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- PDB-6gyz: Crystal structure of GlmM from Staphylococcus aureus -

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Basic information

Entry
Database: PDB / ID: 6gyz
TitleCrystal structure of GlmM from Staphylococcus aureus
ComponentsPhosphoglucosamine mutase
KeywordsISOMERASE / cell wall / phosphoglucosamine mutase / peptidoglycan
Function / homology
Function and homology information


phosphoglucosamine mutase / phosphoglucosamine mutase activity / carbohydrate metabolic process / magnesium ion binding
Similarity search - Function
Phosphoglucosamine mutase, bacterial type / Alpha-D-phosphohexomutase, C-terminal / Phosphoglucomutase/phosphomannomutase, C-terminal domain / Alpha-D-Glucose-1,6-Bisphosphate; Chain A, domain 3 / Alpha-D-Glucose-1,6-Bisphosphate, subunit A, domain 3 / Alpha-D-phosphohexomutase, C-terminal domain / Alpha-D-phosphohexomutase superfamily / Alpha-D-phosphohexomutase, alpha/beta/alpha domain II / Alpha-D-phosphohexomutase, alpha/beta/alpha domain III / Phosphoglucomutase/phosphomannomutase, alpha/beta/alpha domain II ...Phosphoglucosamine mutase, bacterial type / Alpha-D-phosphohexomutase, C-terminal / Phosphoglucomutase/phosphomannomutase, C-terminal domain / Alpha-D-Glucose-1,6-Bisphosphate; Chain A, domain 3 / Alpha-D-Glucose-1,6-Bisphosphate, subunit A, domain 3 / Alpha-D-phosphohexomutase, C-terminal domain / Alpha-D-phosphohexomutase superfamily / Alpha-D-phosphohexomutase, alpha/beta/alpha domain II / Alpha-D-phosphohexomutase, alpha/beta/alpha domain III / Phosphoglucomutase/phosphomannomutase, alpha/beta/alpha domain II / Phosphoglucomutase/phosphomannomutase, alpha/beta/alpha domain III / Alpha-D-phosphohexomutase, conserved site / Phosphoglucomutase and phosphomannomutase phosphoserine signature. / Alpha-D-phosphohexomutase, alpha/beta/alpha domain I / Alpha-D-phosphohexomutase, alpha/beta/alpha I/II/III / Alpha-D-phosphohexomutase, C-terminal domain superfamily / Phosphoglucomutase/phosphomannomutase, alpha/beta/alpha domain I / TATA-Binding Protein / 2-Layer Sandwich / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Phosphoglucosamine mutase
Similarity search - Component
Biological speciesStaphylococcus aureus (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3 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 1.2Feb 20, 2019Group: Advisory / Data collection / Derived calculations
Category: diffrn_source / pdbx_data_processing_status ...diffrn_source / pdbx_data_processing_status / pdbx_validate_close_contact / struct_conn / struct_conn_type
Item: _diffrn_source.pdbx_synchrotron_site
Revision 1.3Jul 10, 2019Group: Data collection / Category: diffrn_source / Item: _diffrn_source.pdbx_synchrotron_site
Revision 1.4Jan 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 / struct_ncs_dom_lim
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Phosphoglucosamine mutase
B: Phosphoglucosamine mutase


Theoretical massNumber of molelcules
Total (without water)99,5662
Polymers99,5662
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: SAXS
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2750 Å2
ΔGint-15 kcal/mol
Surface area36240 Å2
MethodPISA
Unit cell
Length a, b, c (Å)88.653, 93.884, 156.308
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11(chain A and (resseq 3:4 or (resid 5 and (name...
21(chain B and (resseq 3:4 or (resid 5 and (name...

NCS domain segments:

