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7 3 I H I G H L I G H T S 2 0 2 1

Fig. 58: The TR-XSS models show the early stages of AdK activation. a) Open (white) and closed (magenta) crystal structures. b) Ground and excited TR-XSS models.

PRINCIPAL PUBLICATION AND AUTHORS

Tracking the ATP-binding response in adenylate kinase in real time, F. Orädd (a), H. Ravishankar (a), J. Goodman (a), P. Rogne (a), L. Backman (a), A. Duelli (b), M.N. Pedersen (c), M. Levantino (c), M. Wulff (c), M. Wolf-Watz (a), M. Andersson (a), Sci. Adv. 7, eabi5514 (2021); https:/doi.org/10.1126/sciadv.abi5514 (a) Umeå University, Umeå (Sweden) (b) University of Copenhagen, Copenhagen (Denmark) (c) ESRF

REFERENCES

[1] H. Ravishankar et al., Sci. Adv. 6, eaaz0981 (2020). [2] F. Orädd et al., J. Membr. Biol. 254, 51-64 (2021).

methods is that changes in all interatomic distances are measured simultaneously in real time, which enables direct determination of ordering and cooperativity of the structural events. In addition, because the method is non-intrusive, risks associated with the introduction of labelling probes, for example, are minimised.

Time-resolved TR-XSS data covering a temporal range from 20 µs to 25 ms revealed the buildup of an intermediate state over time (Figure 57). As the sample conditions were chosen to induce closure of the two domain lobes upon ATP release, this closing event was modelled in order to find a structural representation of the 4.3-ms intermediate. While the open and closed crystal structures do not generate a satisfactory fit to the data, several structures obtained from molecular dynamics (MD) simulations showed excellent fits to the data. The structural ensemble that best represented the time-resolved data showed similar movements of both domains to close upon the ATP substrate and a high degree of local domain unfolding (Figure 58). The unfolding was also observed in nuclear magnetic resonance (NMR)

experiments. Hence, it was concluded that AdK closing is a cooperative mechanism involving both domains and probably triggered by local unfolding, in agreement with a cracking model that has been proposed to underlie global structural transformation, such as allostery, in proteins.

Time resolved scattering is a powerful tool to determine the structures of transient, high-energy intermediate states of proteins [2]. Since obtaining a structural solution to the low-resolution data relies on the existence of high- resolution structural data, the method is a complement to other structural biology techniques, such as cryogenic electron microscopy (cryo-EM). Also, indirect triggering methods will enable TR-XSS characterisation of a wide selection of protein targets in particular when considering developments of caged compounds, electric-field stimulation and synthetic photoswitches.