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

Abstract

PepT_So is a proton-coupled bacterial symporter, from the major facilitator superfamily (MFS), which transports di-/tripeptide molecules. The recently obtained crystal structure of PepT_So 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.

Figure 1

Conformational landscape of PepT_So. The conformational landscape is generated using the extracellular and intracellular side distances measured between atom pairs Arg32-CZ (TM1)–Asp316-CG (TM7) and Ser131-CO (TM4)–Tyr431-OH (TM10), respectively. The conformational states are depicted as IF (1), partial IF–OC (2), OC (3), partial OC–OF (4), OF (5), and wide open states (6 and 7). The black dots indicate the PepT_So crystal structures available in the Protein Data Bank.

Figure 2

Distinct conformational states of PepT_So are visualized by passing a spherical probe from one side of the protein to the other through (A) the crystal structure IF state, and for the MD simulations predicted structures for (B) OC and (C) OF states, calculated using the HOLE program. The gating residues Arg32 (TM1)–Asp316 (TM7) and Ser131 (TM4)–Tyr431 (TM10) that act as bottlenecks for the conformational transition are indicated. (D) The pore radius along the protein for the three conformational states.

Figure 3

PepT_So is shown in the center with TM 1, 2, 4, 7, 8, and 10 in green, magenta, blue, cyan, yellow, and orange, respectively. The short helices (SH) which join N and C domains are not shown for clarity. The remaining six helices are in gray. All state conformations are the extracellular view of the protein. (A) IF state is stabilized by ion lock at the extracellular side, and the intracellular half is wide open. (B) Inward movement of TM4 and TM10 determines the partial IF–OC state. (C) Further inward movement leads to formation of hydrogen bond interaction between Tyr431-Ser131 in the OC state. (D) Gating residues at the intracellular side weaken the extracellular interaction to form a partial OC–OF state. (E) Helices TM1 and TM7 move far away to 15 Å in the OF state.

Figure 4

MD simulation predicted DEER distance distribution ranges (green) are compared to the experimental DEER distance distribution range (blue).

Figure 5

(A) Cross-validated scores for 4300 possible DEER residue-pair sets. The red line is the score of the MSM using experimental residue distance pairs. (B) The residues used for experimental DEER and (C) highest ranked predicted DEER residue-pairs choices.