Human pantothnate kinase 3

Target ID:

PANK3

Aliases:

FLJ12899MGC16863

Deposition Date:

Thursday 31st August 2006

Families:

Non-protein Kinase

Related Diseases:

NeurobiologyMetabolism

Structure type:

apo

Download Coordinates:

2I7P

Authors:

B.HONG,L.WANG,L.SHEN,W.TEMPEL,P.LOPPNAU,P.FINERTY,C.H.ARROWSMITH,A.M.EDWARDS,M.SUNDSTROM,J.WEIGELT,A.BOCHKAREV,H.PARK,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Toronto

ICM Datapack:

ICM Datapack

2I7P

tabs

Structure Details

Coenzyme A(CoA)is an essential cofactor in all living organisms,
where it functions as an acyl group carrier and a carbonyl-activating group in a number of central biochemical transformation, including the tricarboxylic acid cycle and fatty acid metabolism1).
CoA is involved in over 100 different reactions in intermediary metabolism and the source of 4′-phosphopantetheine, prosthetic group of carrier proteins of fatty acid, polyketide and nonribosomal peptide synthases.

The biosynthesis of CoA from pantothenic acid is an essential and universal pathway in prokaryotes and eukaryotes. CoA is synthesized from pantothenic acid in five steps.
In the first step, pantothenic acid is phosphorylated to 4′-phosphoantothenic acid in a reaction catalyzed by pantothenate kinase(PANK). There are four pantothenate kinase isoforms in human. PanK1 and PanK3 isoforms are most highly expressed in liver, while PanK4 in muscle2,3). Interestingly, PanK2 includes mitochondria-targeting sequence in its N-terminus and localizes to mitochondria. Recently, the mutations observed in catalytic domain of PanK2 was reported to cause the inherited PanK-associated neurodegeneration (PKAN), which accompanies with iron-accumulation in brain. The catalytic domain is highly conserved between PanK isoforms, and its structural studies may provide the basis for understanding the process of PKAN4).

We solved the conserved catalytic domains of two isoforms, PanK1 and PanK3, both of which adopt almost the same architecture as members of actin folding family. Until now, the biological unit of pantothenate kinase is known as dimer in solution2), and our two structures also support the fact that dimer is a active form.
Structurally, each monomer has one small and one large domain, both of which include β-sheet flanked with α-helice.
Two monomers are dimerized through the interaction between the longest helix (red) of secondary elements composed of each monomer
The dimerization of pantothenate kinase was assessed to be important for protein thermostability.
The cleft between two domains are supposed to be a putative ATP binding site. Initially, both PanK structures were solved in complex with AcCoA , although the adenine rings of AcCoA were removed in final models because of poor densities. The one side of bound AcCoA was bound to β-sheet of large domain, while the other side covered by long loop coming from the second monomer.

Materials and Methods

Human pantothenate kinase 3 (PANK3)

PDB:2I7P

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:
Entry Clone Source:synthetic DNA from GenScript
SGC Clone Accession:
Tag:N-terminal hexa histiden tag with thrombin cleavage site: mgsshhhhhhssglvprgs
Host:E.coli BL21 (DE3)

Construct

Prelude:
Sequence:mgsshhhhhhssglvprgsPWFGMDIGGTLVKLSYFEPIDITAEEEQEEVESLKSIRKYLTSNVAYGSTGIRDVHLELKDLTLFGRRGNLHFIRFPTQDLPTFIQMGRDKNFSTLQTVLCATGGGAYKFEKDFRTIGNLHLHKLDELDCLVKGLLYIDSVSFNGQAECYYFANASEPERCQKMPFNLDDPYPLLVVNIGSGVSILAVHSKDNYKRVTGTSLGGGTFLGLCSLLTGCESFEEALEMASKGDSTQADKLVRDIYGGDYERFGLPGWAVASSFGNMIYKEKRESVSKEDLARATLVTITNNIGSVARMCAVNEKINRVVFVGNFLRVNTLSMKLLAYALDYWSKGQLKALFLEHEGYFGAVGALLGLPN
Vector:p28a-LIC

Growth

Medium:
Antibiotics:
Procedure:The target was expressed in E. coli by inoculating 100 mL of overnight culture grown in Luria-Bertani medium into a 1.8 L of Terrific Broth medium in the presence of 50 µg/mL kanamycin at 37ºC. When OD600 was ~2.5 to 3.5, the culture was induced with 1mM IPTG and the temperature was reduced to 18°C, and the cells were allowed to grow overnight before harvesting and flash frozen by liquid N2.

Purification

Procedure
The thawed cell pellets were suspended in 100 mL of the binding buffer (10 mM Tris pH 7.5, 0.5 M NaCl, 5% glycerol, 5 mM imidazole) with a protease inhibitor cocktail (0.1 mM M benzamidine-HCl and 0.1 mM phenylmethyl sulfonyl fluoride), and 0.5% CHAPS. The cells were lysed by liquid fluidizer. The lysate was centrifuged at 15000 rpm for 30 min and the supernatant was passed through DE52 (Whatman) column equilibrated with the binding buffer and then loaded onto 3 mL Ni-NTA column (Qiagen) equilibrated with the same binding buffer at 4 ºC. The Ni-NTA column was washed with 150 mL of the wash buffer (10mM Tris pH 7.5, 0.5 M NaCl, 5% glycerol, 30 mM imidazole) and the protein was eluted with 15 mL of the elution buffer (10mM Tris pH 7.5, 0.5 M NaCl, 5% glycerol, 250 mM imidazole). The protein were further purified and desalted using gel filtration column, Superdex 200 (26/60), which was pre-equilibrated with 20 mM Tris pH 8.0, 0.2 M NaCl, and 10 mM DTT. All proteins were concentrated using an Amicon Ultra centrifugal filter to a final concentration of 25 mg/mL. Protein concentrations were measured using Bradford assay with purity >95% based on SDS-PAGE analysis.

Extraction

Procedure

Concentration:25 mg/mL
Ligand
MassSpec:
Crystallization:Crystallization trials were set up using the sitting drop vapor diffusion method and the protein drop was equilibrated against a reservoir solution with 1:1 volume ratio. PANK3 was crystallized in 0.1M Tris-HCl, 16% PEG, 0.2M DiNH4Citrate, and 0.02M octaethylene glycol dodecyl ether.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

PANK2 mutation screening recommended to confirm diagnosis of pantothenate kinase-associated neurodegeneration.
AJNR Am J Neuroradiol. 2006 May;27(5):951. No abstract available.
Biochemical properties of human pantothenate kinase 2 isoforms and mutations linked to pantothenate kinase-associated neurodegeneration.
J Biol Chem. 2006 Jan 6;281(1):107-14. Epub 2005 Nov 3.
Mitochondrial localization of human PANK2 and hypotheses of secondary iron accumulation in pantothenate kinase-associated neurodegeneration.
Ann N Y Acad Sci. 2004 Mar;1012:282-98.
Altered neuronal mitochondrial coenzyme A synthesis in neurodegeneration with brain iron accumulation caused by abnormal processing, stability, and catalytic activity of mutant pantothenate kinase 2. J Neurosci. 2005 Jan 19;25(3):689-98.