Ens-ID: 1

Dom-IDComponent-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11LYSLYSTYRTYR(chain A and (resseq 3:4 or (resid 5 and (name...AA3 - 43 - 4
12PHEPHEPHEPHE(chain A and (resseq 3:4 or (resid 5 and (name...AA55
13LYSLYSLYSLYS(chain A and (resseq 3:4 or (resid 5 and (name...AA3 - 4513 - 451
14LYSLYSLYSLYS(chain A and (resseq 3:4 or (resid 5 and (name...AA3 - 4513 - 451
15LYSLYSLYSLYS(chain A and (resseq 3:4 or (resid 5 and (name...AA3 - 4513 - 451
16LYSLYSLYSLYS(chain A and (resseq 3:4 or (resid 5 and (name...AA3 - 4513 - 451
17LYSLYSLYSLYS(chain A and (resseq 3:4 or (resid 5 and (name...AA3 - 4513 - 451
18LYSLYSLYSLYS(chain A and (resseq 3:4 or (resid 5 and (name...AA3 - 4513 - 451
19LYSLYSLYSLYS(chain A and (resseq 3:4 or (resid 5 and (name...AA3 - 4513 - 451
110LYSLYSLYSLYS(chain A and (resseq 3:4 or (resid 5 and (name...AA3 - 4513 - 451
21LYSLYSTYRTYR(chain B and (resseq 3:4 or (resid 5 and (name...BB3 - 43 - 4
22PHEPHEPHEPHE(chain B and (resseq 3:4 or (resid 5 and (name...BB55
23LYSLYSASPASP(chain B and (resseq 3:4 or (resid 5 and (name...BB3 - 4503 - 450
24LYSLYSASPASP(chain B and (resseq 3:4 or (resid 5 and (name...BB3 - 4503 - 450
25LYSLYSASPASP(chain B and (resseq 3:4 or (resid 5 and (name...BB3 - 4503 - 450
26LYSLYSASPASP(chain B and (resseq 3:4 or (resid 5 and (name...BB3 - 4503 - 450
27LYSLYSASPASP(chain B and (resseq 3:4 or (resid 5 and (name...BB3 - 4503 - 450
28LYSLYSASPASP(chain B and (resseq 3:4 or (resid 5 and (name...BB3 - 4503 - 450
29LYSLYSASPASP(chain B and (resseq 3:4 or (resid 5 and (name...BB3 - 4503 - 450
210LYSLYSASPASP(chain B and (resseq 3:4 or (resid 5 and (name...BB3 - 4503 - 450

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Components

#1: Protein Phosphoglucosamine mutase


Mass: 49782.922 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus (bacteria)
Gene: glmM, BTN44_13160, EP54_06960, EQ90_12990, HMPREF3211_00023, RK64_11470, RK68_01815, RK73_08340
Production host: Escherichia coli (E. coli) / References: UniProt: A0A0D6H967, phosphoglucosamine mutase

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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: 3.33 Å3/Da / Density % sol: 63.04 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / Details: 2.2M Na-Malonate

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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: Oct 5, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9795 Å / Relative weight: 1
ReflectionResolution: 3→93.88 Å / Num. obs: 26871 / % possible obs: 100 % / Redundancy: 12.5 % / CC1/2: 0.998 / Rmerge(I) obs: 0.158 / Net I/σ(I): 8.4
Reflection shellResolution: 3→3.18 Å / Redundancy: 12.5 % / Rmerge(I) obs: 1.868 / Mean I/σ(I) obs: 2 / Num. unique obs: 4266 / CC1/2: 0.842 / % possible all: 100

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Processing

Software
NameVersionClassification
PHENIX1.10.1_2155refinement
PDB_EXTRACT3.24data extraction
DIALSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3PDK

3pdk


Resolution: 3→80.482 Å / SU ML: 0.43 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 32.79
RfactorNum. reflection% reflection
Rfree0.2727 1339 5.01 %
Rwork0.2219 --
obs0.2244 26709 99.61 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 208.9 Å2 / Biso mean: 109.014 Å2 / Biso min: 53.24 Å2
Refinement stepCycle: final / Resolution: 3→80.482 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6831 0 0 0 6831
Num. residues----893
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0046925
X-RAY DIFFRACTIONf_angle_d0.7569337
X-RAY DIFFRACTIONf_chiral_restr0.051041
X-RAY DIFFRACTIONf_plane_restr0.0051247
X-RAY DIFFRACTIONf_dihedral_angle_d15.9834197
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A3456X-RAY DIFFRACTION11.556TORSIONAL
12B3456X-RAY DIFFRACTION11.556TORSIONAL
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 10

