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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

The POTRA1 and POTRA2 domains of TamA are structurally specialized.(a) NMR solution structure of TamA POTRA1, with backbone representation of the ensemble of the 10 lowest energy structures. Steady-state 1H-15N NOE values are color plotted onto a cartoon ribbon representation of the structure (Red: Lower values corresponding to high mobility, Blue: Higher values corresponding to low mobility). NMR data is detailed in Supplementary Table 4. (b) The closest to average structure of POTRA1. (c) Heteronuclear 1H-15N NOE measurements of the POTRA1 domain demonstrate the inherent flexibility of the loop, with the average value for loop 4 residues 88–91 being 0.45 ± 0.11 compared to the domain average of 0.77 ± 0.19. (d) Multiple sequence alignment in the region corresponding to loop 4. The amino acid composition in this loop from other Gamma-proteobacteria is consistent with a disordered structure seen in the E. coli protein. (e) Surface view of the surface charge features of BamA POTRA1 and TamA POTRA1 created between the two helices (red and blue surfaces represent acidic and basic residues, respectively).
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f3: The POTRA1 and POTRA2 domains of TamA are structurally specialized.(a) NMR solution structure of TamA POTRA1, with backbone representation of the ensemble of the 10 lowest energy structures. Steady-state 1H-15N NOE values are color plotted onto a cartoon ribbon representation of the structure (Red: Lower values corresponding to high mobility, Blue: Higher values corresponding to low mobility). NMR data is detailed in Supplementary Table 4. (b) The closest to average structure of POTRA1. (c) Heteronuclear 1H-15N NOE measurements of the POTRA1 domain demonstrate the inherent flexibility of the loop, with the average value for loop 4 residues 88–91 being 0.45 ± 0.11 compared to the domain average of 0.77 ± 0.19. (d) Multiple sequence alignment in the region corresponding to loop 4. The amino acid composition in this loop from other Gamma-proteobacteria is consistent with a disordered structure seen in the E. coli protein. (e) Surface view of the surface charge features of BamA POTRA1 and TamA POTRA1 created between the two helices (red and blue surfaces represent acidic and basic residues, respectively).

Mentions: Views of the 10 lowest energy structures generated from NOE-based distance constraints and dihedral angle constraints derived from secondary chemical shifts and refined in water (Fig. 3a) reflect that the solution structure is well defined with a backbone heavy atom r.m.s.d. of 0.67 Å (Supplementary Table 4 shows the structural statistics for the ensemble). The closest to average structure (Fig. 3b) highlighted two distinguishing features of TamA POTRA1: (i) a highly-elongated shape accentuated by a dynamic segment of the polypeptide (loop 4) between the second and third β-strands (Fig. 3c), with the length of this loop largely conserved across bacterial species (Fig. 3d), and (ii) a displacement in two α-helices that increases the overall surface area (Supplementary Fig. 2) and created a basic groove surface feature on one face (Fig. 3e). This is in contrast to the hydrophobic groove found on the equivalent face of all five POTRA domains from BamA that were proposed to form the substrate binding site11. These obvious differences in structure taken together with sequence divergence from BamA POTRA domains strongly suggest that TamA POTRA1 fulfils a distinct biological role.


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)

The POTRA1 and POTRA2 domains of TamA are structurally specialized.(a) NMR solution structure of TamA POTRA1, with backbone representation of the ensemble of the 10 lowest energy structures. Steady-state 1H-15N NOE values are color plotted onto a cartoon ribbon representation of the structure (Red: Lower values corresponding to high mobility, Blue: Higher values corresponding to low mobility). NMR data is detailed in Supplementary Table 4. (b) The closest to average structure of POTRA1. (c) Heteronuclear 1H-15N NOE measurements of the POTRA1 domain demonstrate the inherent flexibility of the loop, with the average value for loop 4 residues 88–91 being 0.45 ± 0.11 compared to the domain average of 0.77 ± 0.19. (d) Multiple sequence alignment in the region corresponding to loop 4. The amino acid composition in this loop from other Gamma-proteobacteria is consistent with a disordered structure seen in the E. coli protein. (e) Surface view of the surface charge features of BamA POTRA1 and TamA POTRA1 created between the two helices (red and blue surfaces represent acidic and basic residues, respectively).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: The POTRA1 and POTRA2 domains of TamA are structurally specialized.(a) NMR solution structure of TamA POTRA1, with backbone representation of the ensemble of the 10 lowest energy structures. Steady-state 1H-15N NOE values are color plotted onto a cartoon ribbon representation of the structure (Red: Lower values corresponding to high mobility, Blue: Higher values corresponding to low mobility). NMR data is detailed in Supplementary Table 4. (b) The closest to average structure of POTRA1. (c) Heteronuclear 1H-15N NOE measurements of the POTRA1 domain demonstrate the inherent flexibility of the loop, with the average value for loop 4 residues 88–91 being 0.45 ± 0.11 compared to the domain average of 0.77 ± 0.19. (d) Multiple sequence alignment in the region corresponding to loop 4. The amino acid composition in this loop from other Gamma-proteobacteria is consistent with a disordered structure seen in the E. coli protein. (e) Surface view of the surface charge features of BamA POTRA1 and TamA POTRA1 created between the two helices (red and blue surfaces represent acidic and basic residues, respectively).
Mentions: Views of the 10 lowest energy structures generated from NOE-based distance constraints and dihedral angle constraints derived from secondary chemical shifts and refined in water (Fig. 3a) reflect that the solution structure is well defined with a backbone heavy atom r.m.s.d. of 0.67 Å (Supplementary Table 4 shows the structural statistics for the ensemble). The closest to average structure (Fig. 3b) highlighted two distinguishing features of TamA POTRA1: (i) a highly-elongated shape accentuated by a dynamic segment of the polypeptide (loop 4) between the second and third β-strands (Fig. 3c), with the length of this loop largely conserved across bacterial species (Fig. 3d), and (ii) a displacement in two α-helices that increases the overall surface area (Supplementary Fig. 2) and created a basic groove surface feature on one face (Fig. 3e). This is in contrast to the hydrophobic groove found on the equivalent face of all five POTRA domains from BamA that were proposed to form the substrate binding site11. These obvious differences in structure taken together with sequence divergence from BamA POTRA domains strongly suggest that TamA POTRA1 fulfils a distinct biological role.

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