This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Lactose permease (LacY) is an integral membrane protein (www.ks.uiuc.edu/Research/Categories/Membrane/) that uses the cell s electrochemical proton gradient to actively transport substrates across the cell membrane [129 131]. This protein plays a critical role in transmembrane traffic, and, therefore, is crucial for a healthy metabolism of a wide range of living organisms, including human beings. Malfunction of this transporter is associated with various pathophysiological conditions, such as diabetes and depression in humans [132,133]. Since the first functional characterization of LacY in 1956 [134], various biochemical, biophysical, and structural biological studies [133, 135, 136] have resulted in a transport model that involves two main conformational states of the transporter protein: an outward open state in which the substrate is accessible to the protein only from the periplasmic side, and an inward open state in which the periplasmic entrance is closed, but the cytoplasmic half channel is open, thus, allowing the substrate to diffuse into the cell. Recent crystal structures of LacY from E. coli with [132] and without [137] lactose and a large body of experimental data have laid the basis for computational investigation of the lactose/proton co-transport mechanism of LacY. Using NAMD [44] we have initiated molecular dynamics simulations [138] of LacY investigating key steps of the transport scheme. A model of LacY embedded in a fully hydrated lipid bilayer, which provides a native environment for the protein and, thus, allows the system to evolve conformationally, was used for simulations. In order to properly describe the conformational response of lacY, the protein needs to be modeled in its natural environment, i.e., embedded in a sufficiently hydrated lipid bilayer, which results in system sizes of 130,000 atoms or more. The results analyzed by VMD [49] strongly implicate the residue Glu269 as one of the main proton translocation sites, whose protonation state controls several key steps of the transport process. A critical salt bridge between Glu269 and Arg144 was found to keep the cytoplasmic entrance open, however, via a different mechanism from the currently accepted model. After protonation of Glu269, this salt bridge was found to break, and Arg144 to move away from Glu269 establishing a new salt bridge with Glu126. Furthermore, the displacement of Arg144, and consequently of water molecules from the interdomain region, was seen to initiate the closing of the cytoplasmic entrance (reduction of 4 Angstroms in diameter 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. This is one of the first MD studies of a membrane transporter that challenge membrane protein simulations due to the complexity of their function. Revealing the mechanism of co-transport of protons and sugar molecules in LacY requires long simulations of many intermediate states representing different protonation states that are involved in the transport cycle. To study molecular and energetic details of the transport process, the Resource is currently using SMD [60] to induce the lactose permeation through the protein.
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