Sugar binding and protein conformational changes in lactose permease

Lactose permease is an integral membrane protein that uses the cell membrane's proton gradient for import of lactose. Based on extensive biochemical data and a substrate-bound crystal structure, intermediates involved in lactose/H⁺ co-transport have been suggested. Yet, the transport mechanism, especially the coupling of protonation states of essential residues and protein conformational changes involved in the transport, is not understood. Here we report molecular-dynamics simulations of membrane-embedded lactose permease in different protonation states, both in the presence and in the absence of lactose. The results analyzed in terms of pore diameter, salt-bridge formation, and substrate motion, strongly implicate Glu²⁶⁹ as one of the main proton translocation sites, whose protonation state controls several key steps of the transport process. A critical ion pair (Glu²⁶⁹ and Arg¹⁴⁴) was found to keep the cytoplasmic entrance open, but via a different mechanism than the currently accepted model. After protonation of Glu²⁶⁹, the salt bridge between Glu²⁶⁹ and Arg ¹⁴⁴ was found to break, and Arg¹⁴⁴ to move away from Glu²⁶⁹, establishing a new salt bridge with Glu¹²⁶; furthermore, neutralization of Glu²⁶⁹ and the displacement of Arg¹⁴⁴ and consequently of water molecules from the interdomain region was seen to initiate the closing of the cytoplasmic half channel (2.6-4.0 Å reduction in diameter in the cytoplasmic constriction region in 10 ns) by allowing hydrophobic surfaces of the N- and C-domains to fuse. Charged Glu²⁶⁹ was found to strongly bind the lactose permeant, indicating that proton transfer from water or another residue to Glu²⁶⁹ is a prerequisite for unbinding of lactose from the binding pocket.