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ATP binding and ATP hydrolysis in full-length MsbA monitored via time-resolved Fourier transform infrared spectroscopy

  • Daniel Mann ORCID logo , Kristin Labudda ORCID logo , Sophie Zimmermann , Kai Ulrich Vocke ORCID logo , Raphael Gasper ORCID logo , Carsten Kötting ORCID logo EMAIL logo und Eckhard Hofmann ORCID logo EMAIL logo
Veröffentlicht/Copyright: 27. April 2023
Biological Chemistry
Aus der Zeitschrift Biological Chemistry Band 404 Heft 7

Abstract

The essential Escherichia coli ATPase MsbA is a lipid flippase that serves as a prototype for multi drug resistant ABC transporters. Its physiological function is the transport of lipopolisaccharides to build up the outer membranes of Gram-negative bacteria. Although several structural and biochemical studies of MsbA have been conducted previously, a detailed picture of the dynamic processes that link ATP hydrolysis to allocrit transport remains elusive. We report here for the first time time-resolved Fourier transform infrared (FTIR) spectroscopic measurements of the ATP binding and ATP hydrolysis reaction of full-length MsbA and determined reaction rates at 288 K of k 1 = 0.49 ± 0.28 s−1 and k 2 = 0.014 ± 0.003 s−1, respectively. We further verified these rates with photocaged NPEcgAppNHp where only nucleotide binding was observable and the negative mutant MsbA-H537A that showed slow hydrolysis (k 2 < 2 × 10−4 s−1). Besides single turnover kinetics, FTIR measurements also deliver IR signatures of all educts, products and the protein. ADP remains protein-bound after ATP hydrolysis. In addition, the spectral changes observed for the two variants MsbA-S378A and MsbA-S482A correlated with the loss of hydrogen bonding to the γ-phosphate of ATP. This study paves the way for FTIR-spectroscopic investigations of allocrite transport in full-length MsbA.

Keywords: ABC transporter; ATP hydrolysis; FTIR; integral membrane protein
Corresponding authors: Carsten Kötting, Ruhr University Bochum, Department of Biophysics, Universitätsstraße 150, D-44780 Bochum, Germany; and Ruhr University Bochum, Center for Protein Diagnostics, Biospectroscopy, Universitätsstraße 150, D-44780 Bochum, Germany, E-mail: ; and Eckhard Hofmann, Ruhr University Bochum, Protein Crystallography, Department of Biophysics, Universitätsstraße 150, D-44780 Bochum, Germany, E-mail:

Funding source: Deutsche Forschungsgemeinschaft

Award Identifier / Grant number: SFB642, TP A22 and TP A1

Acknowledgments

We would like to thank Meike Priehn, Fabian Zeipert, Vanessa Granitzka and Jenny Kleinmann who contributed to this project within their practical courses. We thank Prof. Dr. Klaus Gerwert for continuing support during the project.

  1. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

  2. Research funding: We acknowledge financial support from the Deutsche Forschungsgemeinschaft: CK and EH were funded withing the SFB642, TP A1 and TP A22, respectively, and within the Research Training Group GRK2341 “MiCon”. CK also acknowledges funding by grant 321722360.

  3. Conflict of interest statement: The authors declare no conflicts of interest regarding this article.

References

Allin, C. and Gerwert, K. (2001). Ras catalyzes GTP hydrolysis by shifting negative charges from γ- to β-phosphate as revealed by time-resolved FTIR difference spectroscopy. Biochemistry 40: 3037–3046, https://doi.org/10.1021/bi0017024.Suche in Google Scholar PubMed

Andersen, J.P., Vestergaard, A.L., Mikkelsen, S.A., Mogensen, L.S., Chalat, M., and Molday, R.S. (2016). P4-ATPases as phospholipid flippases—structure, function, and enigmas. Front. Physiol. 7: 1–23, https://doi.org/10.3389/fphys.2016.00275.Suche in Google Scholar PubMed PubMed Central

