Mechanism of the All-α to All-β Conformational Transition of RfaH-CTD: Molecular Dynamics Simulation and Markov State Model

Abstract

The C-terminal domain of the bacterial transcription antiterminator RfaH undergoes a dramatic all-α-helix to all-β-barrel transition when released from its N-terminal domain. These two distinct folding patterns correspond to different functions: the all-α state acts as an essential regulator of transcription to ensure RNA polymerase binding, whereas the all-β state operates as an activator of translation by interacting with the ribosomal protein S10 and recruits ribosomal mRNA. Accordingly, this drastic conformational change enables RfaH to physically couple the transcription and translation processes in gene expression. To understand the mechanism behind this extraordinary functionally relevant structural transition, we constructed Markov state models using an adaptive seeding method. The constructed models highlight several parallel folding pathways with heterogeneous molecular mechanisms, which reveal the folding kinetics and atomic details of the conformational transition.

Differential local stability governs the metamorphic fold-switch of bacterial virulence factor RfaH

“…This is supported by previous observations that disruption of the E48:R138 salt bridge located in this region led RfaH to exist in equilibrium between the autoinhibited and active folds (12). However, this view contrasts with conclusions drawn from other computational approaches (18,22), which suggested that contacts involving RfaH CTD residues comprising the strand β 3 are particularly stable and nucleate the β-barrel, as solution dynamics do not display high stability in this region for RfaH or NusG CTD in the β-fold (Fig. S3).…”

“…Since the trigger for RfaH metamorphosis is the complete ops-paused TEC (13,14,16), it is challenging to study this process experimentally. Instead, most of the thermodynamic and kinetic studies have used computational approaches to directly explore this fold-switch by simulating either the isolated CTD (18)(19)(20) or the entire RfaH protein (21,22). Although the RfaH CTD is composed of only 51 residues (residues 112-162), its transition from alpha to beta has not been observed through conventional molecular dynamics (19), but through the use of enhanced sampling techniques (18,20,22) or reduced system granularity (21,23).…”

“…Instead, most of the thermodynamic and kinetic studies have used computational approaches to directly explore this fold-switch by simulating either the isolated CTD (18)(19)(20) or the entire RfaH protein (21,22). Although the RfaH CTD is composed of only 51 residues (residues 112-162), its transition from alpha to beta has not been observed through conventional molecular dynamics (19), but through the use of enhanced sampling techniques (18,20,22) or reduced system granularity (21,23). A way to circumvent such computing barriers is the use of confinement simulations, which rely on a discontinuous thermodynamic integration to estimate the absolute free energy of a clearly defined energy well (24).…”

Molecular dynamics simulations reveal ligand-controlled positioning of a peripheral protein complex in membranes

“…In previous studies, MSMs have been used to model the folding of proteins to a known native state 50 , or to demonstrate activation pathways and metastable intermediates between known active and inactive structures 51–54 . This study expands upon this body of knowledge by presenting a system in which the structure of the active state is experimentally inaccessible, and these MSMs resolve the active state through simulation alone.…”

Multi-Funnel Landscape of the Fold-Switching Protein RfaH-CTD

“…However, probing such fold switching and mapping their energy landscape by experiments or in silico is a challenge. [10][11][12][13][14] Computationally, the problem is that the exploration of the ensemble of possible structures and the conversion between these structures happens on timescales that, on general-purpose computers, are not accessible in all-atom molecular dynamics simulations with explicit solvent. Enhanced sampling techniques such as Replica Exchange Molecular Dynamics (REMD) [15][16][17][18][19][20] promise to overcome this problem by realizing a random walk in temperature which allows the system to escape out of traps and cross barriers by explorations to higher temperatures.…”