The structural and mechanistic basis of K(+) translocation by the KtrAB system
Final Report Abstract
The KtrAB system is a key player in bacterial osmoadaptation. However, its mode of transport was not understood and it was under debate whether KtrAB functions as channel or active transporter. While the KtrB subunit on its own takes up Na+ and K+ independent of each other and of ATP, the KtrAB system translocates K+ in a Na+- and ATP-dependent manner. In this project we aimed at answering which structural impact KtrA has on KtrB and which mechanism controls ion flux through the KtrAB complex. Although we could not solve a structure of the separate KtrB subunit, we could shed light on these two questions by solving a 6.6 Å cryo-EM structure of KtrAB with ADP bound. The structural model uncovered two extended helices of the KtrB dimer that penetrated deeply into the regulatory KtrA subunits. In comparison, in the previously solved ATP-bound structure these extended helices formed helical hairpins, which pointed away from the ion-translocating pathways. Accompanying EPR measurements confirmed the conformational changes and showed that the gates in the KtrB dimer are fixed in a closed conformation in the ADP-bound state while they get unlocked in the presence of ATP. However, a constantly open state could not be stabilized. In summary, based on the structural data we conclude that KtrAB in fact functions as nucleotide-dependent K+ channel for which we could uncover a previously unknown regulatory mechanism for ligand-induced action at a distance. We suggest that the main function of the KtrA subunits is to stably close KtrBs' pores. The establishment of a functional in vitro approach is pending. Furthermore, the investigation of structural and functional role of Na+ and the identification of potential additional factors that stabilize the activated, open conformation is needed.
Publications
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(2015) Functional diversity of the superfamily of K+ transporters to meet various requirements. Biol. Chem., 396: 1003-14
Diskowski, M., Mikusevic, V., Stock, C., Hänelt, I.
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(2017) Helical jackknives control the gates of the double-pore K+ uptake system KtrAB. eLife, 6: e24303
Diskowski, M., Mehdipour, A.R., Wunnicke, D., Mills, D., Mikusevic, V., Bärland, N., Hoffmann, J., Morgner, N., Steinhoff, H.J., Hummer, G., Vonck, J., Hänelt, I.
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(2017) The synergetic effects of combining structural biology and EPR spectroscopy. Crystals, 7: 117
Wunnicke, D. Hänelt, I.
