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- PDB-6gg7: cyanobacterial GAPDH with full-length CP12 -

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Basic information

Entry
Database: PDB / ID: 6gg7
Titlecyanobacterial GAPDH with full-length CP12
Components
  • CP12 polypeptide
  • Glyceraldehyde-3-phosphate dehydrogenase
KeywordsPHOTOSYNTHESIS / Calvin Cycle / Regulation
Function / homology
Function and homology information


negative regulation of reductive pentose-phosphate cycle / Oxidoreductases; Acting on the aldehyde or oxo group of donors; With NAD+ or NADP+ as acceptor / oxidoreductase activity, acting on the aldehyde or oxo group of donors, NAD or NADP as acceptor / glucose metabolic process / NAD binding / NADP binding / metal ion binding
Similarity search - Function
Calvin cycle protein CP12-like / CP12 domain / CP12 domain / CP12 / Glyceraldehyde-3-phosphate dehydrogenase, type I / Glyceraldehyde 3-phosphate dehydrogenase, active site / Glyceraldehyde 3-phosphate dehydrogenase active site. / Glyceraldehyde 3-phosphate dehydrogenase, NAD binding domain / Glyceraldehyde 3-phosphate dehydrogenase, NAD(P) binding domain / Glyceraldehyde 3-phosphate dehydrogenase, catalytic domain ...Calvin cycle protein CP12-like / CP12 domain / CP12 domain / CP12 / Glyceraldehyde-3-phosphate dehydrogenase, type I / Glyceraldehyde 3-phosphate dehydrogenase, active site / Glyceraldehyde 3-phosphate dehydrogenase active site. / Glyceraldehyde 3-phosphate dehydrogenase, NAD binding domain / Glyceraldehyde 3-phosphate dehydrogenase, NAD(P) binding domain / Glyceraldehyde 3-phosphate dehydrogenase, catalytic domain / Glyceraldehyde/Erythrose phosphate dehydrogenase family / Glyceraldehyde 3-phosphate dehydrogenase, C-terminal domain / Glyceraldehyde 3-phosphate dehydrogenase, NAD binding domain / Dihydrodipicolinate Reductase; domain 2 / Dihydrodipicolinate Reductase; domain 2 / NAD(P)-binding domain superfamily / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
NICOTINAMIDE-ADENINE-DINUCLEOTIDE / CP12 polypeptide / Glyceraldehyde-3-phosphate dehydrogenase
Similarity search - Component
Biological speciesThermosynechococcus elongatus (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.32 Å
AuthorsMcFarlane, C.R. / Murray, J.W.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research CouncilBB/J014575/1 United Kingdom
CitationJournal: Proc Natl Acad Sci U S A / Year: 2019
Title: Structural basis of light-induced redox regulation in the Calvin-Benson cycle in cyanobacteria.
Authors: Ciaran R McFarlane / Nita R Shah / Burak V Kabasakal / Blanca Echeverria / Charles A R Cotton / Doryen Bubeck / James W Murray /
Abstract: Plants, algae, and cyanobacteria fix carbon dioxide to organic carbon with the Calvin-Benson (CB) cycle. Phosphoribulokinase (PRK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are essential ...Plants, algae, and cyanobacteria fix carbon dioxide to organic carbon with the Calvin-Benson (CB) cycle. Phosphoribulokinase (PRK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are essential CB-cycle enzymes that control substrate availability for the carboxylation enzyme Rubisco. PRK consumes ATP to produce the Rubisco substrate ribulose bisphosphate (RuBP). GAPDH catalyzes the reduction step of the CB cycle with NADPH to produce the sugar glyceraldehyde 3-phosphate (GAP), which is used for regeneration of RuBP and is the main exit point of the cycle. GAPDH and PRK are coregulated by the redox state of a conditionally disordered protein CP12, which forms a ternary complex with both enzymes. However, the structural basis of CB-cycle regulation by CP12 is unknown. Here, we show how CP12 modulates the activity of both GAPDH and PRK. Using thermophilic cyanobacterial homologs, we solve crystal structures of GAPDH with different cofactors and CP12 bound, and the ternary GAPDH-CP12-PRK complex by electron cryo-microscopy, we reveal that formation of the N-terminal disulfide preorders CP12 prior to binding the PRK active site, which is resolved in complex with CP12. We find that CP12 binding to GAPDH influences substrate accessibility of all GAPDH active sites in the binary and ternary inhibited complexes. Our structural and biochemical data explain how CP12 integrates responses from both redox state and nicotinamide dinucleotide availability to regulate carbon fixation.
History
DepositionMay 2, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 8, 2019Provider: repository / Type: Initial release
Revision 1.1Nov 20, 2019Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jan 17, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Glyceraldehyde-3-phosphate dehydrogenase
C: CP12 polypeptide
A: Glyceraldehyde-3-phosphate dehydrogenase
D: CP12 polypeptide
hetero molecules


Theoretical massNumber of molelcules
Total (without water)92,1356
Polymers90,8084
Non-polymers1,3272
Water10,160564
1
B: Glyceraldehyde-3-phosphate dehydrogenase
C: CP12 polypeptide
A: Glyceraldehyde-3-phosphate dehydrogenase
D: CP12 polypeptide
hetero molecules

B: Glyceraldehyde-3-phosphate dehydrogenase
C: CP12 polypeptide
A: Glyceraldehyde-3-phosphate dehydrogenase
D: CP12 polypeptide
hetero molecules


