**5. Conclusions**

γ We herein reviewed magnetic-resonance approaches (in combination with additional data) to provide information at the atomic level on the binding of mimics of the different ATP forms present during the hydrolysis cycle. We investigated this for two ATP-fuelled proteins, an ABC transporter and a DNA helicase (Table 2), both driven by an ATPase motor domain. We showed that the ATP analogues mainly used for structural studies for such systems, AMPPNP and ATP-γ-S, are not suitable for the systems studied here, since both are hydrolysed by the proteins. Furthermore, we show that analogues which should induce the same state in the hydrolysis cycle can fail to do so, since they result in different conformations. We also discuss that some analogues can interfere with protein function, such as DNA binding for DnaB. NMR, and also EPR, are sensitive tools to assess the impact of different analogues for a given protein, a need that arises through the observation that they can have widely differing effects on different proteins. NMR spectroscopy could be of help in tracing minor differences both in the overall protein conformation and in the state of the phosphate groups. Here we showed

that solid-state NMR enabled revealing notable differences in the structural properties of closely related P-loop fold NTPases, namely the SF4 DnaB helicase and BmrA ABC-transporter, which both belong to the same division of ASCE-NTPases.

**Table 2.** Summary of the ATP analogue efficiencies for the two protein systems BmrA and DnaB. ++ indicates high efficiency, + moderate efficiency, - low efficiency and – not efficient.


**Supplementary Materials:** The following are available online [133–137].

**Author Contributions:** D.L. and R.C. prepared the samples. D.L. and T.W. performed the NMR experiments. N.W. and D.K. recorded the EPR experiments. M.I.K. and A.Y.M. analysed the protein structures. D.L., T.W., M.I.K., N.W., D.K., A.Y.M., B.H.M. and A.B. analysed the data. All authors contributed to the writing of the manuscript. D.L., T.W., B.H.M. and A.B. designed the research, and B.H.M. and A.B. supervised the project. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the ETH Career SEED-69 16-1 (T.W.) and the ETH Research Grant ETH-43 17-2 (T.W.), an ERC Advanced Grant (B.H.M., grant number 741863, FASTER), by the Swiss National Science Foundation (B.H.M., grant number 200020\_159707 and 200020-188711), the French Agence Nationale de Recherche (A.B., ANR-14-CE09-0024B, ANR-19-CE11-0023), the LABEX ECOFECT (A.B., ANR-11-LABX-0048) within the Université de Lyon program Investissements d'Avenir (A.B., ANR-11-IDEX-0007), the EvoCell Program of the Osnabrueck University (M.I.K.) and a grant from the Russian Science Foundation (A.Y.M., 17-14-01314).

**Acknowledgments:** We thank Gunnar Jeschke for his comments on the manuscript. D.L. and T.W. acknowledge helpful discussion with Marco E. Weber.

**Conflicts of Interest:** The authors declare no conflict of interest.