Angiulli, G., Dhupar, H.S., Suzuki, H., Wason, I.S., Hoa, F.D.V., and Walz, T. (2020). New approach for membrane protein reconstitution into peptidiscs and basis for their adaptability to different proteins. eLife 9: e53530, https://doi.org/10.7554/elife.53530.Suche in Google Scholar PubMed PubMed Central

Borbat, P.P., Surendhran, K., Bortolus, M., Zou, P., Freed, J.H., and Mchaourab, H.S. (2007). Conformational motion of the ABC transporter MsbA induced by ATP hydrolysis. PLoS Biol. 5: 2211–2219, https://doi.org/10.1371/journal.pbio.0050271.Suche in Google Scholar PubMed PubMed Central

Bordignon, E., Seeger, M.A., Galazzo, L., and Meier, G. (2020). From in vitro towards in situ: structure-based investigation of ABC exporters by electron paramagnetic resonance spectroscopy. FEBS Lett. 594: 3839–3856, https://doi.org/10.1002/1873-3468.14004.Suche in Google Scholar PubMed

Chen, J., Lu, G., Lin, J., Davidson, A.L., and Quiocho, F.A. (2003). A tweezers-like motion of the ATP-binding cassette dimer in an ABC transport cycle. Mol. Cell 12: 651–661, https://doi.org/10.1016/j.molcel.2003.08.004.Suche in Google Scholar PubMed

Davidson, A.L., Dassa, E., Orelle, C., and Chen, J. (2008). Structure, function, and evolution of bacterial ATP-binding cassette systems. Microbiol. Mol. Biol. Rev. 72: 317–364, https://doi.org/10.1128/mmbr.00031-07.Suche in Google Scholar PubMed PubMed Central

Deng, H., Wang, J., Callender, R., and Ray, W.J. (1998). Relationship between bond stretching frequencies and internal bonding for [16O4]- and [18O4]Phosphates in aqueous solution. J. Phys. Chem. B 102: 3617–3623, https://doi.org/10.1021/jp973314q.Suche in Google Scholar

Diederichs, K., Diez, J., Greller, G., Müller, C., Breed, J., Schnell, C., Vonrhein, C., Boos, W., and Welte, W. (2000). Crystal structure of MalK, the ATPase subunit of the trehalose/maltose ABC transporter of the archaeon Thermococcus litoralis. EMBO J. 19: 5951–5961, https://doi.org/10.1093/emboj/19.22.5951.Suche in Google Scholar PubMed PubMed Central

Dong, J., Yang, G., and Mchaourab, H.S. (2005). Structural basis of energy transduction in the transport cycle of MsbA. Science 308: 1023–1028, https://doi.org/10.1126/science.1106592.Suche in Google Scholar PubMed

Dowhan, W. (1997). Molecular basis for membrane phospholipid diversity: why are there so many lipids? Annu. Rev. Biochem. 66: 199–232, https://doi.org/10.1146/annurev.biochem.66.1.199.Suche in Google Scholar PubMed

Dreier, M.-A., Althoff, P., Norahan, M.J., Tennigkeit, S.A., El-Mashtoly, S.F., Lübben, M., Kötting, C., Rudack, T., and Gerwert, K. (2021). Time-resolved spectroscopic and electrophysiological data reveal insights in the gating mechanism of anion channel rhodopsin. Commun. Biol. 4: 1–10, https://doi.org/10.1038/s42003-021-02101-5.Suche in Google Scholar PubMed PubMed Central

Galazzo, L., Meier, G., Timachi, M.H., Hutter, C.A.J., Seeger, M.A., and Bordignon, E. (2020). Spin-labeled nanobodies as protein conformational reporters for electron paramagnetic resonance in cellular membranes. Proc. Natl. Acad. Sci. U.S.A. 117: 2441–2448, https://doi.org/10.1073/pnas.1913737117.Suche in Google Scholar PubMed PubMed Central

Galazzo, L., Meier, G., Januliene, D., Parey, K., De Vecchis, D., Striednig, B., Hilbi, H., Schäfer, L.V., Kuprov, I., Moeller, A., et al.. (2022a). The ABC transporter MsbA adopts the wide inward-open conformation in E. coli cells. Sci. Adv. 8: eabn6845, https://doi.org/10.1126/sciadv.abn6845.Suche in Google Scholar PubMed PubMed Central

