The Structural And Functional Research Of E. Coli YbgH

Active transport is of great importance for cellular process, including uptake of essential nutrients and ions from the surroundings or excretion of end products of metabolism and toxic substances. Active transport is the movement of substrate molecules across the cell membrane in the direction against their concentration gradient, i.e. moving from a region of lower concentration to a region of higher concentration. Unlike passive transport, active transport uses cellular energy. Based on the different sources of energy, the active transporter can be classified into two groups, primary active transporter and secondary active transporter. The primary active transporter involves the use of chemical energy, for example from ATP hydrolysis. The secondary transporters utilize the electrochemical potential of proton or sodium gradient to achieve ’uphill’ translocation of substrates across the membrane. Major facilitator superfamily (MFS) is the largest family of secondary active transporters. E. coli YbgH (Ec YbgH) belongs to the proton-dependent oligopeptide transporter (POTs) subfamily of MFS. Members of this subfamily are proton driven symporters. The substrates of YbgH includes di-peptides that have a positively charged sidechain at the C-terminal end.We solved the crystal structure of EcYbgH at3.4A resolution and studied its the function using cell-based transport assays. The structure of EcYbgH is stabilized in an inward-facing conformation. We found motif AL5-6(located between the TMs5and6) and conserved Asp44-Arg297salt bridge play important roles in stabilizing EcYbgH in the inward-facing state. Meanwhile, the canonical motif A located between the TMs2and3contributes to stabilization of the outward-facing conformation. When we introduced mutations to break one or two of the three interdomain interactions, the uptake activity of EcYbgH decreased significantly. However, when all three interdomain interactions are disrupted, the uptake activity was rescued to a level even higher than that observed for WT YbgH. These observations strongly support the notion that an energetic balance between the inward and outward states is essential for the transport activity of MFS in general. Over-stabilization of one of the two states would block efficient conformational changes. In addition, our cell-based transport activity assays demonstrate that the most conserved proton-titratable amino acid residue, Glu21, is important for transport activity, and that the inserted inter-domain TM-helix pair (HAB) may regulate the conformational change or EcYbgH.In addition to the YbgH study, I was involved in serveral other membrane protein projects, including YajR, CsgG, PgpB, and LptD/E among others. In these projects, my main contribution is computational work including phasing, refinement, figure preparation and other analyses. In these works, I overcame many difficulties, such as anistropic diffraction and low resolution refinement, and have learnt many useful techniques.