Human palmitoyl-protein thioesterase 1

Target ID:

PPT1

Deposition Date:

Tuesday 31st March 2009

Families:

Miscellaneous

Structure type:

apo

Download Coordinates:

3GRO

Authors:

Dobrovetsky E, Seitova A, Tong Y, Tempel W, Dong A, Arrowsmith CH, Edwards AM, Bountra C, Weigelt J, Bochkarev A, Cossar D, Park H

Site:

Toronto

ICM Datapack:

ICM Datapack

3GRO

tabs

Structure Details

Palmitoyl Protein Thioesterase (EC3.1.2.22) was first isolated from bovine brain as an enzyme which removes palmitate from palmitoylated proteins[1]. The enzyme was cloned from human cDNA and shown to be normally secreted into the extracellular compartment as a glycosylated protein with conserved sequences characteristic of thioesterases[2]. The bovine and human enzymes are highly similar (94% identity, 97% similarity). The enzyme is broadly distributed in human tissues[3]. Mutation of PPT1 causes severe neurodegenerative disease[4] - including Infantile Neuronal Ceroid Lipofuscinosis INCL (substitution R122W)[5]. INCL is characterized as a lysosmal storage disease resulting in the deposition of granules (principally saposins A and D) in neuronal and non-neuronal tissues[6].

The crystal structure of bovine PPT1 has been determined as a classical α/β-hydrolase with a deep substrate-binding cleft and a characteristic (Ser-His-Asp) catalytic triad[7]. In contrast to other lipases, bovine PPT1 is fixed in an "open" configuration with the substrate-binding groove accessible to solvent. The related enzyme PPT2 has also been solved and shown to be structurally highly similar despite significant sequence divergence[8]. Human PPT2 (1PJA) retains the lipase lid which covers the substrate-binding groove. We have solved the structure of human PPT1, expressed in sf9 cells and recovered from the culture supernatant, by molecular replacement on the bovine PPT1 structure (1EI9). The human enzyme shows the same placement of the catalytic residues and the substrate-binding groove, however loop α4 is displaced to occupy a position which covers a significant portion of the substrate-binding cleft - analogous to that observed for PPT2. A disulphide bond (C152-C160) which is argued to fix loop α4 in the "open" conformation in the bovine structure is also present in the closed conformation of human PPT1. In our structure, the enzyme is a homodimer which is consistent with our size-exclusion chromatographic data and that reported by Lyly et al[9]. Ongoing studies with bound substrate should provide insight on the movement of the proposed lid loop (α4).

Materials and Methods

PPT1

PDB:3GRO

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC008426
Entry Clone Source:MGC, AU44-G2,
SGC Clone Accession:PPT1:DCC001-D04:C44672
Tag:C-terminal tag: EFVEHHHHHHHH
Host:Sf9 insect cells

Construct

Prelude:
Sequence:ARALQHLDPPAPLPLVIWHGMGDSCCNPLSMGAIKKMVEKKIPGIYVLSLEIGKTLMEDVENSFFLNVNSQVTTVCQALAKDPKLQQGYNAMGFSQGGQFLRAVAQRCPSPPMINLISVGGQHQGVFGLPRCPGESSHICDFIRKTLNAGAYSKVVQERLVQAEYWHDPIKEDVYRNHSIFLADINQERGINESYKKNLMALKKFVMVKFLNDSIVDPVDSEWFGFYRSGQAKETIPLQETSLYTQDRLGLKEMDNAGQLVFLATEGDHLQLSEEWFYAHIIPFLG
Vector:pFHMSP-LIC-C

Growth

Medium:
Antibiotics:
Procedure:Plasmid transfer vector pFHMSP-LIC-C containing the gene was transformed into DH10Bac E.coli cells (Invitrogen) to obtain recombinant viral DNA. SF9 cells were transfected with Bacmid DNA using Cellfectin reagent (Invitrogen), and recombinant baculovirus was generated. Viral stock was amplified from P1 to P3.

Sf9 cells grown in HyQ® SFX Insect Serum Free Medium (Cat.# SH3027802) at density of 3 million cells per milliliter of media and with viability not less then 97% were infected with 7 mL of P3 viral stock for each 1 L of cell culture. Cell culture medium was collected after 4 days of incubation on a shaker at 100 RPM and 27 °C when cells viability dropped to 25-45%.

