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Conserved features in TamA enable interaction with TamB to drive the activity of the translocation and assembly module.

Selkrig J, Belousoff MJ, Headey SJ, Heinz E, Shiota T, Shen HH, Beckham SA, Bamert RS, Phan MD, Schembri MA, Wilce MC, Scanlon MJ, Strugnell RA, Lithgow T - Sci Rep (2015)

Bottom Line: We show that specific functional features in TamA have been conserved through evolution, including residues surrounding the lateral gate and an extensive surface of the POTRA domains.Quartz crystal microbalance measurements pinpoint which POTRA domain specifically docks the TamB subunit of the nanomachine.We speculate that the POTRA domain of TamA functions as a lever arm in order to drive the activity of the TAM, assembling proteins into bacterial outer membranes.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Microbiology, Monash University, Clayton 3800, Australia [2] Department of Biochemistry and Molecular Biology, Monash University, Clayton 3800, Australia.

ABSTRACT
The biogenesis of membranes from constituent proteins and lipids is a fundamental aspect of cell biology. In the case of proteins assembled into bacterial outer membranes, an overarching question concerns how the energy required for protein insertion and folding is accessed at this remote location of the cell. The translocation and assembly module (TAM) is a nanomachine that functions in outer membrane biogenesis and virulence in diverse bacterial pathogens. Here we demonstrate the interactions through which TamA and TamB subunits dock to bridge the periplasm, and unite the outer membrane aspects to the inner membrane of the bacterial cell. We show that specific functional features in TamA have been conserved through evolution, including residues surrounding the lateral gate and an extensive surface of the POTRA domains. Analysis by nuclear magnetic resonance spectroscopy and small angle X-ray scattering document the characteristic structural features of these POTRA domains and demonstrate rigidity in solution. Quartz crystal microbalance measurements pinpoint which POTRA domain specifically docks the TamB subunit of the nanomachine. We speculate that the POTRA domain of TamA functions as a lever arm in order to drive the activity of the TAM, assembling proteins into bacterial outer membranes.

No MeSH data available.


Related in: MedlinePlus

Specific functions for the POTRA domains of TamA.(a) Cartoon depicting the POTRA deletion series of TamA. The purification of these proteins from detergent-solubilized membranes is detailed in Supplementary Fig. 3. (b) QCM-D measurements show the frequency response, indicative of mass changes, to (1) the attachment of TamA to the gold-surface, (2) reconstitution of the membrane layer with phospholipids, (3) the addition of purified TamB, and (4) addition of urea-denatured Ag43. (c) QCM-D measurements for TamA(ΔPOTRA1). (d) Membranes were isolated from E. coli expressing TamA or TamA(ΔPOTRA1) and subject to BN-PAGE or SDS-PAGE and analysis by immunoblotting. (e) QCM-D measurements for TamA(ΔPOTRA1–2). (f) QCM-D measurements for TamA(ΔPOTRA1–3). Asterisks (*) – wash step (with 20 mM TRIS and 150 mM NaCl). Replicate experiments are presented in Supplementary Figure 4.
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f4: Specific functions for the POTRA domains of TamA.(a) Cartoon depicting the POTRA deletion series of TamA. The purification of these proteins from detergent-solubilized membranes is detailed in Supplementary Fig. 3. (b) QCM-D measurements show the frequency response, indicative of mass changes, to (1) the attachment of TamA to the gold-surface, (2) reconstitution of the membrane layer with phospholipids, (3) the addition of purified TamB, and (4) addition of urea-denatured Ag43. (c) QCM-D measurements for TamA(ΔPOTRA1). (d) Membranes were isolated from E. coli expressing TamA or TamA(ΔPOTRA1) and subject to BN-PAGE or SDS-PAGE and analysis by immunoblotting. (e) QCM-D measurements for TamA(ΔPOTRA1–2). (f) QCM-D measurements for TamA(ΔPOTRA1–3). Asterisks (*) – wash step (with 20 mM TRIS and 150 mM NaCl). Replicate experiments are presented in Supplementary Figure 4.

Mentions: Since TAM function is required for pathogenesis, and this function is ablated with the loss of either TamA or TamB4, the interaction between TamA and TamB is crucial in vivo. To address which TamA POTRA domain is required for the docking of TamB, a deletion series of truncated TamA proteins (Fig. 4a) was assessed in a QCM-D assay system with a histidine-tagged form of TamA attached to a gold surface with a membrane layer reconstituted around the protein14. QCM-D is an extremely sensitive technique that allows for accurate, temporal based monitoring of binding to a surface by measuring sub-nanogram mass changes. This technique is preferable to examine changes in membrane embedded protein systems as it measures the system as a whole, including trapped solvent and changes in lipids.


