SASDB75: Escherichia coli TraE protein: A VirB8 homolog from plasmid pKM101

Basic information

• Entry: Database: SASBDB / ID: SASDB75

Sample

Escherichia coli TraE protein: A VirB8 homolog from plasmid pKM101

• Escherichia coli TraE protein (VirB8 homolog) (protein), TraE, Escherichia coli

• Function / homology: Type IV secretion system protein VirB8/PtlE / Bacterial virulence protein VirB8 / VirB8 protein / protein secretion by the type IV secretion system / NTF2-like domain superfamily / membrane => GO:0016020 / TraE protein
• Biological species: Escherichia coli (E. coli)
• Citation: Journal: Proc Natl Acad Sci U S A / Year: 2018; Title: VirB8 homolog TraE from plasmid pKM101 forms a hexameric ring structure and interacts with the VirB6 homolog TraD.; Authors: Bastien Casu / Charline Mary / Aleksandr Sverzhinsky / Aurélien Fouillen / Antonio Nanci / Christian Baron / ; Abstract: Type IV secretion systems (T4SSs) are multiprotein assemblies that translocate macromolecules across the cell envelope of bacteria. X-ray crystallographic and electron microscopy (EM) analyses have increasingly provided structural information on individual T4SS components and on the entire complex. As of now, relatively little information has been available on the exact localization of the inner membrane-bound T4SS components, notably the mostly periplasmic VirB8 protein and the very hydrophobic VirB6 protein. We show here that the membrane-bound, full-length version of the VirB8 homolog TraE from the plasmid pKM101 secretion system forms a high-molecular-mass complex that is distinct from the previously characterized periplasmic portion of the protein that forms dimers. Full-length TraE was extracted from the membranes with detergents, and analysis by size-exclusion chromatography, cross-linking, and size exclusion chromatography (SEC) multiangle light scattering (MALS) shows that it forms a high-molecular-mass complex. EM and small-angle X-ray scattering (SAXS) analysis demonstrate that full-length TraE forms a hexameric complex with a central pore. We also overproduced and purified the VirB6 homolog TraD and show by cross-linking, SEC, and EM that it binds to TraE. Our results suggest that TraE and TraD interact at the substrate translocation pore of the secretion system.

Contact author

• Bastien Casu (Département de biochimie, Université de Montréal, Montréal, Canada)

Models

• Model #557: ; Type: dummy / Radius of dummy atoms: 1.90 A / Symmetry: P6 / Chi-square value: 1.16 / P-value: 0.100000; Search similar-shape structures of this assembly by Omokage search (details)

Sample

• Sample: Name: Escherichia coli TraE protein: A VirB8 homolog from plasmid pKM101
• Buffer: Name: 50 mM sodium phosphate 300 mM NaCl 40 mM imidazole 0.15 % octyl glucose neopentyl glycol (OGNG); Concentration: 50.00 mM / pH: 7.4 ; Composition: 300 mM NaCl, 40 mM imidazole, 0.15 % octyl glucose neopentyl glycol (OGNG)

Entity #358

Name: TraE / Type: protein / Description: Escherichia coli TraE protein (VirB8 homolog) / Formula weight: 28.537 / Num. of mol.: 6 / Source: Escherichia coli / References: UniProt: Q46703
Sequence:

MGSSHHHHHH ENLYFQGGTK ANKKTGLTRE AIKEFNESRK GLEVDLMDEV LKSRRTAWMV ATGSAVVTVF ALSLVGYVVH KYSQPIPAHL LTLNEATHEV QQVKLTRDQT SYGDEIDKFW LTQYVIHRES YDFYSVQVDY TAVGLMSTPN VAESYQSKFK GRNGLDKVLG DSETTRVKIN SVILDKPHGV ATIRFTTVRR VRSNPVDDQP QRWIAIMGYE YKSLAMNAEQ RYVNPLGFRV TSYRVNPEVN

Experimental information

• Beam: Instrument name: Cornell High Energy Synchrotron Source (CHESS) G1; City: Ithaca, NY / 国: USA / Type of source: X-ray synchrotronSynchrotron
• Detector: Name: Finger Lakes CCD / Type: CCD

Scan

Title: TraE protein / Measurement date: Jun 2, 2016 / Cell temperature: 20 °C / Exposure time: 2 sec. / Number of frames: 70 / Unit: 1/nm /

	Min	Max
Q	0.0915	2.6113

Distance distribution function P(R)

Sofotware P(R): GNOM 4.6 / Number of points: 441 /

	Min	Max
Q	0.009152	0.2606
P(R) point	1	441
R	0	137

Result

Type of curve: single_conc
Comments: The MW of the TraE/OGNG complex was additionally confirmed using SEC-MALLS (248kDa).

	Experimental	Porod
MW	264.2 kDa	223 kDa
Volume	-	360 nm3

	P(R)	P(R) error	Guinier	Guinier error
Forward scattering, I0	0.0094	3.0E-5	0.0094	6.0E-5
Radius of gyration, Rg	4.44 nm	0.013	4.44 nm	0.13

	Min	Max
D	-	13.7
Guinier point	10	35