Galazzo, L., Teucher, M., and Bordignon, E. (2022b). Chapter Four – orthogonal spin labeling and pulsed dipolar spectroscopy for protein studies. In: Britt, R.D. (Ed.), Methods in enzymology, advances in biomolecular EPR. Academic Press: Cambridge, pp. 79–119.10.1016/bs.mie.2022.02.004Suche in Google Scholar PubMed

Gerwert, K. (1999). Molecular reaction mechanisms of proteins monitored by time-resolved FTIR-spectroscopy. Biol. Chem. 380: 931–935, https://doi.org/10.1515/bc.1999.115.Suche in Google Scholar PubMed

Göddeke, H., Timachi, M.H., Hutter, C.A.J., Galazzo, L., Seeger, M.A., Karttunen, M., Bordignon, E., and Schäfer, L.V. (2018). Atomistic mechanism of large-scale conformational transition in a heterodimeric ABC exporter. J. Am. Chem. Soc. 140: 4543–4551, https://doi.org/10.1021/jacs.7b12944.Suche in Google Scholar PubMed

Higgins, C.F. (1995). The ABC of channel regulation. Cell 82: 693–696, https://doi.org/10.1016/0092-8674(95)90465-4.Suche in Google Scholar PubMed

Ho, H., Miu, A., Alexander, M.K., Garcia, N.K., Oh, A., Zilberleyb, I., Reichelt, M., Austin, C.D., Tam, C., Shriver, S., et al.. (2018). Structural basis for dual-mode inhibition of the ABC transporter MsbA. Nature 557: 196–201, https://doi.org/10.1038/s41586-018-0083-5.Suche in Google Scholar PubMed

Hohl, M., Hürlimann, L.M., Böhm, S., Schöppe, J., Grütter, M.G., Bordignon, E., and Seeger, M.A. (2014). Structural basis for allosteric cross-talk between the asymmetric nucleotide binding sites of a heterodimeric ABC exporter. Proc. Natl. Acad. Sci. U.S.A. 111: 11025–11030, https://doi.org/10.1073/pnas.1400485111.Suche in Google Scholar PubMed PubMed Central

Jones, P.M., O’Mara, M.L., and George, A.M. (2009). ABC transporters: a riddle wrapped in a mystery inside an enigma. Trends Biochem. Sci. 34: 520–531, https://doi.org/10.1016/j.tibs.2009.06.004.Suche in Google Scholar PubMed

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., et al.. (2020). Structural kinetics of MsbA investigated by stopped-flow time-resolved small-angle X-ray scattering. Structure 28: 348.e3–354.e3, https://doi.org/10.1016/j.str.2019.12.001.Suche in Google Scholar PubMed

Kaur, H., Abreu, B., Akhmetzyanov, D., Lakatos-Karoly, A., Soares, C.M., Prisner, T., and Glaubitz, C. (2018). Unexplored nucleotide binding modes for the ABC exporter MsbA. J. Am. Chem. Soc. 140: 14112–14125, https://doi.org/10.1021/jacs.8b06739.Suche in Google Scholar PubMed

Kehlenbeck, D.-M., Traore, D.A.K., Josts, I., Sander, S., Moulin, M., Haertlein, M., Prevost, S., Forsyth, V.T., and Tidow, H. (2022). Cryo-EM structure of MsbA in saposin-lipid nanoparticles (Salipro) provides insights into nucleotide coordination. FEBS J. 289: 2959–2970, https://doi.org/10.1111/febs.16327.Suche in Google Scholar PubMed

Kötting, C. and Gerwert, K. (2005). Proteins in action monitored by time-resolved FTIR spectroscopy. ChemPhysChem 6: 881–888, https://doi.org/10.1002/cphc.200400504.Suche in Google Scholar PubMed

Kötting, C., Kallenbach, A., Suveyzdis, Y., Wittinghofer, A., and Gerwert, K. (2008). The GAP arginine finger movement into the catalytic site of Ras increases the activation entropy. Proc. Natl. Acad. Sci. U.S.A. 105: 6260–6265, https://doi.org/10.1073/pnas.0712095105.Suche in Google Scholar PubMed PubMed Central

