore). The final concentration of the LmNMT was 9.2 mg/ml and stored at 4 ºC. For long term storage, the protein was flash frozen and stored at -80 ºC.

Extraction

Procedure
The culture was harvested by centrifugation. Pellets from 2 L of culture were resuspended to approximately 40 mL/L of cell culture in Binding Buffer with the addition of protease inhibitors (1 mM benzamidine and 1 mM phenylmethyl sulfonyl fluoride (PMSF)). Resuspended pellets stored at -80 ºC were thawed overnight at 4 °C on the day before purification. Prior to sonication, each pellet from 1 L of culture was pretreated with 0.5 % CHAPS and 500 units of benzonase for 40 minutes at room temperature. After 6 minutes sonication, the cell lysate was centrifuged using a Beckman JA-25.25 rotor at 24,000 rpms for 20 minutes at 4 ºC.
Concentration:9.2 mg/ml.
Ligand
MassSpec:
Crystallization:The protein was crystallized at 20 degC in LmNMT was crystallized using hanging drop vapor diffusion. Crystallization buffer is: 25.9% PEG1500, 0.2M NaCl, 0.1M NaCacodylate pH5.6, at 20 degC. using the sitting drop vapor diffusion method.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Boutin, J. A. Myristoylation. Cell Signal 9, 15-35, 1997
Panethymitak, C. et al. Characterization and selective inhibition of myristoyl-CoA:protein N-myristoyltransferase from Trypanosoma brucei and Leishmania major. Biochem. J. 396, 277-285, 2006
Weston, S. A., et al. Crystal structure of the anti-fungal target N-myristoyl transferase. Nature Struct. Biol. 5, 213-221, 1998
Bhatnagar R. S. et al. Structure of N-myristoyltransferase with bound myristoylCoA and peptide substrate analogs. Nature Struct. Biol. 5, 1091-1097, 1998