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Nonameric structures of the cytoplasmic domain of FlhA and SctV in the context of the full-length protein.

ABSTRACT: Type three secretion is the mechanism of protein secretion found in bacterial flagella and injectisomes. At its centre is the export apparatus (EA), a complex of five membrane proteins through which secretion substrates pass the inner membrane. While the complex formed by four of the EA proteins has been well characterised structurally, little is known about the structure of the membrane domain of the largest subunit, FlhA in flagella, SctV in injectisomes. Furthermore, the biologically relevant nonameric assembly of FlhA/SctV has been infrequently observed and differences in conformation of the cytoplasmic portion of FlhA/SctV between open and closed states have been suggested to reflect secretion system specific differences. FlhA has been shown to bind to chaperone-substrate complexes in an open state, but in previous assembled ring structures, SctV is in a closed state. Here, we identify FlhA and SctV homologues that can be recombinantly produced in the oligomeric state and study them using cryo-electron microscopy. The structures of the cytoplasmic domains from both FlhA and SctV are in the open state and we observe a conserved interaction between a short stretch of residues at the N-terminus of the cytoplasmic domain, known as FlhAL/SctVL, with a groove on the adjacent protomer's cytoplasmic domain, which stabilises the nonameric ring assembly.

SUBMITTER: Kuhlen L

PROVIDER: S-EPMC8213127 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Saccharomyces cerevisiae, the budding yeast, must remodel initial cell shape and cell wall integrity during vegetative growth and pheromone-induced morphogenesis. The cell wall remodeling is monitored and regulated by the cell wall integrity (CWI) signaling pathway. Wsc1p, together with Wsc2p and Wsc3p, belongs to a family of highly O-glycosylated cell surface proteins that function as stress sensors of the cell wall in S. cerevisiae. These cell surface proteins have the main role of activating the CWI signaling pathway by stimulating the small G-protein Rho1p, which subsequently activates protein kinase C (Pkc1p) and a mitogen activated protein (MAP) kinase cascade that activates downstream transcription factors of stress-response genes. Wsc1p, Wsc2p, and Wsc3p possess a cytoplasmic domain where two conserved regions of the sequence have been assessed to be important for Rom2p interaction. Meanwhile, other research groups have also proposed that these transmembrane proteins could support protein-protein interactions with Ras2p. Molecular structures of the cytoplasmic domain of Wsc1p, Wsc2p and Wsc3p were generated using the standard and fully-automated ORCHESTAR procedures provided by the Sybyl-X 2.1.1 program. The tridimensional structure of full length Ras2p was also generated with Phyre2. These protein models were validated with Procheck-PDBsum and ProSA-web tools and subsequently used in docking-based modeling of protein-protein and protein-compound interfaces for extensive structural and functional characterization of their interaction. The results retrieved from STRING 10.5 suggest that the Wsc-family is involved in protein-protein interactions with each other and with Ras2p. Docking-based studies also validated the existence of protein-protein interactions mainly between Motif I (Wsc3p > Wsc1p > Wsc2p) and Ras2p, in agreement with the data provided by STRING 10.5. Additionally, it has shown that Calcofluor White preferably binds to Wsc1p (-9.5 kcal/mol), meanwhile Caspofungin binds to Wsc3p (-9.1 kcal/mol), Wsc1p (-9.1 kcal/mol) and more weakly Wsc2p (-6.9 kcal/mol). Thus, these data suggests Caspofungin as a common inhibitor for the Wsc-family. MTiOpenScreen database has provided a list of new compounds with energy scores higher than those compounds used in our docking studies, thus suggesting these new compounds have a better affinity towards the cytoplasmic domains and Ras2p. Based on these data, there are new and possibly more effective compounds that should be considered as therapeutic agents against yeast infection.

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Nonameric structures of the cytoplasmic domain of FlhA and SctV in the context of the full-length protein.

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