Lactose Permease: Breaching the Barrier

Lactose permeaseFig. 1 Lactose Permease embedded in a lipid bilayer.

Lactose permease (LacY) is an integral protein that facilitate the passage of lactose, one of the essential nutrients for all life forms, across the otherwise impermeable phospholipid bilayers that surround all cells and organelles. The active transport uses the energy of the electrochemical proton gradient, i.e. one H+ is transported in with each sugar (co-transport). The proteins play a critical role in transmembrane traffic, and, therefore, are critical for a healthy metabolism of a wide range of living organism, including human being. Malfunction of these transporters is associated with various pathophysiological conditions, such as diabetes and depression. Solved in 2003, the crystal structure of LacY of E. coli exhibits 12 transmembrane helices. Two halves of the protein (N-domain and C-domain, respectively) form a hydrophilic cavity opening to the cytoplasm, where the substrate is bound in its binding pocket. The periplasmic side of the protein is closed and the substrate is ready to diffuse into the cell through the opening of the cavity.

The crystal structure of LacY reprents the inward open state of the protein, in which the substrate is accessible only from the cytoplamsic side. Apparently, the accessibility of the substrate from the periplasm is necessary for the import of lactose from outside of the cell. One of the most important unknowns in the mechanism of sugar transport in LacY is the nature of protein conformational changes that switch substrate accessibility from the cytoplasmic part to the periplasmic one.

Lactose permease

Fig. 2 Substrate transport in LacY.

Beginning from a putative outward open conformation, in which the substrate is accessible from the periplasm, the right figure provides the schematic representation of a possible lactose/H+ co-transport mechanism. Intermediates d, e, and f correspond to inward open conformation as seen in the crystal structure; intermediates a and b correspond to the outward open conformation that has not yet been resolved structurally by observation. A histidine (His in the right Figure) in the periplasmic side of LacY is postulated as a possible proton acceptor. It is protonated in the ground state (intermediate a) of LacY considering that the periplasm is rich in protons. The putative transport process shown is composed of eight steps: (I) Lactose binds to ground state LacY, in which a Glutamate E1 is protonated. (II) The periplasmic side of LacY closes, induced by the movement of the lactose to the binding pocket. (III) A proton is transfered from E1 to Glutamate E3, and Arginine R+ in the N-domain and unprotonated E1 forms a salt bridge which induces the inward open conformation d. Meanwhile lactose moves toward the cytoplasmic side. (IV) Lactose is released to the cytoplasm. (V) The proton on E3 is released to the cytoplasm; (VI) A histidine in the periplasmic half (His) transfers its proton to E1 and LacY closes at the cytoplasm. (VII) His becomes protonated. (VIII) LacY opens at the periplasm.

Protonation-Induced Transition

Salt bridge and conformational changes

Fig. 3 Coupling of the protein conformational change and salt bridge breakage.

Our molecular dynamics simulations using NAMD has observed the reverse of step III, in which a proton transfer induces a conformational transition from intermediate d to c. According to the mechanism in the above figure, the reverse of step III includes: proton transfer from E3 to E, breakage of a salt bridge between E1 and R+, closing of the protein at the cytoplasm entrance and opening at the periplasmic side, as well as substrate movement towards the periplasmic side. To mimic the effect of proton translocation, we simulated two systems: sysa with E1 protonated and E3deprotonated, and sysb E1 deprotonated and E3 protonated. Fig. 3a and b show the conformational changes between the crystal structure (cyan: N-domain; pink: C-domain) and final structure (blue: N-domain; red: C-domain) of the two systems after 10 nanosecond simulation. The cytoplasmic halves of the N-domain helices in the final structure of sysb show a significant inward motion resulting in a partial closure of the cytoplasmic cavity In contrast, no noticeable conformational changes were observed in system sysa. In Fig. 3c,d,e, and f more details of the interaction of the interdomain helices are shown. In sysb, the hydrophobic faces of the interdomain helices are in a much closer contact than that in sysa. The approach of the these hydrophobic faces appears to be controlled by water occupancy in the interdomain region.

The breakage of the salt bridge between E1 and R+ is correlated with the conformational change of the protein. When E1 is deprotonated, this salt bridge in sysa remain stable during the 10 nanosecond equilibration. This same salt bridge breaks immediately after protonation of E1 in sysb.
After the breakage of salt bridge E1-R+, R+ establishes a salt bridge with another acidic residue, glutamate E2, which sits in the N-domain binding site.


Publications Database Sugar binding and protein conformational changes in lactose permease. Ying Yin, Morten Ø. Jensen, Emad Tajkhorshid, and Klaus Schulten. Biophysical Journal, 91:3972-3985, 2006. Publications Database Sugar transport across lactose permease probed by steered molecular dynamics. Morten Ø. Jensen, Ying Yin, Emad Tajkhorshid, and Klaus Schulten. Biophysical Journal, 93:92-102, 2007.


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