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- W4211006247 abstract "Aim: To investigate the mechanism of action of the Vitamin B3 transporter PnuC by Cryo-EM and protein engineering. Background: Salmonella typhimurium and Neisseria mucosa are species that can cause a series of human infections, e.g. typhoid, endocarditis. As these organisms are NAD auxotrophs which strictly depend on external Vitamin B3, their unique transporter PnuC were considered as a potential drug target. However, it remains unclear about PnuC’s mechanism of action. Results: High resolution of the PnuC (27 kDa) in occluded conformation with (2.4 A) and without substrate (3.6A) were solved by Cryo-EM, binding residue VAL 57, TRP 95 identified. By limiting the trace element (FeCl3, etc), we were able to control NR level in E. coli cells and finally got substrate-free PnuC sample. However, the protein dissociated into monomers, indicating more flexible conformation without substrate. Site-directed mutagenesis also confirmed the trimer was unstable and dissociated into monomers. Cross-link assays also confirmed that PnuC trimers without substrate may already dissociated in native membrane. Adding the substrate NR back reversed monomers into trimers again, indicating a clear substrate-induced conformation change. ITC results showed Kd value of 330 nM. Finally, we engineered a fusion trimer PnuCT (84 kDa) and protomer disactivated trimers PnuCT1-3. Substrate binding assay revealed that the NR binding dropped from 100% to 20%, 10%, and 0%, indicating a clear evidence of cooperativity effect between each protomers in a single trimer.Conclusion: Here we show that the whole trimer of PnuC transporter, besides the alternating conformational states cycle, also undergoes associated -> dissociated -> associated cycle. Moreover, engineered fusion-trimer test clearly showed cooperativity effect among each protomers in a single trimer. These new findings may facilitate more rational development of therapeutics toward related pathogens." @default.
- W4211006247 created "2022-02-13" @default.
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- W4211006247 date "2022-02-01" @default.
- W4211006247 modified "2023-09-26" @default.
- W4211006247 title "The Vitamin B3 transporter PnuC: substrate induced conformation change and trimerization cooperativity mechanism" @default.
- W4211006247 doi "https://doi.org/10.1016/j.bpj.2021.11.519" @default.
- W4211006247 hasPublicationYear "2022" @default.
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