Conserved features in TamA enable interaction with TamB to drive the activity of the translocation and assembly module.

Selkrig J, Belousoff MJ, Headey SJ, Heinz E, Shiota T, Shen HH, Beckham SA, Bamert RS, Phan MD, Schembri MA, Wilce MC, Scanlon MJ, Strugnell RA, Lithgow T - Sci Rep (2015)

Specific functions for the POTRA domains of TamA.(a) Cartoon depicting the POTRA deletion series of TamA. The purification of these proteins from detergent-solubilized membranes is detailed in Supplementary Fig. 3. (b) QCM-D measurements show the frequency response, indicative of mass changes, to (1) the attachment of TamA to the gold-surface, (2) reconstitution of the membrane layer with phospholipids, (3) the addition of purified TamB, and (4) addition of urea-denatured Ag43. (c) QCM-D measurements for TamA(ΔPOTRA1). (d) Membranes were isolated from E. coli expressing TamA or TamA(ΔPOTRA1) and subject to BN-PAGE or SDS-PAGE and analysis by immunoblotting. (e) QCM-D measurements for TamA(ΔPOTRA1–2). (f) QCM-D measurements for TamA(ΔPOTRA1–3). Asterisks (*) – wash step (with 20 mM TRIS and 150 mM NaCl). Replicate experiments are presented in Supplementary Figure 4.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4525385&req=5

f4: Specific functions for the POTRA domains of TamA.(a) Cartoon depicting the POTRA deletion series of TamA. The purification of these proteins from detergent-solubilized membranes is detailed in Supplementary Fig. 3. (b) QCM-D measurements show the frequency response, indicative of mass changes, to (1) the attachment of TamA to the gold-surface, (2) reconstitution of the membrane layer with phospholipids, (3) the addition of purified TamB, and (4) addition of urea-denatured Ag43. (c) QCM-D measurements for TamA(ΔPOTRA1). (d) Membranes were isolated from E. coli expressing TamA or TamA(ΔPOTRA1) and subject to BN-PAGE or SDS-PAGE and analysis by immunoblotting. (e) QCM-D measurements for TamA(ΔPOTRA1–2). (f) QCM-D measurements for TamA(ΔPOTRA1–3). Asterisks (*) – wash step (with 20 mM TRIS and 150 mM NaCl). Replicate experiments are presented in Supplementary Figure 4.
Mentions: Since TAM function is required for pathogenesis, and this function is ablated with the loss of either TamA or TamB4, the interaction between TamA and TamB is crucial in vivo. To address which TamA POTRA domain is required for the docking of TamB, a deletion series of truncated TamA proteins (Fig. 4a) was assessed in a QCM-D assay system with a histidine-tagged form of TamA attached to a gold surface with a membrane layer reconstituted around the protein14. QCM-D is an extremely sensitive technique that allows for accurate, temporal based monitoring of binding to a surface by measuring sub-nanogram mass changes. This technique is preferable to examine changes in membrane embedded protein systems as it measures the system as a whole, including trapped solvent and changes in lipids.

Bottom Line: We show that specific functional features in TamA have been conserved through evolution, including residues surrounding the lateral gate and an extensive surface of the POTRA domains.Quartz crystal microbalance measurements pinpoint which POTRA domain specifically docks the TamB subunit of the nanomachine.We speculate that the POTRA domain of TamA functions as a lever arm in order to drive the activity of the TAM, assembling proteins into bacterial outer membranes.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Microbiology, Monash University, Clayton 3800, Australia [2] Department of Biochemistry and Molecular Biology, Monash University, Clayton 3800, Australia.

ABSTRACT
The biogenesis of membranes from constituent proteins and lipids is a fundamental aspect of cell biology. In the case of proteins assembled into bacterial outer membranes, an overarching question concerns how the energy required for protein insertion and folding is accessed at this remote location of the cell. The translocation and assembly module (TAM) is a nanomachine that functions in outer membrane biogenesis and virulence in diverse bacterial pathogens. Here we demonstrate the interactions through which TamA and TamB subunits dock to bridge the periplasm, and unite the outer membrane aspects to the inner membrane of the bacterial cell. We show that specific functional features in TamA have been conserved through evolution, including residues surrounding the lateral gate and an extensive surface of the POTRA domains. Analysis by nuclear magnetic resonance spectroscopy and small angle X-ray scattering document the characteristic structural features of these POTRA domains and demonstrate rigidity in solution. Quartz crystal microbalance measurements pinpoint which POTRA domain specifically docks the TamB subunit of the nanomachine. We speculate that the POTRA domain of TamA functions as a lever arm in order to drive the activity of the TAM, assembling proteins into bacterial outer membranes.

No MeSH data available.


Related in: MedlinePlus