Human dipeptidyl peptidase 7 in complex with inhibitor GSK237826A

Target ID:

DPP7

Deposition Date:

Friday 14th May 2010

Families:

Proteases

Structure type:

protein-ligand

Download Coordinates:

3N0T

Authors:

Dobrovetsky E, Dong A, Seitova A, Crombett L, Paganon S, Cossar D, Edwards AM, Arrowsmith CH, Bountra C, Weigelt J, Hassel A, Shewchuk L, Haffner C, Bochkarev A

Site:

Toronto

ICM Datapack:

ICM Datapack

3N0T

tabs

Structure Details

DPP7 is an aminopeptidase with orthologs in >50 species, including lower eukaryotes, cleaving peptides at the C-terminal side of Proline residues. DPP7 is also known as DPP2 and QPP (quiescent cell proline dipeptidase) and has substrate specificity similar to DPP4 (Underwood et al, 1999), though there is only low sequence identity. The protein is reported to be essential for maintaining lymphocytes and fibroblasts, and its inhibition results in apoptosis (Mele et al, 2009). Danilova et al (2009) have shown in mouse knockdown experiments that DPP7 has a role in glucose homeostasis which is apparently opposite to that of DPP4. Physiological studies have shown that neuropeptides like casomorphin and bradykinin and their fragments are cleaved by brain DPP7 (Mentlein and Struckhoff, 1989). No natural DPP7 activators or inhibitors have been reported. A significant fraction of synthetic inhibitors reported for the enzyme were initially designed as inhibitors for DPP4 (Maes et al, 2007). DPP7 possesses a slightly acidic pH optimum, between 5.5 and 7.0, and is localized to intracellular vesicles (Leiting et al, 2003; Danilova et al, 2007). DPP7 is the first reported protease that contains a leucine zipper motif forming the functional homodimer (Chiravuri et al, 2000).

We have purified rHuDPP7 from insect cells infected with virus carrying the human DNA sequence. The protein is secreted into the culture medium and is glycosylated. The high-resolution crystal structure has been determined, revealing a glycosylated (Asn 86 and Asn 363) homodimer. The fold is similar to DPP4 (PDB:1TK3) having a classical catalytic domain with α/β hydrolase fold (residues 28-190 and 400-476). This structure of DPP7 presents a novel α-helical cap domain which is inserted between residues 190 and 400 of the hydrolase domain. This cap domain is equivalent to the non-catalytic 8-bladed β-propeller domain (residues 39-511) of DPP4. The predicted catalytic triad, Ser 162, Asp 418 and His 443, is located in the interior of the protein, deep inside the cleft between the two domains.

Materials and Methods

DPP7

PDB:3N0T

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC011907
Entry Clone Source:MGC
SGC Clone Accession:DPP7:SDC134-D09:C41107
Tag:N-terminal tag: APEHHHHHHDYDIPTTENLYFQGAMD
Host:Sf9 insect cells

Construct

Prelude:
Sequence:gamdAPGFQERFFQQRLDHFNFERFGNKTFPQRFLVSDRFWVRGEGPIFFYTGNEGDVWAFANNSGFVAELAAERGALLVFAEHRYYGKSLPFGAQSTQRGHTELLTVEQALADFAELLRALRRDLGAQDAPAIAFGGSYGGMLSAYLRMKYPHLVAGALAASAPVLAVAGLGDSNQFFRDVTADFEGQSPKCTQGVREAFRQIKDLFLQGAYDTVRWEFGTCQPLSDEKDLTQLFMFARNAFTVLAMMDYPYPTDFLGPLPANPVKVGCDRLLSEAQRITGLRALAGLVYNASGSEHCYDIYRLYHSCADPTGCGTGPDARAWDYQACTEINLTFASNNVTDMFPDLPFTDELRQRYCLDTWGVWPRPDWLLTSFWGGDLRAASNIIFSNGNLDPWAGGGIRRNLSASVIAVTIQGGAHHLDLRASHPEDPASVVEARKLEATIIGEWVKAARREQQPALRGGPRLSL
Vector:pFHMSP-LIC-N

Growth

Medium:
Antibiotics:
Procedure:Plasmid transfer vector pFHMSP-LIC-N 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 100 mL gravity column, washed with 100 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 DPP7 confirmed by SDS-PAGE analysis. The protein was then TEV cleaved to remove the poly histidine tag. TEV was added in the ration of 50:1 DPP7:TEV. The reaction was incubated at 4°C for ~2 days. Cleavage was confirmed by SDS-PAGE analysis and the TEV and tag removed by passing the sample through a 1mL HisTrap FF crude column which had been equilibrated with GF buffer.

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 3.5mg/mL. Average yield was about 2 mg/L.
Ligand
MassSpec:
Crystallization:Protein was incubated with inhibitor with 1 to 50 (protein/inhibitor) molar ratio prior to crystallization.
Crystal used for structure determination were grown in: 2M NH4SO4 ,0.2M NaAc 0.1M Hepes 7.5, 5% MPDCryo solution: 25% Glycerol, 2M NH4SO4 ,0.2M NaAc 0.1M Hepes 7.5, 5% MPD
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Robert Underwood, Murali Chiravuri, Henry Lee, Tracy Schmitz, Alisa K. Kabcenell, Kurt Yardley, and Brigitte T. Huber (1999). Sequence, Purification, and Cloning of an Intracellular Serine Protease, Quiescent Cell Proline Dipeptidase. J. Biol. Chem. 274(48):34053-34058.

Deanna A. Mele, Pradeep Bista, Diana Velez Baez, Brigitte T. Huber (2009) Dipeptidyl peptidase 2 is an essential survival factor in the regulation of cell quiescence. Cell Cycle 8:2425-2434.

Olga V. Danilova, Albert K. Tai, Deanna A. Mele, Martin Beinborn, Andrew B. Leiter, Andrew S. Greenberg, James W. Perfield II, Jason DeFuria, Praful S. Singru, Ronald M. Lechan, and Brigitte T. Huber (2009). Neurogenin 3-Specific Dipeptidyl Peptidase-2 Deficiency Causes Impaired Glucose Tolerance, Insulin Resistance, and Visceral Obesity. Endocrinol. 150(12):5240-5248.

Murali Chiravuri, Henry Lee, Suzanne L. Mathieu, and Brigitte T. Huber (2000). Homodimerization via a Leucine Zipper Motif Is Required for Enzymatic Activity of Quiescent Cell Proline Dipeptidase. J. Biol. Chem. 275(35):26994-26999.

Olga Danilova, Bei Li, A. Katrin Szardenings,Brigitte T. Huber, and Jonathan S. Rosenblum (2007). Synthesis and activity of a potent, specific azabicyclo[3.3.0]-octane-based DPP II inhibitor. Bioorg. Med. Chem. Lett. 17:507-510

Barbara Leiting, Kellyann D. Pryor, Joseph K. Wu, Frank Marsilio, Reshma A. Patel, Charles S. Craik.,Jonathan A. Ellman., Richard T. Cummings And Nancy A. Thornberry (2003). Catalytic properties and inhibition of proline-specific dipeptidyl peptidases II, IV and VII. Biochem. J. 371:525-532.

Rolf Mentlein and Gernot Struckhoff (1989). Purification of Two Dipeptidyl Aminopeptidases II from Rat Brain and Their Action on Proline-Containing Neuropeptides. J. Neurochem. 52(4):1284-1293

Marie-Berthe Maes, Simon Scharpé, Ingrid De Meester (2007). Dipeptidyl peptidase II (DPPII), a review.. Clin. Chim. Acta 380:31-49