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- PDB-6gyw: Crystal structure of DacA from Staphylococcus aureus -

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Basic information

Entry
Database: PDB / ID: 6gyw
TitleCrystal structure of DacA from Staphylococcus aureus
ComponentsDiadenylate cyclase
KeywordsTRANSFERASE / c-di-AMP / diadenylate cyclase
Function / homologyYojJ-like (1 / DNA integrity scanning protein, DisA, N-terminal domain / 3-Layer(aba) Sandwich / Alpha Beta / :
Function and homology information
Biological speciesStaphylococcus aureus (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.7 Å
AuthorsTosi, T. / Freemont, P.S. / Grundling, A.
CitationJournal: PLoS Pathog / Year: 2019
Title: Inhibition of the Staphylococcus aureus c-di-AMP cyclase DacA by direct interaction with the phosphoglucosamine mutase GlmM.
Authors: Tommaso Tosi / Fumiya Hoshiga / Charlotte Millership / Rahul Singh / Charles Eldrid / Delphine Patin / Dominique Mengin-Lecreulx / Konstantinos Thalassinos / Paul Freemont / Angelika Gründling /
Abstract: c-di-AMP is an important second messenger molecule that plays a pivotal role in regulating fundamental cellular processes, including osmotic and cell wall homeostasis in many Gram-positive organisms. ...c-di-AMP is an important second messenger molecule that plays a pivotal role in regulating fundamental cellular processes, including osmotic and cell wall homeostasis in many Gram-positive organisms. In the opportunistic human pathogen Staphylococcus aureus, c-di-AMP is produced by the membrane-anchored DacA enzyme. Inactivation of this enzyme leads to a growth arrest under standard laboratory growth conditions and a re-sensitization of methicillin-resistant S. aureus (MRSA) strains to ß-lactam antibiotics. The gene coding for DacA is part of the conserved three-gene dacA/ybbR/glmM operon that also encodes the proposed DacA regulator YbbR and the essential phosphoglucosamine mutase GlmM, which is required for the production of glucosamine-1-phosphate, an early intermediate of peptidoglycan synthesis. These three proteins are thought to form a complex in vivo and, in this manner, help to fine-tune the cellular c-di-AMP levels. To further characterize this important regulatory complex, we conducted a comprehensive structural and functional analysis of the S. aureus DacA and GlmM enzymes by determining the structures of the S. aureus GlmM enzyme and the catalytic domain of DacA. Both proteins were found to be dimers in solution as well as in the crystal structures. Further site-directed mutagenesis, structural and enzymatic studies showed that multiple DacA dimers need to interact for enzymatic activity. We also show that DacA and GlmM form a stable complex in vitro and that S. aureus GlmM, but not Escherichia coli or Pseudomonas aeruginosa GlmM, acts as a strong inhibitor of DacA function without the requirement of any additional cellular factor. Based on Small Angle X-ray Scattering (SAXS) data, a model of the complex revealed that GlmM likely inhibits DacA by masking the active site of the cyclase and preventing higher oligomer formation. Together these results provide an important mechanistic insight into how c-di-AMP production can be regulated in the cell.
History
DepositionJul 2, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 23, 2019Provider: repository / Type: Initial release
Revision 1.1Jan 30, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _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 / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jan 17, 2024Group: Advisory / Data collection ...Advisory / 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 / pdbx_unobs_or_zero_occ_atoms
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
A: Diadenylate cyclase
B: Diadenylate cyclase


Theoretical massNumber of molelcules
Total (without water)38,7982
Polymers38,7982
Non-polymers00
Water2,666148
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: SAXS
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1460 Å2
ΔGint-8 kcal/mol
Surface area13960 Å2
MethodPISA
Unit cell
Length a, b, c (Å)54.799, 68.190, 76.796
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein Diadenylate cyclase / DAC / Cyclic-di-AMP synthase / c-di-AMP synthase


Mass: 19399.061 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus (bacteria) / Gene: dacA, C7J94_12115 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A2P7CAT2, diadenylate cyclase
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 148 / 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: 2.17 Å3/Da / Density % sol: 43.2 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / Details: 0.2M LiCl, 0.1M Na-Cacodylate pH 6.5, 30% Peg400

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I03 / Wavelength: 0.9795 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: May 17, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9795 Å / Relative weight: 1
ReflectionResolution: 1.7→68.19 Å / Num. obs: 32376 / % possible obs: 99.9 % / Redundancy: 12.2 % / CC1/2: 0.999 / Rmerge(I) obs: 0.078 / Net I/σ(I): 13.9
Reflection shellResolution: 1.7→1.73 Å / Redundancy: 10 % / Rmerge(I) obs: 0.627 / Mean I/σ(I) obs: 2 / Num. unique obs: 1659 / CC1/2: 0.968 / % possible all: 99

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Processing

Software
NameVersionClassification
PHENIX1.9_1692refinement
PDB_EXTRACT3.24data extraction
DIALSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4RV7

