Tuning the Continuum of Structural States in the Native Ensemble of a Regulatory Protein

Abstract

The mesoscale nature of proteins allows for an efficient coupling between environmental cues and conformational changes, enabling their function as molecular transducers. Delineating the precise structural origins of such a connection and the expected spectroscopic response has, however, been challenging. In this work, we perform a combination of urea–temperature double perturbation experiments and theoretical modeling to probe the conformational landscape of Cnu, a natural thermosensor protein. We observe unique ensemble signatures that point to a continuum of conformational substates in the native ensemble and that respond intricately to perturbations upon monitoring secondary and tertiary structures, distances between an intrinsic FRET pair, and hydrodynamic volumes. Binding assays further reveal a weakening of the Cnu functional complex with temperature, highlighting the molecular origins of signal transduction critical for pathogenic response in enterobacteriaceae.

A binding cooperativity switch driven by synergistic structural swelling of an osmo-regulatory protein pair

“…The increased structural heterogeneity with decreasing salt should lead to a concomitant structural swelling or an increased hydrodynamic volume. In fact, structural modulations of Cnu with temperature, urea, and mutation all reveal a proportionate increase in hydrodynamic volume demonstrated earlier through a combination of size-exclusion chromatography (SEC) and analytical ultracentrifugation (AUC) techniques 32,36,37 . However, it is challenging to discern the same at low ionic strength employing SEC 38 , light scattering 39 , or AUC 40 due to non-specific interactions with the column matrix, strong inter-particle interactions, and primary charge effects, respectively.…”

“…The binding interface of Cnu involves precisely those residues in the fourth helix (and the associated regions in the third helix; Fig. 1a) that display complex conformational behavior with temperature and salt as observed for all members of the Hha-family 23,31,32,37 . Titration experiments reveal that the binding of H-NS (residues 1–59 of the N-terminal domain, see Methods and Supplementary Fig.…”

“…However, it is challenging to discern the same at low ionic strength employing SEC 38 , light scattering 39 , or AUC 40 due to non-specific interactions with the column matrix, strong inter-particle interactions, and primary charge effects, respectively. On the other hand, we had earlier shown that an increase in hydrodynamic volume is accompanied by an increase in distance between a natural intra-molecular tyrosine–tryptophan donor–acceptor pair 37 . Y40 (located in the third helix) and W67 (fourth helix) lie within 12 Å of each other ( r 0 for this pair) providing a direct avenue to monitor relative distances with ionic strength.…”

Switching Protein Conformational Substates by Protonation and Mutation

“…The protocols for protein purification and the majority of the spectroscopic experiments can be found in references 18 and 19. The buffers used in the pH-dependent study were glycine-HCl (pH 2–3), sodium acetate (pH 3.5–5.5), and sodium phosphate (pH 6–8).…”

“…One such example in the enterobacteriaceae is the four-helix Hha-family of proteins that regulates the availability of the transcription factor H-NS 11–15. Mutational studies16,17 and spectroscopic experiments provide strong evidence that Cnu (YdgT), a member of the Hha-family, exhibits graded structural polymorphism with temperature18 that could be critical for the expression of virulence genes in enterobacteriaceae by modulating the binding affinity with H-NS 19…”