Titel: Structural and functional insights into a mycobacterial type VII secretion system
Sprache: Englisch
Autor*in: Ritter, Christina
GND-Schlagwörter: ProteinsekretionGND
Erscheinungsdatum: 2022
Tag der mündlichen Prüfung: 2023-03-22
With over 1.5 million deaths per year, mycobacterial pathogens, including Mycobacterium tuberculosis, belong to the deadliest pathogens worldwide. Essential to their complex infection mechanism is the secretion of virulence proteins across their otherwise almost impermeable cell envelope. The transport of these heterodimeric protein complexes is mediated by a specialized secretion system, the Type VII or ESX secretion system. Slow-growing, often pathogenic mycobacteria, code for up to five different ESX systems, termed ESX-1 to ESX-5, which fulfill a diverse set of functions in the mycobacterial life cycle. Although the systems are considered promising drug targets, the molecular mechanism by which substrates are transported across the mycobacterial membrane remains elusive so far.
This thesis presents the structure of the inner membrane spanning ESX-5 system from Mycobacterium xenopi. Integrating data from cryo-EM, crosslinking mass spectrometry and homology models enabled a detailed view on the hexameric secretion system with central secretion pore. The complex is formed by six protomeric units that comprise the protein components EccB5 :EccC5 :EccD5 and EccE5 in a 1:1:2:1 stoichiometry. While the transmembrane segment was overall well defined within the cryo-EM density, the secretion pore, formed by two helices of EccC5, showed inherent flexibility, which was hypothesized to be caused by a conserved proline residue in the center of the helix. In addition, analysis of the pore diameter suggested a role of phenylalanine residues in a gating mechanism. A mutational analysis carried out in this thesis revealed that the integrity of these pore residues and the integrity of the pore scaffold, formed by EccB5, is essential for secretion. A cryo-EM structure further confirmed that mutation of the central proline residue leads to the rigidification of the pore helices, indicating that plasticity of the central transmembrane pore is necessary to allow transport of different folded substrates across the cell envelope.
This plasticity was also observed within the cytosol for the three consecutive ATPase domains of EccC5 and on the periplasmic side for the periplasmic domain of EccB5. As shown by cryo-EM, addition of ATPγS slightly stabilized two EccC5 conformations within the cytoplasm, probably corresponding to different ATPγS bound states. It is hypothesized that the remaining intrinsic flexibility could bestow dynamics to the system that are essential to carry out huge conformational changes in the translocation cycle.
Along with other recently published structures of ESX-5 and ESX-3 systems and improved structure prediction methods, the presented results have paved the way for structural comparison of the different systems and for a deeper understanding of the underlying mechanism of secretion.
URL: https://ediss.sub.uni-hamburg.de/handle/ediss/10384
URN: urn:nbn:de:gbv:18-ediss-110874
Dokumenttyp: Dissertation
Betreuer*in: Wilmanns, Matthias
Enthalten in den Sammlungen:Elektronische Dissertationen und Habilitationen

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