Unraveling structural transitions and kinetics along the fold-switching pathway of the RfaH C-terminal domain using exchange-based NMR

• 成果类型: Article
• 署名作者: Cai, Mengli; Ying, Jinfa; Lopez, Juan M.; Huang, Ying; Clore, G. Marius
• 署名单位: National Institutes of Health (NIH) - USA; NIH National Institute of Diabetes & Digestive & Kidney Diseases (NIDDK); Pontificia Universidad Catolica del Peru; National Institutes of Health (NIH) - USA; NIH National Institute of Diabetes & Digestive & Kidney Diseases (NIDDK)
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-13574
• DOI: 10.1073/pnas.2506441122
• 发表日期: 2025-05-14
• 关键词: model-free approach; chemical-exchange; transcription factor; backbone dynamics; macromolecules; relaxation; shift; sensitivity; RESOLUTION; EVOLUTION

摘要

The bacterial transcriptional regulator RfaH comprises structurally and functionally distinct N- (NTD) and C- (CTD) terminal domains. The latter switches from a helical hairpin packed against the NTD to a five-stranded beta-roll upon displacement by RNA polymerase binding. Here, we use exchange-based NMR to probe fold-switching intermediates sampled by the isolated CTD. In addition to the predominant (similar to 76 to 77%), semistable beta-roll conformation (state A), we identify four structurally and kinetically distinct states: A ', B, B ', and B ''. State B is NMR observable with an occupancy of similar to 23%, exchanges slowly (tau(ex) similar to 300 ms) with the major A species, and comprises a largely unfolded ensemble with transient occupancy of helical (alpha 5*) and beta-hairpin (beta 1*/beta 2*) elements. Backbone chemical shift-based structure predictions using the program CS-ROSETTA suggest that the two transient structural elements within the B state may interact with one another to form a semicompact structure. A ' (similar to 0.35%) is an off-pathway state that exchanges rapidly (tau(ex) similar to 1 ms) with state A and likely entails a minor localized conformational change in the beta 1/beta 2 loop. State B ' (similar to 0.3%) exchanges rapidly (tau(ex) similar to 1.2 ms) with state B and exhibits downfield N-15 backbone shifts (relative to B) in the alpha 5* region indicative of reduced helicity. Finally state B '' (similar to 0.05%) exchanges rapidly (tau(ex) similar to 0.8 to 1 ms) with either B ' (linear model) or B (branched model), displays significant differences in absolute N-15 chemical shift from states B and B ', and likely represents a further intermediate with increased helicity along the fold-switching pathway.