Structural investigation of Ca2+ signaling and transport across biological membranes
Final Report Abstract
Plasma-membrane Ca2+-ATPases expel Ca2+ from the cytoplasm and are key regulators of Ca2+ homeostasis in eukaryotes. They are autoinhibited under low Ca2+ concentrations. Calmodulin (CaM)- binding to a unique regulatory domain releases the autoinhibition and activates the pump. However, the structural basis for this activation, including the overall structure of this calcium pump and its complex with calmodulin, is unknown. We previously determined the high-resolution structure of calmodulin in complex with the regulatory domain of the plasma-membrane Ca2+-ATPase ACA8 and revealed a bimodular mechanism of calcium control in eukaryotes. During this Emmy Noether project, we showed that activation of ACA8 by CaM involves large conformational changes. Combining advanced modeling of neutron scattering data acquired from stealth nanodiscs and native mass spectrometry with detailed dissection of binding constants, we developed a structural model for the full-length ACA8 Ca2+ pump in its calmodulin-activated state illustrating a displacement of the regulatory domain from the core enzyme. In addition, we identified novel calmodulin-binding regions within the P2X7 ballast domain and found that binding of Ca2+-calmodulin (Ca2+-CaM) and GDP to P2X7 ballast domain are mutually exclusive and have opposite effects on the stability of the ballast domain. SAXS experiments indicate that CaM- binding disrupts the trimeric state of P2X7 ballast domain. Our results provide a possible framework for the regulation of the P2X7 receptor.
Publications
- (2016) Crystal Structure of a Group I Energy Coupling Factor Vitamin Transporter S Component in Complex with Its Cognate Substrate, Cell Chem Biol 23, 827-836
Josts, I., Almeida Hernandez, Y., Andreeva, A., and Tidow, H.
(See online at https://doi.org/10.1016/j.chembiol.2016.06.008) - (2017) Lipid-like Peptides can Stabilize Integral Membrane Proteins for Biophysical and Structural Studies, Chembiochem 18, 1735-1742
Veith, K., Martinez Molledo, M., Almeida Hernandez, Y., Josts, I., Nitsche, J., Low, C., and Tidow, H.
(See online at https://doi.org/10.1002/cbic.201700235) - (2018) Conformational States of ABC Transporter MsbA in a Lipid Environment Investigated by Small-Angle Scattering Using Stealth Carrier Nanodiscs, Structure 26, 1072-1079 e1074
Josts, I., Nitsche, J., Maric, S., Mertens, H. D., Moulin, M., Haertlein, M., Prevost, S., Svergun, D. I., Busch, S., Forsyth, V. T., and Tidow, H.
(See online at https://doi.org/10.1016/j.str.2018.05.007) - (2018) Structural basis for activation of plasma-membrane Ca(2+)-ATPase by calmodulin, Commun Biol 1, 206
Nitsche, J., Josts, I., Heidemann, J., Mertens, H. D., Maric, S., Moulin, M., Haertlein, M., Busch, S., Forsyth, V. T., Svergun, D. I., Uetrecht, C., and Tidow, H.
(See online at https://doi.org/10.1038/s42003-018-0203-7) - (2019) Novel CaM-binding motif in its NudT9H domain contributes to temperature sensitivity of TRPM2, Biochim Biophys Acta Mol Cell Res 1866, 1162-1170
Gattkowski, E., Johnsen, A., Bauche, A., Mockl, F., Kulow, F., Garcia Alai, M., Rutherford, T. J., Fliegert, R., and Tidow, H.
(See online at https://doi.org/10.1016/j.bbamcr.2018.12.010) - (2019) Ternary structure of the outer membrane transporter FoxA provides insights into TonB-mediated siderophore uptake, Elife 8
Josts, I., Veith, K., and Tidow, H.
(See online at https://doi.org/10.7554/elife.48528) - (2020) Structural Kinetics of MsbA Investigated by Stopped-Flow Time-Resolved Small-Angle X-Ray Scattering. Structure, 28(3): 348-354
Josts, I., Gao, Y., Monteiro, D.C.F., Niebling, S., Nitsche, J., Veith, K., Gräwert, T.W., Blanchet, C.E., Schroer, M.A., Huse, N., Pearson, A.R., Svergun, D.I., and Tidow, H.
(See online at https://doi.org/10.1016/j.str.2019.12.001) - (2021) Massive X-ray screening reveals two allosteric drug binding sites of SARS-CoV-2 main protease. Science. 2021 Apr 2:eabf7945
Günther, S., Reinke, P.Y.A., …, Tidow, H., …, and Meents, A.
(See online at https://doi.org/10.1126/science.abf7945) - (2021) Structural insights into a novel family of integral membrane siderophore reductases. Proc Natl Acad Sci USA, 118(34):e2101952118
Josts, I., Veith, K., Normant, V., Schalk, I.J., and Tidow, H.
(See online at https://doi.org/10.1073/pnas.2101952118)
