SASDE78: Solution structure of the diadenylate cyclase/phosphoglucosamine ...

Basic information

• Entry: Database: SASBDB / ID: SASDE78

Sample

Solution structure of the diadenylate cyclase/phosphoglucosamine mutase (DacA/GlmM) complex from Staphylococcus aureus

• Diadenylate cyclase (protein), DacA, Staphylococcus aureus (strain USA300)
• Phosphoglucosamine mutase (protein), GlmM, Staphylococcus aureus (strain USA300)

Function / homology

Function:

phosphoglucosamine mutase / phosphoglucosamine mutase activity / diadenylate cyclase / diadenylate cyclase activity / cAMP biosynthetic process / adenylate cyclase activity / carbohydrate metabolic process / magnesium ion binding / ATP binding / plasma membrane

Domain/homology:

Phosphoglucosamine mutase, bacterial type / : / Diadenylate cyclase CdaA, N-terminal domain / CdaA N-terminal transmembrane domain / Diadenylate cyclase CdaA / Diadenylate cyclase / : / DNA integrity scanning protein, DisA, N-terminal / Alpha-D-phosphohexomutase, C-terminal / DNA integrity scanning protein, DisA, N-terminal domain superfamily / DisA bacterial checkpoint controller nucleotide-binding / Diadenylate cyclase (DAC) domain profile. / Phosphoglucomutase/phosphomannomutase, 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

Component:

Diadenylate cyclase / Phosphoglucosamine mutase

• Biological species: Staphylococcus aureus (strain USA300) (bacteria)
• Citation: Journal: 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. 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.

Contact author

• Tommaso Tosi (Imperial College London)

Models

• Model #2646: ; Type: dummy / Software: (5.0) / Radius of dummy atoms: 3.75 A / Chi-square value: 1.283; Search similar-shape structures of this assembly by Omokage search (details)

Sample

• Sample: Name: Solution structure of the diadenylate cyclase/phosphoglucosamine mutase (DacA/GlmM) complex from Staphylococcus aureus; Specimen concentration: 12 mg/ml / Entity id: 1391 / 1392
• Buffer: Name: 30 mM Tris, 150 mM NaCl / pH: 7.5

Entity #1391

Name: DacA / Type: protein / Description: Diadenylate cyclase / Formula weight: 19.374 / Num. of mol.: 2 / Source: Staphylococcus aureus (strain USA300) / References: UniProt: A0A0H2XIU4
Sequence:

GSHMASFLKR YTSNTYSKDE EKLIQSVSKA VQYMAKRRIG ALIVFEKETG LQDYIETGIA MDSNISQELL INVFIPNTPL HDGAMIIQGT KIAAAASYLP LSDSPKISKS LGTRHRAAVG ISEVSDAFTV IVSEETGDIS VTFDGKLRRD ISNEIFEELL AEHWFGTRFQ KKGVK

Entity #1392

Name: GlmM / Type: protein / Description: Phosphoglucosamine mutase / Formula weight: 49.731 / Num. of mol.: 2 / Source: Staphylococcus aureus (strain USA300) / References: UniProt: Q2FEX1
Sequence:

MGKYFGTDGV RGVANQELTP ELAFKLGRYG GYVLAHNKGE KHPRVLVGRD TRVSGEMLES ALIAGLISIG AEVMRLGIIS TPGVAYLTRD MGAELGVMIS ASHNPVADNG IKFFGSDGFK LSDEQENEIE ALLDQENPEL PRPVGNDIVH YSDYFEGAQK YLSYLKSTVD VNFEGLKIAL DGANGSTSSL APFLFGDLEA DTETIGCSPD GYNINEKCGS THPEKLAEKV VETESDFGLA FDGDGDRIIA VDENGQIVDG DQIMFIIGQE MHKNQELNND MIVSTVMSNL GFYKALEQEG IKSNKTKVGD RYVVEEMRRG NYNLGGEQSG HIVMMDYNTT GDGLLTGIQL ASVIKMTGKS LSELAGQMKK YPQSLINVRV TDKYRVEENV DVKEVMTKVE VEMNGEGRIL VRPSGTEPLV RVMVEAATDE DAERFAQQIA DVVQDKMGLD KLVPR

Experimental information

• Beam: Instrument name: Diamond Light Source B21 / City: Oxfordshire / 国: UK / Shape: 1 x 5 mm / Type of source: X-ray synchrotron / Wavelength: 0.1 Å / Dist. spec. to detc.: 4.014 mm
• Detector: Name: Pilatus 2M

Scan

Title: Solution structure of the diadenylate cyclase/phosphoglucosamine mutase (DacA/GlmM) complex from Staphylococcus aureus
Measurement date: May 7, 2018 / Storage temperature: 4 °C / Cell temperature: 20 °C / Exposure time: 3 sec. / Unit: 1/A /

	Min	Max
Q	0.0062	0.3144

Distance distribution function P(R)

Sofotware P(R): GNOM 5.0 / Number of points: 906 /

	Min	Max
Q	0.00616707	0.207531
P(R) point	1	906
R	0	120.7

Result

Type of curve: sec /

	Experimental	Porod
MW	130.9 kDa	128 kDa
Volume	-	204 nm3

	P(R)	Guinier	Guinier error
Forward scattering, I0	0.09991	0.1	9.0E-5
Radius of gyration, Rg	3.88 nm	3.87 nm	0.01

	Min	Max
D	-	12.07
Guinier point	6	122