Structural Biology and Molecular Biophysics

Atomic-Level Free Energy Landscape Reveals Cooperative Symport Mechanism of Melibiose Transporter

Ruibin Liang author has email address
Lan Guan author has email address

Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, United States
Department of Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, School of Medicine, Lubbock, United States

https://doi.org/10.7554/eLife.103421.1

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Figures and data

The transport cycle of the MelB symporter. The current study describes the melibiose (Mel, black double square) translocation between the periplasm and cytoplasm in the presence or absence of a bound coupling cation (Na⁺, red dot). The translocation of melibiose into cytoplasm starts from the OF_F state ([2]) and proceeds to the IF_F state ([6]) through several intermediate states such as the OF_B ([3]), OC_B ([4]), and IF_B ([5]). Reversal of this process results in melibiose translocation into periplasm. These key intermediate states are highlighted in bold text, with the protein depicted in solid colors. See the main text for a detailed definition of the states.

The potential of mean force (PMF) for the translocation of the melibiose across the WT MelB_St in the Na⁺-bound state along the minimum free energy pathway (represented as a string of images 0 to 32). Top insets: representative snapshots of the MelB_St (blue ribbons) and melibiose (spheres) in varied intermediate states. The bottom and top sides of each protein structure indicate the periplasmic and cytoplasmic sides of the membrane, respectively. The Asp19 and Asp124 residues, which are the major melibiose binding residues, are depicted as orange balls and sticks. The atoms in melibiose molecule are depicted as spheres.

Free energy barriers (ΔG′s) and net free energy changes (ΔG^‡′s).
Free energy barriers and net free energy changes (in kcal/mol) for melibiose translocation in the WT Na⁺ bound, WT Na⁺ unbound, and the D59C mutant Na⁺ unbound systems were analyzed for the overall translocation process and several key steps in this process.

Free energy surfaces (FES) for the transport of the melibiose through the WT MelB_St in the Na⁺ bound state. (A) FES spanned by the first principle component of the backbone (PC1) vs. melibiose transport coordinate Z. An illustrative structure indicating the scale of the Z coordinate is shown on the right, and key residues in the periplasmic gate, binding site and cytoplasmic gate are highlighted in red, orange and green, respectively (see main text). (B) FES spanned by interhelical distance between helices I and VII on the periplasmic side. An illustrative structure indicating the helices I (red) and VII (green) is shown on the right. (C) FES spanned by interhelical distance between helices IV and X on the cytoplasmic side. An illustrative structure indicating the helices IV (red) and X (green) is shown on the right. The approximate minimum free energy pathways (MFEP) tracking the major basins on the FES are indicated as black lines, and the regions corresponding to the intermediate states are labeled by arrows. All FES plots share the same color bar as (A).

Change of pore radius profile coupled to melibiose transport through WT MelB_St in the Na⁺ bound state. (A): Pore radii profile across the MelB as a function of the Z coordinate (relative to the COM of Asp124 and Asp19) along the entire protein (x-axis) for each different state during the melibiose translocation. As the melibiose is translocated from the periplasm to cytoplasm, the system transitions from the OF_F (red) to IF_F (blue) states through the OF_B (orange), OC_B (black), IF_B (green), and IF_C (brown) intermediate states, each of which features different pore radius profiles. The IF_C state corresponds to the melibiose passing through the cytoplasmic constricted region near Z=11 Å. An illustrative structure indicating the scale of the Z coordinate is shown on the right. (B) and (C): Translocation of melibiose from the periplasmic to cytoplasmic sides of MelB_St induces the change of pore radii measured at Z=-2.5 Å (periplasmic sugar-binding site) and Z=11 Å (cytoplasmic constricted region). The melibiose translocation process is represented as the transitioning of the system from the OF_F to IF_F states through multiple intermediate states along the x-axes. The protein structures below the plots serve as visual guides for the location of the melibiose in different states.

PMFs for the transport of the melibiose across MelB_St along the MFEPs represented as strings of images (0 to 33). The PMFs are calculated in the WT, Na⁺ bound (red) and unbound (blue) states, and the D59C mutant in the Na⁺ unbound state (green).

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