Theoretical massNumber of molelcules
Total (without water)184,26912
Polymers181,6158
Non-polymers2,6544
Water1448
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation8_555-y,-x,-z+1/21
Buried area29220 Å2
ΔGint-162 kcal/mol
Surface area43110 Å2
MethodPISA
Unit cell
Length a, b, c (Å)141.052, 141.052, 75.931
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number96
Space group name H-MP43212
Components on special symmetry positions
IDModelComponents
11B-1109-

HOH

21A-1126-

HOH

31A-1300-

HOH

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Components

#1: Protein Glyceraldehyde-3-phosphate dehydrogenase


Mass: 36792.734 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermosynechococcus elongatus (strain BP-1) (bacteria)
Strain: BP-1 / Gene: tll1466 / Plasmid: pRSETA modified / Details (production host): thrombin cleavable his-tag / Production host: Escherichia coli KRX (bacteria)
References: UniProt: Q8DIW5, Oxidoreductases; Acting on the aldehyde or oxo group of donors; With NAD+ or NADP+ as acceptor
#2: Protein CP12 polypeptide


Mass: 8611.127 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermosynechococcus elongatus (strain BP-1) (bacteria)
Strain: BP-1 / Gene: cp12 / Plasmid: pRSETA modified / Details (production host): thrombin cleavable his-tag / Production host: Escherichia coli KRX (bacteria) / References: UniProt: Q8DHX3
#3: Chemical ChemComp-NAD / NICOTINAMIDE-ADENINE-DINUCLEOTIDE


Mass: 663.425 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C21H27N7O14P2 / Comment: NAD*YM
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 564 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.4 Å3/Da / Density % sol: 48.96 %
Crystal growTemperature: 290 K / Method: vapor diffusion
Details: Tryptone CM1(A6) 1% Tryptone, 25% PEG, 100 mM Hepes pH 8.

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I24 / Wavelength: 0.96861 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Feb 27, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.96861 Å / Relative weight: 1
ReflectionResolution: 1.32→60.42 Å / Num. obs: 178371 / % possible obs: 99.99 % / Redundancy: 12.8 % / Biso Wilson estimate: 13.62 Å2 / CC1/2: 0.988 / Rmerge(I) obs: 0.1266 / Rrim(I) all: 0.132 / Net I/σ(I): 11.43
Reflection shellResolution: 1.32→1.367 Å / Redundancy: 12.6 % / Rmerge(I) obs: 0.9185 / Num. unique obs: 17624 / CC1/2: 0.586 / Rrim(I) all: 0.9573 / % possible all: 100

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Processing

Software
NameVersionClassification
PHENIX(1.13_2998: ???)refinement
xia2data reduction
xia2data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4BOY

4boy


Resolution: 1.32→60.416 Å / SU ML: 0.13 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 15.65
RfactorNum. reflection% reflection
Rfree0.1735 8791 4.93 %
Rwork0.1454 --
obs0.1468 178371 99.98 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 1.32→60.416 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5516 0 88 564 6168
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0055908
X-RAY DIFFRACTIONf_angle_d0.9218081
X-RAY DIFFRACTIONf_dihedral_angle_d13.5173333
X-RAY DIFFRACTIONf_chiral_restr0.078916
X-RAY DIFFRACTIONf_plane_restr0.0061049
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.3197-1.33470.28353030.26115563X-RAY DIFFRACTION99
1.3347-1.35040.27252970.25015539X-RAY DIFFRACTION100
1.3504-1.36680.26832940.24325592X-RAY DIFFRACTION100
1.3668-1.38410.26282940.22845611X-RAY DIFFRACTION100
1.3841-1.40240.24952760.21325599X-RAY DIFFRACTION100
1.4024-1.42160.2233270.18835553X-RAY DIFFRACTION100
1.4216-1.44190.22442970.17885612X-RAY DIFFRACTION100
1.4419-1.46340.21132810.17285592X-RAY DIFFRACTION100
1.4634-1.48630.21323080.16325584X-RAY DIFFRACTION100
1.4863-1.51060.17722730.15125633X-RAY DIFFRACTION100
1.5106-1.53670.19352790.14595616X-RAY DIFFRACTION100
1.5367-1.56460.17422440.1445625X-RAY DIFFRACTION100
1.5646-1.59470.17882890.13555639X-RAY DIFFRACTION100
1.5947-1.62730.172990.12725603X-RAY DIFFRACTION100
1.6273-1.66270.16242910.12175623X-RAY DIFFRACTION100
1.6627-1.70140.16313200.12365603X-RAY DIFFRACTION100
1.7014-1.74390.16673030.11985627X-RAY DIFFRACTION100
1.7439-1.79110.1423050.11765610X-RAY DIFFRACTION100
1.7911-1.84380.16522900.11985646X-RAY DIFFRACTION100
1.8438-1.90330.16242830.12285672X-RAY DIFFRACTION100
1.9033-1.97130.15062930.12985633X-RAY DIFFRACTION100
1.9713-2.05020.15362950.13055667X-RAY DIFFRACTION100
2.0502-2.14360.15382630.12995671X-RAY DIFFRACTION100
2.1436-2.25660.17232900.12815687X-RAY DIFFRACTION100
2.2566-2.3980.16513050.13725684X-RAY DIFFRACTION100
2.398-2.58310.1653000.1455707X-RAY DIFFRACTION100
2.5831-2.84310.17253060.1495723X-RAY DIFFRACTION100
2.8431-3.25440.17982810.15815769X-RAY DIFFRACTION100
3.2544-4.10010.15912980.13685826X-RAY DIFFRACTION100
4.1001-60.4790.16693070.14816071X-RAY DIFFRACTION100

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