Figure 1: Structure of the ligand bound oligopeptide transporter (POT) in the inward open conformation (Guettou et al., 2013).
Using biophysical and biochemical methods, the Löw group aim to enhance understanding of the structural basis for substrate recognition in peptide transporters.
Previous and current research
Cell membranes compartmentalise metabolic processes and present a selective barrier for permeation. Therefore, nutrient transport through the plasma membrane is essential to maintain homeostasis within the cell. Many proton-coupled secondary active transporters of the major facilitator superfamily (MFS) are involved in the accumulation of nutrients above extracellular levels in the cell. Structural and functional analyses of MFS transporters suggest an alternating-access mechanism for the transport of substrates across the membrane. Here the transporter adopts different conformational states, allowing the substrate binding site to face either side of the membrane. A full transport cycle involves at least three different conformational states – inward open, occluded and outward open –, with each of them in a ligand-bound and ligand-free state.
Proton coupled oligopeptide transporters of the PepT family (also known as the POT family) are responsible for the uptake of a range of different di- and tripeptides, derived from the digestion of dietary proteins, and are highly conserved in all kingdoms of life. The best studied members of this family include the two human peptide transporters, PepT1 and PepT2. Both are also of great pharmacological and pharmaceutical interest as they accept a number of drugs and amino acid-conjugated pro-drugs as substrates. A detailed understanding of the structural basis for substrate recognition can therefore help to convert pharmacologically active compounds into substrates for PepT1 and PepT2 and thus improve their absorption in the small intestine and subsequent distribution in the body. To this end we will study the proton-dependent oligopeptide transporter (POT) family using a combination of biochemical and biophysical methods. POTs share the canonical fold of MFS transporters with 12 predicted transmembrane helices each. There are currently no crystal structures available for any of the human PepT transporters, but the first bacterial PepT structures have recently been reported (including one from our lab).
Future projects and goals
- Structural and functional studies of the reaction cycle of bacterial POTs using X-ray crystallography.
- Structural and dynamic insights into the binding mode of POTs to peptides, drugs, and inhibitors.
- Functional expression, purification, biochemical characterisation and crystallisation of eukaryotic POTs.
Integral membrane proteins are a challenging class of proteins in terms of their structural and functional characterisation. Over the years we have developed and established new tools and a workflow for protein production and quality control of membrane proteins including functional assays (in whole cells or in reconstituted systems) with the major focus on nutrient uptake systems. The PETRA III synchrotron presents an excellent facility for us to continue this work.
Figure 2: Structural differences between the inward open and occluded state structures of the sugar transporter XylE. The structures were overlaid and the transmembrane helices (TM) are labelled and arrows designate changes in the position of the helices upon opening of the transporter towards the cytoplasm.