Resolving mechanistic details of peptide transport across membranes using crystallographic and non-crystallographic structural biology approaches

Cells are enveloped by a membrane barrier composed of lipids and proteins that keep useful materials inside the cell and exclude harmful, toxic compounds from entering. Some of the proteins that residue in the membrane have evolved to function as transport machines, shuttling essential nutrients into the cell and exporting waste products. Understanding how these transport proteins (transporters) function is of major biotechnological and medical significance, as many of these proteins function abnormally in diseases such as cancer, which require cells to take up many more nutrients than surrounding tissue.

Proteins adopt a variety of different states which enable them to carry out their specific tasks in cells. However, to date the biomedical science community has largely focused their efforts on determining the three-dimensional structure of transporters using the well-established technique of X-ray protein crystallography. The structures represent static snapshots but fail to provide information on the dynamics of these proteins.

Our research project aims at addressing a major conceptual gap in the field, by understanding the dynamics of transport and how lipids present in the membrane impact on the structure and function of transporters. We will use the latest techniques in biological spectroscopy to map out the variety of structural states adopted by an important family of nutrient transporters responsible for the uptake of peptides into the cell. Our methodology will be to label these proteins at selected positions and to measure the distance between the labels in native lipid environments. Using the crystal structures we have already obtained, and new ones to be resolved here, we will measure the changes in these distances as the proteins move peptides across the membrane. We will be able to model the structural changes taking place during function, to understand in much more detail how nutrients and small molecules can be selectively transported into the cell for further use in metabolism and cell function.

This work has significant implications for not only metabolic processes, especially in disease conditions, of which there are many, but also in the use of these proteins to deliver drugs into a cell as well as use these proteins in biotechnological ways to allow cells to make selected compounds for use in industry and pharamacology, which are long term aims.

Structural biology in a functionally supporting environment is often a technical challenge, especially for membrane proteins. Here, we plan to resolve conformational changes within a membrane-embedded secondary transporter under the influence of a proton motive force (pmf). Starting with crystal structures, we will reconstitute a transporter into membranes with different lipid composition and generate pmfs and substrate gradients to measure transport activity, as already shown for one example by us (Parker et al., (2014) eLife, Dec. 2:3), and then use ESR DEER to determine conformational changes within the protein during the transport cycle using spectroscopy - one example of this approach has just been published by us in a collaborative project for DETERGENT solubilized transporter (Fowler, et al., (2015) Structure, 23:290:301). Additionally, the substrate binding site environment will be described electronically using NMR to probe occluded and transporting substrate from NMR labels on the substrate and from labels at specific residues in the putative binding site environment, from crystal structures - backbone to substrate distances and dynamic (flexibility) details will also be deduced.

We will use our knowledge (some crystal structures) of the POT family to address specifically the role that lipids and the proton electrochemical gradient have on directing the dynamics and conformational excursions of these proteins. We have taken a technically demanding route, employing crystallography and site-directed (cysteine) spin label EPR spectroscopy to map out the conformational dynamics of these proteins in defined lipid environments. Q-band ESR DEER is relatively new (not many instruments exist), giving technically a major improvement (x10 over X-band) in s/n, and much longer T2s. Underpinned by data simulation and MD (Brazil), quantitative kinetics and conformational models will be generated for membrane embedded transporters, and any lipid specificity explained.

Here, we will resolve the molecular mechanism of peptide transport across biomembranes under the influence of a proton motive force and in different lipid environments, using static crystal structures as starting points and functional assays. Conformational changes within a transporter will be determined using spectroscopic methods (ESR DEER), and the substrate binding site interactions resolved using solid state NMR for bound substrate. The dynamic equilibrium between various transport cycle intermediates, will also be determined, and molecular dynamics will underpin the descriptions driven by experimental data, and dynamic and conformational analyses of spectroscopic data simulated to yield quantitative descriptions spatially and temporally. With the pivotal role these, and homologues transporters play in nutrition, as well as their potential for delivery selectively of drugs and their biotechnological potential, this work falls very well within the priority of areas of "health and welfare", "molecules cells and industrial biotechnology" and "animal disease".

Commercial Sector:
Even though transporters are pivotal to cellular biochemistry and human metabolism, we know very little about how they function at a molecular level. Thus, understanding the implications for human health when they malfunction is essential for therapies. Here, we will impact on those biotechnology and drug design activities that require molecular detail for addressing metabolic targets. In particular, the promiscuity of transporters is something that can be exploited, and peptide transporters in particular, have the potential to be drug transporters. So drug delivery is significantly impacted by this new information about mechanism.

Health Sector:
Current prevalence of obesity and diabetes can be a consequence of metabolic disorder at various levels. Transporters are the major way in which nutrition is taken up, and so this work has a bearing on both therapies and management of metabolism and uptake of nutrients.

Antimicrobial resistance:
Finally, bacterial transporters have the potential to be targets for antibiotics, if selective uptake can be designed into the antibiotic. Although promiscuous in substrate uptake by nature, understanding the nature of this promiscuity may well shed light on how to exploit it for antimicrobial delivery. Where relevant, IP protection will be sought via ISIS
Innovation (a wholly owned subsidiary of the University of Oxford) to protect potentially sensitive information.


Wider Public:
Increased public understanding is an important benefit for UK society as a whole. We will communicate our findings to the public and target as diverse an audience as possible by performing outreach activities related to the project in schools, museums and science open days. We will also engage with the press via the Oxford University press office.