Free Energy Landscape of the Complete Transport Cycle in a Key Bacterial Transporter

Abstract

PepTSo is a proton-coupled bacterial symporter, from the major facilitator superfamily (MFS), which transports di-/tripeptide molecules. The recently obtained crystal structure of PepTSo provides an unprecedented opportunity to gain an understanding of functional insights of the substrate transport mechanism. Binding of the proton and peptide molecule induces conformational changes into occluded (OC) and outward-facing (OF) states, which we are able to characterize using molecular dynamics (MD) simulations. The structural knowledge of the OC and OF state is important to fully understand the major energy barrier associated with the transport cycle. In order to gain functional insight into the interstate dynamics, we performed extensive all atom MD simulations. The Markov state model was constructed to identify the free energy barriers between the states, and kinetic information on intermediate pathways was obtained using the transition pathway theory (TPT). TPT shows that the OF state is obtained by the movement of TM1 and TM7 at the extracellular side approximately 12–16 Å away from each other, and the inward movement of TM4 and TM10 at the intracellular halves to 3–4 Å characterizes the OC state. Helix distance distributions obtained from MD simulations were compared with experimental double electron–electron resonance spectroscopy and were found to be in excellent agreement with previous studies. We also predicted the optimal positions for placement of methane thiosulfonate spin label probes to capture the slowest protein dynamics. Our finding sheds light on the conformational cycle of this key membrane transporter and the functional relationships between the multiple intermediate states.

How antiporters exchange substrates across the cell membrane? An atomic-level description of the complete exchange cycle in NarK

“…We projected simulation data onto these two metrics as the opening and closure of the pore channel strictly determines the specific state such as IF, OC, and OF. 30,31,34 Free energy landscape reveals the thermodynamic and kinetic information about the antiporter transport mechanism (Fig.1A). The canonical L-shaped free energy landscape displays three distinct free energy basins corresponding to IF, OC, and OF states, in line with the rocker-switch mechanism.…”

Proton-control of transitions in an amino-acid transporter

“…It is clear that very long simulation times are often required, as recently demonstrated for the semiSWEET transporter (36) or the GluT1 transporter (37). Indeed, to study the complete conformation cycle, it would seem likely that the approach of Markov State Modelling (MSM) might be the most appropriate as recently demonstrated with PepT so transporter (38). In this work however, we were interested in fully characterizing only one part of the full transition pathway, facilitating the use of shorter timescales that enabled the application of more rigorous methods.…”

FingerprintContacts: Predicting Alternative Conformations of Proteins from Coevolution

“…Proteins are dynamic entities that undergo significant conformational changes while performing diverse cellular functions such as drug binding, 1-4 enzyme catalysis, [5][6][7] and nutrient transport. [8][9][10] Conformational changes often involve transitions among two or more alternative structures. Characterization of the conformational ensemble is critical for deciphering the relationship between protein structure and functional mechanisms.…”