Kuhne, J., Eisenhauer, K., Ritter, E., Hegemann, P., Gerwert, K., and Bartl, F. (2014). Early Formation of the ion-conducting pore in channelrhodopsin-2. Angew. Chem., Int. Ed. 4953–4957, https://doi.org/10.1002/anie.201410180.Suche in Google Scholar PubMed

Kunjachan, S., Rychlik, B., Storm, G., Kiessling, F., and Lammers, T. (2013). Multidrug resistance: physiological principles and nanomedical solutions. Adv. Drug Deliv. Rev. Nanotechnol. Drug Resist. 65: 1852–1865, https://doi.org/10.1016/j.addr.2013.09.018.Suche in Google Scholar PubMed PubMed Central

Mann, D., Güldenhaupt, J., Schartner, J., Gerwert, K., and Kötting, C. (2018). The protonation states of GTP and GppNHp in Ras proteins. J. Biol. Chem. 293: 3871–3879, https://doi.org/10.1074/jbc.ra117.001110.Suche in Google Scholar

Mann, D., Höweler, U., Kötting, C., and Gerwert, K. (2017). Elucidation of single hydrogen bonds in GTPases via experimental and theoretical infrared spectroscopy. Biophys. J. 112: 66–77, https://doi.org/10.1016/j.bpj.2016.11.3195.Suche in Google Scholar PubMed PubMed Central

Mann, D., Teuber, C., Tennigkeit, S.A., Schröter, G., Gerwert, K., and Kötting, C. (2016). Mechanism of the intrinsic arginine finger in heterotrimeric G proteins. Proc. Natl. Acad. Sci. U.S.A. 113: E8041–E8050, https://doi.org/10.1073/pnas.1612394113.Suche in Google Scholar PubMed PubMed Central

Meer, G.van (2011). Dynamic transbilayer lipid asymmetry. Cold Spring Harb. Perspect. Biol. 3: a004671, https://doi.org/10.1101/cshperspect.a004671.Suche in Google Scholar PubMed PubMed Central

Mi, W., Li, Y., Yoon, S.H., Ernst, R.K., Walz, T., and Liao, M. (2017). Article Structural basis of MsbA-mediated lipopolysaccharide transport. Nature 549: 233–237, https://doi.org/10.1038/nature23649.Suche in Google Scholar PubMed PubMed Central

Padayatti, P.S., Lee, S.C., Stanfield, R.L., Wen, P.-C., Tajkhorshid, E., Wilson, I.A., and Zhang, Q. (2019). Structural insights into the lipid A transport pathway in MsbA. Structure 27: 1114.e3–1123.e3, https://doi.org/10.1016/j.str.2019.04.007.Suche in Google Scholar PubMed PubMed Central

Park, C.H. and Givens, R.S. (1997). New photoactivated protecting groups. 6. p-Hydroxyphenacyl: a phototrigger for chemical and biochemical Probes1, 2. J. Am. Chem. Soc. 119: 2453–2463, https://doi.org/10.1021/ja9635589.Suche in Google Scholar

Prieß, M., Göddeke, H., Groenhof, G., and Schäfer, L.V. (2018). Molecular mechanism of ATP hydrolysis in an ABC transporter. ACS Cent. Sci. 4: 1334–1343, https://doi.org/10.1021/acscentsci.8b00369.Suche in Google Scholar PubMed PubMed Central

Raetz, C.R.H., Reynolds, C.M., Trent, M.S., and Bishop, R.E. (2007). Lipid A modification systems in gram-negative bacteria. Annu. Rev. Biochem. 76: 295–329, https://doi.org/10.1146/annurev.biochem.76.010307.145803.Suche in Google Scholar PubMed PubMed Central

Rempel, S., Gati, C., Nijland, M., Thangaratnarajah, C., Karyolaimos, A., Gier, J.W.de, Guskov, A., and Slotboom, D.J. (2020). A mycobacterial ABC transporter mediates the uptake of hydrophilic compounds. Nature 580: 409–412, https://doi.org/10.1038/s41586-020-2072-8.Suche in Google Scholar PubMed

