6RFM

Bordetella pertussis adenylate cyclase toxin transmembrane segment 411-490 in DPC micelles

Summary for 6RFM

• Entry DOI: 10.2210/pdb6rfm/pdb
• NMR Information: BMRB: 34393
• Descriptor: Bifunctional hemolysin/adenylate cyclase (1 entity in total)
• Functional Keywords: adenylate cyclase, bordetella pertussis, membrane protein, calcium binding, toxin
• Biological source: Bordetella pertussis
• Total number of polymer chains: 1
• Total formula weight: 8226.12

Authors

Masin, J.,Bumba, L.,Veverka, V. (deposition date: 2019-04-15, release date: 2020-05-13, Last modification date: 2024-06-19)

Primary citation

Sukova, A.,Bumba, L.,Srb, P.,Veverka, V.,Stanek, O.,Holubova, J.,Chmelik, J.,Fiser, R.,Sebo, P.,Masin, J.
Negative charge of the AC-to-Hly linking segment modulates calcium-dependent membrane activities of Bordetella adenylate cyclase toxin.
Biochim Biophys Acta Biomembr, 1862:183310-183310, 2020

PubMed Abstract: Two distinct conformers of the adenylate cyclase toxin (CyaA) appear to accomplish its two parallel activities within target cell membrane. The translocating conformer would deliver the N-terminal adenylyl cyclase (AC) enzyme domain across plasma membrane into cytosol of cells, while the pore precursor conformer would assemble into oligomeric cation-selective pores and permeabilize cellular membrane. Both toxin activities then involve a membrane-interacting 'AC-to-Hly-linking segment' (residues 400 to 500). Here, we report the NMR structure of the corresponding CyaA polypeptide in dodecylphosphocholine micelles and show that it consists of two α-helices linked by an unrestrained loop. The N-terminal α-helix (Gly418 to His439) remained solvent accessible, while the C-terminal α-helix (His457 to Phe485) was fully enclosed within detergent micelles. CyaA weakly bound Ca ions (apparent K 2.6 mM) and permeabilized negatively charged lipid vesicles. At high concentrations (10 μM) the CyaA polypeptide formed stable conductance units in artificial lipid bilayers with applied voltage, suggesting its possible transmembrane orientation in the membrane-inserted toxin. Mutagenesis revealed that two clusters of negatively charged residues within the 'AC-to-Hly-linking segment' (Glu419 to Glu432 and Asp445 to Glu448) regulate the balance between the AC domain translocating and pore-forming capacities of CyaA in function of calcium concentration.

PubMed: 32333856
DOI: 10.1016/j.bbamem.2020.183310

Experimental method

SOLUTION NMR