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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

Molecular blueprint of the TAM nanomachine.A composite structure of TamA, incorporating information from the NMR structure of POTRA1 (pdb 2LY3) and the SAXS structural information superimposed as a meshwork onto the crystal structure (pdb 4C00). The β-barrel domain is represented as a ribbon diagram for clarity, and the inset details the contacts between residues in the POTRA3 domain and the β-barrel domain, as previously documented by Gruss et al.5. Given the discovery that TamA POTRA1 mediates contact with TamB, the dotted line denotes where TamB applies resistance to the TamA lever arm. The scale bar demonstrates the extent of movement that the TamA lever is capable of, given MCNR measurements14 showing that it can extend to ~77 Å from the internal surface of the outer membrane in response to the presence of a substrate protein. This movement would necessarily require rearrangement of the POTRA3:β-barrel domain contacts, and we speculate that restoring these contacts could be a basis for bringing the POTRA domain lever arm back to the resting position, for continued rounds of protein assembly in vivo.
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f5: Molecular blueprint of the TAM nanomachine.A composite structure of TamA, incorporating information from the NMR structure of POTRA1 (pdb 2LY3) and the SAXS structural information superimposed as a meshwork onto the crystal structure (pdb 4C00). The β-barrel domain is represented as a ribbon diagram for clarity, and the inset details the contacts between residues in the POTRA3 domain and the β-barrel domain, as previously documented by Gruss et al.5. Given the discovery that TamA POTRA1 mediates contact with TamB, the dotted line denotes where TamB applies resistance to the TamA lever arm. The scale bar demonstrates the extent of movement that the TamA lever is capable of, given MCNR measurements14 showing that it can extend to ~77 Å from the internal surface of the outer membrane in response to the presence of a substrate protein. This movement would necessarily require rearrangement of the POTRA3:β-barrel domain contacts, and we speculate that restoring these contacts could be a basis for bringing the POTRA domain lever arm back to the resting position, for continued rounds of protein assembly in vivo.

Mentions: The POTRA domains of TamA have distinguishing structural characteristics: they lack the conformational flexibility seen in BamA, and instead adopt a conformation in solution that is consistent with what we propose to be a lever-arm in the TAM. In SAXS measurements, less than 10 Å movement was possible between POTRA1 and POTRA2, and none was observed between POTRA2 and POTRA3. This is consistent with extensive crystal contacts observed between the POTRA domains in TamA5. Taken together, this suggests that major movements, measured previously at ~30 Å relative to the membrane surface14 would be contributed through a fulcrum at the POTRA3:β-barrel domains (Fig. 5).


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)

Molecular blueprint of the TAM nanomachine.A composite structure of TamA, incorporating information from the NMR structure of POTRA1 (pdb 2LY3) and the SAXS structural information superimposed as a meshwork onto the crystal structure (pdb 4C00). The β-barrel domain is represented as a ribbon diagram for clarity, and the inset details the contacts between residues in the POTRA3 domain and the β-barrel domain, as previously documented by Gruss et al.5. Given the discovery that TamA POTRA1 mediates contact with TamB, the dotted line denotes where TamB applies resistance to the TamA lever arm. The scale bar demonstrates the extent of movement that the TamA lever is capable of, given MCNR measurements14 showing that it can extend to ~77 Å from the internal surface of the outer membrane in response to the presence of a substrate protein. This movement would necessarily require rearrangement of the POTRA3:β-barrel domain contacts, and we speculate that restoring these contacts could be a basis for bringing the POTRA domain lever arm back to the resting position, for continued rounds of protein assembly in vivo.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Molecular blueprint of the TAM nanomachine.A composite structure of TamA, incorporating information from the NMR structure of POTRA1 (pdb 2LY3) and the SAXS structural information superimposed as a meshwork onto the crystal structure (pdb 4C00). The β-barrel domain is represented as a ribbon diagram for clarity, and the inset details the contacts between residues in the POTRA3 domain and the β-barrel domain, as previously documented by Gruss et al.5. Given the discovery that TamA POTRA1 mediates contact with TamB, the dotted line denotes where TamB applies resistance to the TamA lever arm. The scale bar demonstrates the extent of movement that the TamA lever is capable of, given MCNR measurements14 showing that it can extend to ~77 Å from the internal surface of the outer membrane in response to the presence of a substrate protein. This movement would necessarily require rearrangement of the POTRA3:β-barrel domain contacts, and we speculate that restoring these contacts could be a basis for bringing the POTRA domain lever arm back to the resting position, for continued rounds of protein assembly in vivo.
Mentions: The POTRA domains of TamA have distinguishing structural characteristics: they lack the conformational flexibility seen in BamA, and instead adopt a conformation in solution that is consistent with what we propose to be a lever-arm in the TAM. In SAXS measurements, less than 10 Å movement was possible between POTRA1 and POTRA2, and none was observed between POTRA2 and POTRA3. This is consistent with extensive crystal contacts observed between the POTRA domains in TamA5. Taken together, this suggests that major movements, measured previously at ~30 Å relative to the membrane surface14 would be contributed through a fulcrum at the POTRA3:β-barrel domains (Fig. 5).

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