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
3-3.10730.45491150.36522491260699
3.1073-3.23170.33891230.31422505262899
3.2317-3.37870.35911250.28672501262699
3.3787-3.55690.3421140.27242506262099
3.5569-3.77970.33361290.237425012630100
3.7797-4.07160.25331200.213825572677100
4.0716-4.48130.25891800.208724922672100
4.4813-5.12960.22221530.189825182671100
5.1296-6.46240.2961370.23325922729100
6.4624-80.51110.24521430.193527072850100
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)
13.1278-2.01811.08273.342-0.42193.98920.20020.48060.0619-0.435-0.3927-0.60640.64041.1020.16970.75060.02570.15760.82650.00490.941419.1362-27.514544.4383
22.6462-1.21961.18582.477-0.71742.99650.23430.02840.1538-0.1378-0.3043-0.33630.11730.3710.26850.7406-0.01670.16530.7103-0.02510.74116.2985-22.924742.3479
34.6023-0.56072.89581.1188-0.41483.3003-0.017-0.77760.16030.4145-0.1097-0.4292-0.0545-0.1936-0.23210.9871-0.02460.09340.96680.01170.944716.2307-16.526649.2448
43.5166-0.8049-0.28043.04910.73714.58230.41040.00750.0839-0.1769-0.09750.06390.1426-0.1261-0.150.9002-0.01340.06590.53450.02240.6466-5.772-19.544528.7227
53.72481.17010.6477.59111.94323.65460.19330.2279-0.22950.14140.2551-0.70510.44991.159-0.17171.22290.14150.30821.03540.01670.82368.8196-16.841711.3962
61.16190.1322-0.10572.96060.96471.93630.06010.46670.0146-0.7174-0.0068-0.28930.27630.5347-0.18491.03430.10230.24540.7780.06150.70856.9666-18.545819.4026
72.2729-0.3204-0.14463.1112-3.74885.49760.03680.6699-0.0758-0.9875-0.1779-0.46910.6448-0.88780.26361.35860.24210.23331.1899-0.0841.039119.0867-41.21944.5717
87.1424-0.3110.0416.9331-0.67160.6210.7926-0.2710.78870.579-0.8062-0.5405-0.2687-0.49390.20771.08210.18160.25750.8306-0.06241.282210.6868-35.89489.9765
91.131-0.57910.36584.935-3.82413.65561.6327-0.2907-0.8739-2.5226-1.0455-0.14870.5292-0.4239-0.12481.3320.17840.080.9566-0.00241.146310.087-45.93946.2384
104.2611.80670.33173.5677-1.3493.6547-0.4796-0.5305-0.0854-0.14610.0899-0.84670.36270.81360.15270.78380.237-0.11570.8905-0.02390.918820.2861-35.416965.7732
111.538-0.5048-0.81822.9450.39665.30020.1327-0.42320.26960.2803-0.0303-0.3351-0.3440.09120.00070.80290.0508-0.07430.6055-0.03110.74048.7874-29.674967.0613
123.54850.1379-1.41812.6848-0.24344.0308-0.3912-1.461-0.1628-0.15390.1679-0.45320.81431.04470.25520.99960.1868-0.0441.07670.03280.872915.3998-39.666772.2046
131.99060.53180.63323.5050.09464.3008-0.2278-0.51470.44420.0140.2424-0.2402-1.06810.07760.06831.04550.1397-0.13270.8148-0.16450.85897.8146-17.786776.881
143.39420.06840.99232.8388-1.28133.2568-0.1437-1.6212-0.00130.23070.0506-0.3397-0.4895-0.3688-0.12521.03370.164-0.12051.4042-0.10520.760710.8339-29.479294.3962
152.1167-0.6466-0.10561.0193-0.66971.4998-0.4243-1.15870.41920.41130.4115-0.4027-0.4354-0.85-0.2641.13420.2888-0.07591.4127-0.45060.959213.9514-20.271497.1102
160.88740.0474-0.47781.63312.228.8435-1.1519-1.92460.0584-0.23080.3624-0.22381.49610.0815-0.02291.6187-0.1648-0.13021.2669-0.09480.84939.2388-40.4563100.7214
173.8036-0.8939-0.2576.04881.04085.67840.228-1.21951.7093-0.55410.1783-0.0256-0.6976-0.6705-0.59340.995-0.0029-0.08881.0442-0.34721.108436.4851-31.413599.1283
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 3 through 52 )A3 - 52
2X-RAY DIFFRACTION2chain 'A' and (resid 53 through 134 )A53 - 134
3X-RAY DIFFRACTION3chain 'A' and (resid 135 through 168 )A135 - 168
4X-RAY DIFFRACTION4chain 'A' and (resid 169 through 274 )A169 - 274
5X-RAY DIFFRACTION5chain 'A' and (resid 275 through 299 )A275 - 299
6X-RAY DIFFRACTION6chain 'A' and (resid 300 through 378 )A300 - 378
7X-RAY DIFFRACTION7chain 'A' and (resid 379 through 402 )A379 - 402
8X-RAY DIFFRACTION8chain 'A' and (resid 403 through 428 )A403 - 428
9X-RAY DIFFRACTION9chain 'A' and (resid 429 through 451 )A429 - 451
10X-RAY DIFFRACTION10chain 'B' and (resid 3 through 35 )B3 - 35
11X-RAY DIFFRACTION11chain 'B' and (resid 36 through 107 )B36 - 107
12X-RAY DIFFRACTION12chain 'B' and (resid 108 through 134 )B108 - 134
13X-RAY DIFFRACTION13chain 'B' and (resid 135 through 241 )B135 - 241
14X-RAY DIFFRACTION14chain 'B' and (resid 242 through 342 )B242 - 342
15X-RAY DIFFRACTION15chain 'B' and (resid 343 through 379 )B343 - 379
16X-RAY DIFFRACTION16chain 'B' and (resid 380 through 402 )B380 - 402
17X-RAY DIFFRACTION17chain 'B' and (resid 403 through 450 )B403 - 450

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