Purification

Procedure
IMAC purification: A 1.6 L volume of medium was mixed with 20 mL pre-equilibrated NiNTA Superflow beads and stirred (Talboys/Troemner) for 1 hour. The resin was transferred to a 50 mL gravity column, washed with 50 mL of Washing Buffer, and the protein was eluted with 10 mL of Elution Buffer. A second round of NiNTA batch absorption has been performed for increased protein yield.

Bound protein was eluted from the IMAC columns with Elution Buffer and loaded onto the Gelfiltration (GF) column. The chromatogram from gel filtration showed one major protein peak that consisted of PPT1 confirmed by SDS-PAGE analysis.

Extraction

Procedure
The cultured medium was centrifuged at 14,000 xg for 15 minutes, and the pH of the supernatant was adjusted to 7.5 at room temperature by adding 10x Buffer_A. Protease inhibitors were added to final concentrations of 1 mM phenylmethanesulfonyl fluoride (PMSF, Bioshop) and 2 mM benzamidine hydrochloride (Sigma).
Concentration:Purified protein was concentrated using 15 mL concentrators with an appropriate molecular weight cut-off (Amicon Ultra-15 10,000 MWCO, Millipore) to a final value of 10 mg/mL. Average yield was about 2.5 mg/L.
Ligand
MassSpec:
Crystallization:Crystallization was setup using sitting drops with Red Wings and SGC-I screens initially. Small crystals were seen at condition: 0.1M NaCacodylate, pH 5.50, 25% PEG3350, 0.2M (NH4)2SO4, protein concentration 10 mg/mL.Crystal used for structure determination were grown in: 0.1M NaCacodylate, pH 5.40, 23.64% PEG3350, 0.2M (NH4)2SO4, protein concentration 10 mg/mL.Cryoprotectant used 0.9V well solution plus 0.1V 80% Glycerol.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Camp L.A. and Hofmann S.L. (1993). Purification and properties of a Palmitoyl-Protein Thioesterase that cleaves palmitate from H-Ras. J.Biol.Chem. 268(30): 22566-22574.
Camp L.A., Verkruyse L.A., Afendis S.J., Slaughter C.A., and Hofmann S.L. (1994). Molecular cloning and expression of Palmitoyl-Protein Thioesterase. J.Biol.Chem. 269(37): 23212-23219.
Schriner J.E., Yi W., and Hofmann S.L. (1996). cDNA and genomic cloning of human Palmitoyl-Protein Thioesterase (PPT), the enzyme defective in Infantile Neuronal Ceroid Lipofuscinosis. Genomics 34: 317-322.
Salonene T., Jarvela I., Peltonen L., and Jalanko A. (2000). Detection of eight novel Palmitoyl-Protein Thioesterase (PPT) mutations underlying Infantile Neuronal Ceroid Lipfuscinosis (INCL; CLN1). Human Mutation 15: 273-279.
Vesa J., Hellsten E., Verkruyse L.A., Camp L.A., Hofmann, S.L., and Peltonen L. (1995). Mutations in the Palmitoyl-Protein Thioesterase gene causing Infantile Neuronal Ceroid Lipofuscinosis. Nature 376: 584-587.
Lu J.-Y, and Hofmann S.L. (2006). Lysosomal metabolism of lipid-modified proteins. J. Lipid Res. 47: 1352-1357.
Bellizzi J.J., Widom J., Kemp C., Ly J.-Y., Das A.K., Hofmann S.L., and Clardy J. (2000). The crystal structure of Palmitoyl-Protein Thioesterase 1 and the molecular basis of Infantile Neuronal Ceroid Lipofuscinosis. Proc. Natl. Acad. Sci. 97(9): 4573-4578.
Calero G., Gupta P., Nonato M.C., Tandel S., Biehl E.R., Hofmann S.L., and Clardy J. (2003). The crystal structure of Palmitoyl-Protein Thioesterase-2 (PPT2) reveals the basis for divergent substrate specificities of the two lysosomal thioesterases, PPT1 and PPT2. J. Biol. Chem. 278(39): 37957-37964.
Lyly A., von Schantz C., Salonen T., Kopra O., Saarela J., Jauhiainen M., Kyttala A, and Jalanko A. (2007). Glycosylation, transport, and complex formation of Palmitoyl-Protein Thioesterase 1 (PPT1) - distinct characteristics in neurons. BMC Cell Biology 8: 22-35.