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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

Selective pressure has maintained specific residues in TamA through the course of evolution.(a) Topology of TamA and TamB, spanning the bacterial outer membrane (OM) and inner membrane (IM). The TAM drives the insertion of substrate proteins (blue) such as Ag43 into the membrane414. Rate4Site analysis of TamA sequences (detailed in Supplementary Table 1) plotted onto the structural coordinates of TamA (pdb 4C00). The crenel in the β-barrel domain between the first and last β-strands is indicated with an arrow; POTRA domains are labeled P1, P2 and P3. (b) POTRA domain sequences from TamA and BamA of representative species (Supplementary Table 2) were extracted and subjected to CLANS19. Lines are shown connecting similar sequences with an P-value cut-off of 1e-2. Sequences derived from each POTRA domain form a distinct group, color-coded for the corresponding three TamA POTRA domains and the five BamA POTRA domains. (c) Representation of the LSQ fits between TamA POTRA 1 (green) and TamA POTRA 2 (cyan) and the BamA POTRA 1 (yellow) and BamA POTRA2 (orange), calculated using PyMOL (www.pymol.com/pymol). A complete comparison is shown in Supplementary Figure 2.
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f1: Selective pressure has maintained specific residues in TamA through the course of evolution.(a) Topology of TamA and TamB, spanning the bacterial outer membrane (OM) and inner membrane (IM). The TAM drives the insertion of substrate proteins (blue) such as Ag43 into the membrane414. Rate4Site analysis of TamA sequences (detailed in Supplementary Table 1) plotted onto the structural coordinates of TamA (pdb 4C00). The crenel in the β-barrel domain between the first and last β-strands is indicated with an arrow; POTRA domains are labeled P1, P2 and P3. (b) POTRA domain sequences from TamA and BamA of representative species (Supplementary Table 2) were extracted and subjected to CLANS19. Lines are shown connecting similar sequences with an P-value cut-off of 1e-2. Sequences derived from each POTRA domain form a distinct group, color-coded for the corresponding three TamA POTRA domains and the five BamA POTRA domains. (c) Representation of the LSQ fits between TamA POTRA 1 (green) and TamA POTRA 2 (cyan) and the BamA POTRA 1 (yellow) and BamA POTRA2 (orange), calculated using PyMOL (www.pymol.com/pymol). A complete comparison is shown in Supplementary Figure 2.

Mentions: Rate4Site17 can be used to measure sequence conservation through evolution, and we mapped the Rate4Site scores onto the structure of TamA to highlight its highly-conserved features. The most highly-conserved face of the TamA β-barrel domain covers the lateral region where the first and last β-strands meet and the extension of β-strand 1 that forms an “exit pore” (Fig. 1a), with the equivalent region of BamA shown to be critically important for activity1218, further supporting the suggestion that the β-barrel domain of BamA and TamA function by a similar mechanism5. Only two aromatic residues are among the conserved residues of TamA, and sequence logos were constructed to highlight these amino acids (Supplementary Fig. 1). The analysis revealed that the proposed active site of the β-barrel domain in TamA5 is characterized by a dearth of stabilizing aromatic residues amidst the otherwise highly-conserved sequences, in keeping with the mobility required by these strands according to the current models for the mechanism by which BamA and TamA act12185. In addition, the Rate4Site analysis illustrated the conservation of discrete surface patches in TamA POTRA1 and TamA POTRA2 (Fig. 1a).


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)

Selective pressure has maintained specific residues in TamA through the course of evolution.(a) Topology of TamA and TamB, spanning the bacterial outer membrane (OM) and inner membrane (IM). The TAM drives the insertion of substrate proteins (blue) such as Ag43 into the membrane414. Rate4Site analysis of TamA sequences (detailed in Supplementary Table 1) plotted onto the structural coordinates of TamA (pdb 4C00). The crenel in the β-barrel domain between the first and last β-strands is indicated with an arrow; POTRA domains are labeled P1, P2 and P3. (b) POTRA domain sequences from TamA and BamA of representative species (Supplementary Table 2) were extracted and subjected to CLANS19. Lines are shown connecting similar sequences with an P-value cut-off of 1e-2. Sequences derived from each POTRA domain form a distinct group, color-coded for the corresponding three TamA POTRA domains and the five BamA POTRA domains. (c) Representation of the LSQ fits between TamA POTRA 1 (green) and TamA POTRA 2 (cyan) and the BamA POTRA 1 (yellow) and BamA POTRA2 (orange), calculated using PyMOL (www.pymol.com/pymol). A complete comparison is shown in Supplementary Figure 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Selective pressure has maintained specific residues in TamA through the course of evolution.(a) Topology of TamA and TamB, spanning the bacterial outer membrane (OM) and inner membrane (IM). The TAM drives the insertion of substrate proteins (blue) such as Ag43 into the membrane414. Rate4Site analysis of TamA sequences (detailed in Supplementary Table 1) plotted onto the structural coordinates of TamA (pdb 4C00). The crenel in the β-barrel domain between the first and last β-strands is indicated with an arrow; POTRA domains are labeled P1, P2 and P3. (b) POTRA domain sequences from TamA and BamA of representative species (Supplementary Table 2) were extracted and subjected to CLANS19. Lines are shown connecting similar sequences with an P-value cut-off of 1e-2. Sequences derived from each POTRA domain form a distinct group, color-coded for the corresponding three TamA POTRA domains and the five BamA POTRA domains. (c) Representation of the LSQ fits between TamA POTRA 1 (green) and TamA POTRA 2 (cyan) and the BamA POTRA 1 (yellow) and BamA POTRA2 (orange), calculated using PyMOL (www.pymol.com/pymol). A complete comparison is shown in Supplementary Figure 2.
Mentions: Rate4Site17 can be used to measure sequence conservation through evolution, and we mapped the Rate4Site scores onto the structure of TamA to highlight its highly-conserved features. The most highly-conserved face of the TamA β-barrel domain covers the lateral region where the first and last β-strands meet and the extension of β-strand 1 that forms an “exit pore” (Fig. 1a), with the equivalent region of BamA shown to be critically important for activity1218, further supporting the suggestion that the β-barrel domain of BamA and TamA function by a similar mechanism5. Only two aromatic residues are among the conserved residues of TamA, and sequence logos were constructed to highlight these amino acids (Supplementary Fig. 1). The analysis revealed that the proposed active site of the β-barrel domain in TamA5 is characterized by a dearth of stabilizing aromatic residues amidst the otherwise highly-conserved sequences, in keeping with the mobility required by these strands according to the current models for the mechanism by which BamA and TamA act12185. In addition, the Rate4Site analysis illustrated the conservation of discrete surface patches in TamA POTRA1 and TamA POTRA2 (Fig. 1a).

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