SASDAS4: I27-PimA (I27-PimA Fusion protein)

Basic information

• Entry: Database: SASBDB / ID: SASDAS4

Sample

I27-PimA

• I27-PimA Fusion protein (protein), I27-PimA

• Citation: Journal: J Biol Chem / Year: 2013; Title: Conformational plasticity of the essential membrane-associated mannosyltransferase PimA from mycobacteria.; Authors: David Giganti / Jorge Alegre-Cebollada / Saioa Urresti / David Albesa-Jové / Ane Rodrigo-Unzueta / Natalia Comino / Michael Kachala / Sonia López-Fernández / Dmitri I Svergun / Julio M Fernández / Marcelo E Guerin / ; Abstract: Phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential glycosyltransferase (GT) that initiates the biosynthetic pathway of phosphatidyl-myo-inositol mannosides, lipomannan, and lipoarabinomannan, which are key glycolipids/lipoglycans of the mycobacterial cell envelope. PimA belongs to a large family of peripheral membrane-associated GTs for which the understanding of the molecular mechanism and conformational changes that govern substrate/membrane recognition and catalysis remains a major challenge. Here we used single molecule force spectroscopy techniques to study the mechanical and conformational properties of PimA. In our studies, we engineered a polyprotein containing PimA flanked by four copies of the well characterized I27 protein, which provides an unambiguous mechanical fingerprint. We found that PimA exhibits weak mechanical stability albeit displaying β-sheet topology expected to unfold at much higher forces. Notably, PimA unfolds following heterogeneous multiple step mechanical unfolding pathways at low force akin to molten globule states. Interestingly, the ab initio low resolution envelopes obtained from small angle x-ray scattering of the unliganded PimA and the PimA·GDP complexed forms clearly demonstrate that not only the "open" and "closed" conformations of the GT-B enzyme are largely present in solution, but in addition, PimA experiences remarkable flexibility that undoubtedly corresponds to the N-terminal "Rossmann fold" domain, which has been proved to participate in protein-membrane interactions. Based on these results and on our previous experimental data, we propose a model wherein the conformational transitions are important for the mannosyltransferase to interact with the donor and acceptor substrates/membrane.

Contact author

• Michael Kachala (EMBL-Hamburg, European Molecular Biology Laboratory (EMBL) - Hamburg outstation, Notkestraße 85, Geb. 25A, 22607 Hamburg, Deutschland, Germany)

Models

• Model #249: ; Type: dummy / Software: MONSA (1.44) / Radius of dummy atoms: 1.90 A / Chi-square value: 1.008016; Search similar-shape structures of this assembly by Omokage search (details)
• Model #248: ; Type: dummy / Software: MONZA (1.44) / Radius of dummy atoms: 1.90 A / Symmetry: P1 / Chi-square value: 1.653796; Search similar-shape structures of this assembly by Omokage search (details)

Sample

• Sample: Name: I27-PimA / Specimen concentration: 1.40-2.50
• Buffer: Name: Tris-HCl / Concentration: 50.00 mM / pH: 7.5 / Composition: 150 mM NaCl
• Entity #147: Name: I27-PimA / Type: protein / Description: I27-PimA Fusion protein / Formula weight: 48.4 / Num. of mol.: 1

Experimental information

• Beam: Instrument name: PETRA III P12 / City: Hamburg / 国: Germany / Type of source: X-ray synchrotron / Wavelength: 0.12 Å / Dist. spec. to detc.: 3.1 mm
• Detector: Name: Pilatus 2M

Scan

Title: PimA / Measurement date: May 17, 2014 / Storage temperature: 10 °C / Cell temperature: 10 °C / Exposure time: 1 sec. / Number of frames: 50 / Unit: 1/nm /

	Min	Max
Q	0.0671	6.0241

Distance distribution function P(R)

Sofotware P(R): GNOM 4.5a / Number of points: 446 /

	Min	Max
Q	0.1833	2.619
P(R) point	43	488
R	0	10.69

Result

Type of curve: single_conc / Standard: BSA /

	Experimental	Porod
MW	45 kDa	-
Volume	-	86 nm3

	P(R)	Guinier
Forward scattering, I0	43.63	43.44
Radius of gyration, Rg	-	3.08 nm

	Min	Max
D	-	10.7
Guinier point	24	111