Human Guanosine monophosphate reductase 2 in complex with NADPH/IMP

Target ID:

GMPR2A

Aliases:

GMPR2MGC15084MGC830

Deposition Date:

Friday 11th November 2005

Families:

Oxidoreductases

Related Diseases:

Metabolism

Structure type:

apo

Download Coordinates:

2C6Q

Authors:

P.KURSULA,P.STENMARK,C.ARROWSMITH,H.BERGLUND,A.EDWARDS,M.EHN,S.GRASLUND,M.HAMMARSTROM,B.M.HALLBERG,T.KOTENYOVA,P.NILSSON-EHLE,D.OGG,C.PERSSON,J.SAGEMARK,H.SCHULER,M.SUNDSTROM,A.THORSELL,J.WEIGELT,P.NORDLUND

Site:

Stockholm

ICM Datapack:

ICM Datapack

2C6Q

tabs

Structure Details

Guanosine monophosphate reductase is a tetrameric protein that catalyzes the irreversible NADPH-dependent deamination of GMP to IMP.
Since GMPR2 is the first step in the conversion of guanosine to adenine nucleotides, it is important in maintaining the intracellular balance between these nucleotides.

Guanosine 5'-phosphate + NADPH + H+ => Inosine 5'-phosphate + NH3 + NADP+

Together with Inosine-monophosphate dehydrogenase (IMPDH), this metabolic conversion
constitutes an important control point in maintaining intracellular purine nucleotide ratios
during cellular differentiation.

The NADPH/IMP complex reveals that:

Binding of NADPH to GMPR2 stabilizes an otherwise disordered part of the protein and induces a large movement of a loop in the NADPH binding pocket. A dynamic binding site thus seem to be required for NADPH binding.
Part of the NADPH binding site is formed by a loop segment from a neighbouring monomer, hinting to a mechanism for crosstalk between different binding sites in the tetramer.
IMP has an unusually short distance to a conserved cysteine in the active site (2.1 Å). This short distance is observed in both structures of GMPR2. It is possible that the active site cysteine makes a covalent intermediate with the substrate during catalysis. Our ternary complex further corroborates this hypothesis by showing how GMP, NADPH and active site cysteine would be lined up for the reaction to take place.

Together with the two first SGC structures of

GMPR and

GMPR2, the structure of GMPR2 in complex with NADPH and IMP completes the picture.
SGC has provided structures of the enzyme(s) in complex with substrate and product, respectively,
as well as a ternary complex with product and cofactor.
The NADPH structure in particular suggests a possible reaction mechanism
and highlights the importance of a dynamic binding site to allow co-factor binding.

Materials and Methods

GMPR2 + NADPH/IMP

PDB:2C6Q

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC008021
Entry Clone Source:MGC
SGC Clone Accession:
Tag:M G S S H H H H H H S S G L V P R G S
Host:BL21(DE3)

Construct

Prelude:
Sequence:mgsshhhhhhssglvprgsLDFKDVLLRPKRSTLKSRSEVDLTRSFSFRNSKQTYSGVPIIAANMDTVGTFEMAKVLCKFSLFTAVHKHYSLVQWQEFAGQNPDCLEHLAASSGTGSSDFEQLEQILEAIPQVKYICLDVANGYSEHFVEFVKDVRKRFPQHTIMAGNVVTGEMVEELILSGADIIKVGIGPGSVCTTRKKTGVGYPQLSAVMECADAAHGLKGHIISDGGCSCPGDVAKAFGAGADFVMLGGMLAGHSESGGELIERDGKKYKLFYGMSSEMAMKKYAGGVAEYRASEGKTVEVPFKGDVEHTIRDILGGIRSTCTYVGAAKLKELSRRTTFIRVTQQVN
Vector:p28a-LIC

Growth

Medium:
Antibiotics:
Procedure:10 mL TB media supplemented with 4 g/L glycerol and 50 µg/mL Kanamycin was inoculated with BL21 DE3 cells. Cells were grown overnight at 30 ºC in 100 mL shake flasks at 200 rpm. 750 mL TB media supplemented with 4 g/L glycerol and 50 µg/mL kanamycin was inoculated with 10 mL of the over night culture. The large scale cultivations were grown in TunAir shake flasks at 37 °C, 225 RPM. When OD reached approximately 1.3. the temperature in the incubator was lowered to 18°C during 30 min. Then the, protein expression was induced by addition of IPTG to a final concentration of 0.5 mM. The protein expression was continued over night.

Purification

Procedure
Columns:
HisTrap HP 1 mL (IMAC)
HiLoadÂ 16/60 Superdex 200 Prep Grade (Gel filtration)

Procedure:
Purification was conducted automatically on an AKTA xpress system operated by UNICORN software at a flow of 0.8 mL/min. Prior to purification columns were equilibrated with IMAC Bind/Wash1 Buffer (HisTrap HP) and Gel filtration buffer (Superdex 200). The protein sample was loaded on the HisTrap HP column that was washed with IMAC Bind/Wash1 Buffer followed by IMAC Wash2 Buffer. Bound protein was eluted from the IMAC columns with 7.5 mL of IMAC Elution Buffer and loaded onto the Gel filtration column. The chromatogram from Gel filtration showed one major protein peak that mainly consisted of GMPR2 of high purity as shown by SDS-PAGE analysis. TCEP was added to the pooled protein peak to a final concentration of 2 mM. The protein was concentrated to 15 mg/mL and stored at -80ºC.

Extraction

Procedure
Cells were harvested by centrifugation and pellets were resuspended in 50mM HEPES pH 7.5, 500mM NaCl, 10% glycerol, 0.5 mM TCEP and 1 Complete EDTA-free protease inhibitor tablet was added. The pellet was then freezed at -80. Cells were thawed and disrupted by sonication (50%, 2s-2s pulse on-off) for three minutes. The sample was centrifuged for 30 min at 49000×g. The soluble fraction was filtered through 0.22 µm prior to loading onto the column.
Concentration:
Ligand
MassSpec:
Crystallization:Purified His-tagged GMPR2 was crystallized using the hanging drop vapour diffusion method with 1µl protein solution (7.5 mg/mL or 5 mg/mL) + 1µl reservoir solution. 7.5 mM GMP was added to the protein prior to setting the drops. The drops were equilibrated against a reservoir solution containing 9% PEG3350, 0.1 M Sodium Citrate pH 5.5. Crystals formed after several days. The crystal was soaked in 6mM NADPH for 1 hour, cryoprotected in well solution containing 20% glycerol and 6 mM NADPH and then flash frozen in liquid nitrogen.
NMR Spectroscopy:
Data Collection:
Data Processing: