Coupling Chemical Energy with Protein Conformational Changes to Translocate Small Molecules Across Membranes

Technical Report · Sat Sep 21 00:00:00 EDT 2019

DOI:https://doi.org/10.2172/1563079· OSTI ID:1563079

Vermaas, Josh ^[1]; Tajkhorshid, Emad ^[1]; Rempe, Susan ^[2]

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1. Univ. of Illinois at Urbana-Champaign, IL (United States)
2. Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)

EmrE is a small, homodimeric membrane transporter that exploits the established pH gradient across the E. coli inner membrane to export polyaromatic cations that might otherwise inhibit cellular growth. While herculean efforts through experimental studies have established many fundamental facts about the specificity and rate of substrate transport in EmrE, the low resolution of the available structures have hampered efforts to tie those findings to the EmrE coupling mechanism between proton and small molecule substrates. Here we present a full three-dimensional structure of EmrE optimized against available cyro-EM data to delineate the critical interactions by which EmrE regulates its conformation. We use the generated structural model to conduct equilibrium and nonequilibrium molecular dynamics simulations to probe EmrE dynamics under different substrate loading states, representing different states in the transport cycle. The model is stable under extended simulation, and reveals that water dynamics within the EmrE lumen change substantially with the loading state. The water dynamics cause hydrogen bonding networks to shift radically when the protonation states change for a pair of solvent-exposed glutamate residues (E14) within the lumen of the transporter, which are proposed to act as proton binding sites during the transport cycle. One specific hydrogen bond from a tyrosine (Y60) of one monomer to a glutamate (E14) on the opposite monomer is especially critical, as it locks the protein conformation when the glutamate is deprotonated. Furthermore, the hydrogen bond provided by Y60 lowers the pK_a of the interacting glutamate relative to its partner on the opposite monomer such that it will protonate second, establishing the need for both glutamates to be protonated for the hydrogen bond to break and a substrate-free transition to take place.

• Research Organization: Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)
• Sponsoring Organization: USDOD; USDOE Laboratory Directed Research and Development (LDRD) Program
• DOE Contract Number: AC04-94AL85000
• OSTI ID: 1563079
• Report Number(s): SAND-2016-10448R; 648363
• Country of Publication: United States
• Language: English