4rv7


Resolution: 1.7→50.99 Å / SU ML: 0.18 / Cross valid method: THROUGHOUT / σ(F): 1.38 / Phase error: 21.5
RfactorNum. reflection% reflection
Rfree0.2004 1616 5.01 %
Rwork0.1735 --
obs0.1749 32265 99.74 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 138.4 Å2 / Biso mean: 42.8617 Å2 / Biso min: 19.11 Å2
Refinement stepCycle: final / Resolution: 1.7→50.99 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2324 0 0 148 2472
Biso mean---45.69 -
Num. residues----301
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0062362
X-RAY DIFFRACTIONf_angle_d0.9623191
X-RAY DIFFRACTIONf_chiral_restr0.038375
X-RAY DIFFRACTIONf_plane_restr0.004408
X-RAY DIFFRACTIONf_dihedral_angle_d13.595873
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 12

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
1.7001-1.75010.25281210.23142502262398
1.7501-1.80660.25011340.21324862620100
1.8066-1.87120.22611260.186825172643100
1.8712-1.94610.22931270.180725392666100
1.9461-2.03470.21171190.174225422661100
2.0347-2.14190.21571420.173625342676100
2.1419-2.27610.18881370.16525432680100
2.2761-2.45190.19451480.169425072655100
2.4519-2.69860.2371340.176725742708100
2.6986-3.0890.24651360.18925692705100
3.089-3.89170.18361470.175626042751100
3.8917-51.01280.1761450.160427322877100
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
13.8660.405-0.1623.11950.07054.67290.1263-0.05840.3826-0.01260.02560.2026-0.5712-0.1456-0.13570.22410.03260.02150.182-0.01220.166420.543510.474514.9016
23.31211.44442.37563.13690.06582.90680.15320.0267-0.5042-0.23240.06620.00230.3805-0.1664-0.20380.2396-0.01-0.00680.2596-0.02870.248418.5896-2.507619.662
34.57911.5713-0.11593.49810.32765.5795-0.03270.2472-0.0402-0.27890.01790.1317-0.0867-0.56060.02870.22150.0279-0.05160.3258-0.01560.265912.23312.947810.2552
48.2439-0.0208-2.14235.4681-0.07789.7931-0.0848-0.4962-0.90030.2680.07390.1250.935-0.05340.05090.3095-0.0284-0.09930.21090.00430.438236.2977-7.669534.6619
57.1438-0.8375-0.7546.70921.11861.1062-0.11350.6928-0.5199-0.5152-0.0129-0.431-0.19490.493-0.00460.21880.016-0.01210.3051-0.06490.263243.02030.344927.6754
65.61080.50992.20292.82390.28253.5247-0.23090.12550.3352-0.04210.02050.0042-0.2142-0.14090.15830.23030.0327-0.05610.2028-0.03380.246734.13896.782930.5392
76.74381.23441.91852.33930.40042.8948-0.16340.90570.4635-0.19880.0667-0.8247-0.17160.89070.26580.3862-0.0655-0.05450.41350.01880.556151.578812.267530.2128
88.61961.16852.16095.25282.01398.8509-0.5565-0.1250.54130.11020.07190.0867-0.00180.29030.16580.3027-0.0888-0.12790.19540.02320.271949.127210.649132.7583
95.2703-0.54570.3384.41622.31056.1776-0.24070.35960.1328-0.1474-0.0394-0.1828-0.29420.39020.25410.2123-0.0162-0.06320.24730.0210.221547.60632.998533.6078
108.0345-1.14260.51872.16441.78165.6786-0.1098-0.12040.42640.3588-0.1123-0.3627-0.18220.4766-0.00120.29160.0066-0.0880.307-0.00310.28552.73981.618738.2333
112.3644-3.7374-0.05496.2094-0.39644.9265-0.1628-0.8929-0.78640.30980.2120.27040.43480.03940.05060.3171-0.0303-0.05130.36670.04660.319441.762-4.253242.2604
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 110 through 139 )A110 - 139
2X-RAY DIFFRACTION2chain 'A' and (resid 140 through 206 )A140 - 206
3X-RAY DIFFRACTION3chain 'A' and (resid 207 through 260 )A207 - 260
4X-RAY DIFFRACTION4chain 'B' and (resid 111 through 130 )B111 - 130
5X-RAY DIFFRACTION5chain 'B' and (resid 131 through 139 )B131 - 139
6X-RAY DIFFRACTION6chain 'B' and (resid 140 through 188 )B140 - 188
7X-RAY DIFFRACTION7chain 'B' and (resid 189 through 206 )B189 - 206
8X-RAY DIFFRACTION8chain 'B' and (resid 207 through 217 )B207 - 217
9X-RAY DIFFRACTION9chain 'B' and (resid 218 through 237 )B218 - 237
10X-RAY DIFFRACTION10chain 'B' and (resid 238 through 246 )B238 - 246
11X-RAY DIFFRACTION11chain 'B' and (resid 247 through 261 )B247 - 261

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