Rudack, T., Jenrich, S., Brucker, S., Vetter, I.R., Gerwert, K., and Kötting, C. (2015). Catalysis of GTP hydrolysis by small GTPases at atomic detail by integration of X-ray crystallography, experimental, and theoretical IR spectroscopy. J. Biol. Chem. 290: 24079–24090, https://doi.org/10.1074/jbc.m115.648071.Suche in Google Scholar

Ruiz, N., Kahne, D., and Silhavy, T.J. (2009). Transport of lipopolysaccharide across the cell envelope: the long road of discovery. Nat. Rev. Microbiol. 7: 677–683, https://doi.org/10.1038/nrmicro2184.Suche in Google Scholar PubMed PubMed Central

Schröter, G., Mann, D., Kötting, C., and Gerwert, K. (2015). Integration of fourier transform infrared spectroscopy, fluorescence spectroscopy, steady-state kinetics and molecular dynamics simulations of Gαi1 distinguishes between the GTP hydrolysis and GDP release mechanism. J. Biol. Chem. 290: 17085–17095, https://doi.org/10.1074/jbc.m115.651190.Suche in Google Scholar

Schultz, K.M., Merten, J.A., and Klug, C.S. (2011). Characterization of the E506Q and H537A dysfunctional mutants in the E. coli ABC transporter MsbA. Biochemistry 50: 3599–3608, https://doi.org/10.1021/bi101666p.Suche in Google Scholar PubMed PubMed Central

Sebastian, T.T., Baldridge, R.D., Xu, P., and Graham, T.R. (2012). Phospholipid flippases: building asymmetric membranes and transport vesicles. Biochim. Biophys. Acta Mol. Cell Biol. Lipids 1821: 1068–1077, https://doi.org/10.1016/j.bbalip.2011.12.007.Suche in Google Scholar PubMed PubMed Central

Sharom, F.J. (2011). The P-glycoprotein multidrug transporter. Essays Biochem. 50: 161–178, https://doi.org/10.1042/bse0500161.Suche in Google Scholar PubMed

Smith, P.C., Karpowich, N., Millen, L., Moody, J.E., Rosen, J., Thomas, P.J., and Hunt, J.F. (2002). ATP binding to the motor domain from an ABC transporter drives formation of a nucleotide sandwich dimer. Mol. Cell 10: 139–149, https://doi.org/10.1016/s1097-2765(02)00576-2.Suche in Google Scholar PubMed PubMed Central

Spadaccini, R., Kaur, H., Becker-Baldus, J., and Glaubitz, C. (2018). The effect of drug binding on specific sites in transmembrane helices 4 and 6 of the ABC exporter MsbA studied by DNP-enhanced solid-state NMR. Biochim. Biophys. Acta Biomembr. 1860: 833–840, https://doi.org/10.1016/j.bbamem.2017.10.017.Suche in Google Scholar PubMed

Syberg, F., Suveyzdis, Y., Kötting, C., Gerwert, K., and Hofmann, E. (2012). Time-resolved fourier transform infrared spectroscopy of the nucleotide-binding domain from the ATP-binding cassette transporter MsbA ATP hydrolysis is the rate-limiting step in the catalytic cycle. J. Biol. Chem. 287: 23923–23931, https://doi.org/10.1074/jbc.m112.359208.Suche in Google Scholar

Szöllősi, D., Rose-Sperling, D., Hellmich, U.A., and Stockner, T. (2018). Comparison of mechanistic transport cycle models of ABC exporters. Biochim. Biophys. Acta Biomembr. 1860: 818–832, https://doi.org/10.1016/j.bbamem.2017.10.028.Suche in Google Scholar PubMed PubMed Central

Thélot, F.A., Zhang, W., Song, K., Xu, C., Huang, J., and Liao, M. (2021). Distinct allosteric mechanisms of first-generation MsbA inhibitors. Science 374: 580–585, https://doi.org/10.1126/science.abi9009.Suche in Google Scholar PubMed PubMed Central

