Structure and dynamics of Yersinia Adhesin A in a membrane by nuclear magnetic resonance and electron paramagnetic resonance spectroscopy

Yersinia enterocolitica (Y. entercolotica) is a bacterium involved in a number of food-borne diseases, including diarrhea, which is estimated to kill up to 1.5 million children each year in the developing world. In order for infection to occur, Y. enterocolitica produces a protein, Yersinia Adhesin A (YadA), which forms sticky, hair-like structures on the surface of the cell. These sticky molecules can attach to our gut, promoting bacterial colonization and infection. In this project, we are trying to understand WHAT YadA looks like, as well as HOW it reaches the cell surface. To do this, we are combining cutting edge techniques across all scientific disciplines (biology, chemistry, and physics) to study this protein, for the first time, within the cell at atomic detail. Understanding both what YadA looks like as well as how it reaches the cell surface is useful for rational drug design - we know Y. enterocolitica requires YadA to infect a human host, and disabling it can clear an infection. We have now genetically established how YadA reaches the cell surface by introducing point mutations that disrupt the folding of YadA so that it no longer maintains its 'sticky' nature, and does not reach the surface of the cell. We have also published a high impact paper on strategies to study the YadA structure at the resolution necessary for drug design, in a real cell membrane with a team of international collaborators based both here and in Germany. These projects have been been published in 4 major scientific articles and book chapters, and, and 5 presentations have been given on results related to this work at national and international conferences, as well as several three local presentations at UiO. We have also published both Norwegian and English versions on part of this work in the UiO newsletter Titan, where we have also liased with a scientific artist to provide graphical representations of how one can imagine YadA adheres to other molecules and surfaces in the human body (https://titan.uio.no/node/3331). This results of this project contributes specifically to the medical needs of combatting Y. enterocolitica diseases, and promoting childhood health within the developing world. In the longterm, a better understanding of bacterial adhesin has major implications in other fields of medical research (such as medical implants), as well in industry (fishing and shipping), where 'sticky bacteria' are a nuisance. Finally, the overarching goal of this project, to develop techniques to solve protein structures within the cellular milieu, has the potential to revolutionize how protein structures are solved, with major implications across all areas of medical and health sciences where understanding how a protein looks (and designing a molecule that can interact with it) is important.

We have published three papers, one book chapter, and given 4 talks at national and international conferences, and have disseminated our results in popular publications, in both norwegian and english. We have a current manuscript prepared for submission, and are in the late stages of final structure calculations of YadAM in the outer membrane, and have also begun site-specific dynamic measurements. We expect these projects to result in high-impact publications, and hope to expand with a methods paper to show generally how OMPs may be recombinantly expressed and measured in situ using the general methods that we have developed for YadAM, to increase the scope even more, showing how the techniques here are also valuable for instance in EM. During the course of this project we have also picked up several new collaborators both locally and internationally, as well as having expanded upon existing collaborations.

Membrane proteins are of particular pharmacological importance, with 30% of all drugs targeting them. In order to rationally design drugs to target a specific protein, its structure must first be solved at atomic level resolution. Nuclear magnetic resonance methods (NMR) are a way by which protein samples can be studied at the atomic level, either in solution (sNMR) or in the solid state (ssNMR). Yersinia Adhesin A (YadA) is a protein found in the membrane of Yersinia Enterocolitica, which is involved in a number of food-borne diseases including enterocolitis, acute enteritis, diarrhea, and mesenteric limphadentisis. Diarrhea alone is estimated to kill 1.5 million children under the age of five globally each year. YadA plays an important role in the ability of Y. Enterocolitica to colonize in a host, by aiding in the autotransport of a head domain to the cell surface that can stick to host tissues. Thus, rational drug design to inhibit host colonization by targeting the functional role of YadA could be vital to childrens's health, particularly in the developing world. A recent structure of the YadA anchor domain region was solved with the protein in the crystalline form. A 4-amino acid stretch of residues was identified that are thought to crucial in the mechanism by which YadA promotes colonization, and may serve as a potential pharmaceutical target. We wish to determine the mechanism by which autotransport occurs, by solving the structure of YadA in native or native-like lipid environment using sNMR and ssNMR. A YadA mutant that is proposed to inhibit the ability of Y. Enterocolotica to colonize host tissues will also be studied. A primary R&D challenge will be obtaining high quality NMR data in native and native-like membrane environments, and the preliminary amino-acid assignment of the NMR data. The assignment should be aided by existing ssNMR data of Yada in the crystalline form.