Impact of N-Terminal Domain Conformation and Domain Interactions on RfaH Fold Switching.

Area of Science:

• Structural biology
• Molecular biophysics
• Protein dynamics

Background:

• RfaH is a metamorphic protein essential for transcription regulation and translation initiation.
• It undergoes coupled domain dissociation and fold switching for its functions.
• The N-terminal domain (NTD) and C-terminal domain (CTD) change conformation upon binding to RNA polymerase (RNAP).

Purpose of the Study:

• To investigate the role of the NTD's beta-hairpin (β3-β4) in triggering RfaH domain dissociation.
• To simulate the thermal unfolding and domain separation of RfaH and a modified variant (H1).
• To analyze the reverse fold switch from an open to a closed state.

Main Methods:

• Homology modeling to create the H1 variant with a remodeled β3-β4 hairpin.
• All-atom physics-based simulations with a structure-based potential.
• Simulations of domain separation driven by CTD thermal unfolding.
• Analysis of temperature-dependent fold switching kinetics.

Main Results:

• The H1 variant showed decreased CTD stability and domain dissociation at a lower temperature compared to free RfaH.
• Complete refolding to the all-β state was not observed, suggesting the hairpin change aids but doesn't solely drive dissociation.
• Fold switching kinetics are temperature-dependent, with an optimal temperature for rapid switching where both folds' stabilities are reduced.

Conclusions:

• Conformational changes in the NTD's β3-β4 hairpin are important for initiating RfaH domain dissociation.
• Inter-domain interactions and NTD conformational changes collectively regulate RfaH's functional structural transitions.
• Optimal temperatures exist for efficient RfaH fold switching, balancing kinetic trapping and instability.