Verma, V.A., Wang, L., Labadie, S.S., Liang, J., Sellers, B.D., Wang, J., Dong, L., Wang, Q., Zhang, S., Xu, Z., et al.. (2022). Discovery of inhibitors of the lipopolysaccharide transporter MsbA: from a screening hit to potent wild-type gram-negative activity. J. Med. Chem. 65: 4085–4120, https://doi.org/10.1021/acs.jmedchem.1c01909.Suche in Google Scholar PubMed

Wang, J.H., Xiao, D.G., Deng, H., Callender, R., and Webb, M.R. (1998). Vibrational study of phosphate modes in GDP and GTP and their interaction with magnesium in aqueous solution. Biospectroscopy 4: 219–227, https://doi.org/10.1002/(sici)1520-6343(1998)4:4<219::aid-bspy1>3.0.co;2-y.10.1002/(SICI)1520-6343(1998)4:4<219::AID-BSPY1>3.0.CO;2-YSuche in Google Scholar

Ward, A., Reyes, C.L., Yu, J., Roth, C.B., and Chang, G. (2007). Flexibility in the ABC transporter MsbA: alternating access with a twist. Proc. Natl. Acad. Sci. U.S.A. 104: 19005–19010, https://doi.org/10.1073/pnas.0709388104.Suche in Google Scholar

Wilkens, S. (2015). Structure and mechanism of ABC transporters. F1000Prime Rep 7: 7–14, https://doi.org/10.12703/P7-14.Suche in Google Scholar

Zaitseva, J., Jenewein, S., Jumpertz, T., Holland, I.B., and Schmitt, L. (2005). H662 is the linchpin of ATP hydrolysis in the nucleotide-binding domain of the ABC transporter HlyB. EMBO J. 24: 1901–1910, https://doi.org/10.1038/sj.emboj.7600657.Suche in Google Scholar

Supplementary Material

This article contains supplementary material (https://doi.org/10.1515/hsz-2023-0122).

Received: 2023-02-05
Accepted: 2023-04-07
Published Online: 2023-04-27
Published in Print: 2023-06-27

© 2023 Walter de Gruyter GmbH, Berlin/Boston

Artikel in diesem Heft

  1. Frontmatter
  2. Highlight: Membrane Proteins from Structure to Function
  3. The Rauischholzhausen Transport Colloquium: membrane proteins from structure to function
  4. Determination of membrane protein orientation upon liposomal reconstitution down to the single vesicle level
  5. Interaction of RTX toxins with the host cell plasma membrane
  6. Interactions of Na+/taurocholate cotransporting polypeptide with host cellular proteins upon hepatitis B and D virus infection: novel potential targets for antiviral therapy
  7. Mycobacterial type VII secretion systems
  8. Lipid exchange among electroneutral Sulfo-DIBMA nanodiscs is independent of ion concentration
  9. Membrane-anchored substrate binding proteins are deployed in secondary TAXI transporters
  10. ATP binding and ATP hydrolysis in full-length MsbA monitored via time-resolved Fourier transform infrared spectroscopy
https://doi.org/10.1515/hsz-2023-0122
Schlagwörter für diesen Artikel
ABC transporter; ATP hydrolysis; FTIR; integral membrane protein

Artikel in diesem Heft

  1. Frontmatter
  2. Highlight: Membrane Proteins from Structure to Function
  3. The Rauischholzhausen Transport Colloquium: membrane proteins from structure to function
  4. Determination of membrane protein orientation upon liposomal reconstitution down to the single vesicle level
  5. Interaction of RTX toxins with the host cell plasma membrane
  6. Interactions of Na+/taurocholate cotransporting polypeptide with host cellular proteins upon hepatitis B and D virus infection: novel potential targets for antiviral therapy
  7. Mycobacterial type VII secretion systems
  8. Lipid exchange among electroneutral Sulfo-DIBMA nanodiscs is independent of ion concentration
  9. Membrane-anchored substrate binding proteins are deployed in secondary TAXI transporters
  10. ATP binding and ATP hydrolysis in full-length MsbA monitored via time-resolved Fourier transform infrared spectroscopy
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