M A T T E R A T E X T R E M E S

S C I E N T I F I C H I G H L I G H T S

2 2 H I G H L I G H T S 2 0 2 2 I

In the present study, high hydrostatic pressure crystallography was applied to the protein Ras, a small GTPase involved in a large number of cancers, making it a high-priority therapeutic target. Ras acts as a molecular switch between a GTP-bound active state and a GDP- bound inactive state, and possesses many conformational states allowing its interaction with many effectors and regulators. The structure of Ras in its dominant ground state corresponds to its state when it binds to an effector and is termed state 2(T). The structures of the different excited states were obtained mainly using mutants of the protein, thus prone to artifacts. Different states have been described, the state 1(T) when Ras interacts with guanine nucleotide exchange factors (GEF), the state 2(T)*-RBD when Ras interacts with the Ras-binding domain of GEF, and the state 1(0) for nucleotide-free Ras. In addition, Ras binds to the cell membrane, inducing allosteric modifications. This allosteric process was studied by the addition of exogenous chemical components, thus also subject to artifacts.

Data collection under high hydrostatic pressure using a diamond anvil cell specially designed for macromolecular crystallography with a large aperture of almost 90° was performed on beamlines ID15B and ID27. These experiments made it possible to reveal that pressurising Ras crystals to more than 300 MPa (3,000 atm) induces a transition from one state to another within the crystal itself, leading to more stable conformers. Analyses of the crystallographic structures of Ras at different pressures above the transition pressure allow to locate the segments that change from one state to another. Ras structure is similar to 2(T)*-RDB conformation at 500 MPa except one small segment (D30-T35) that adopts this conformation

Fig. 10: The representation of the three- dimensional structure of Ras at different pressures, coloured according to the states reached. The segments in the dominant ground state, the state 2(T), are coloured in green, those which change to the state 2(T)*-RBD in purple, to the state 1(T) in cyan, and to the state 1(0) in pink. The very mobile segment that corresponds to the intermediate states is coloured in red.

Fig. 11: The helix α2 of Ras under different pressures, with the tyrosine residue 71 in stick representation, coloured in grey at 0.1 MPa, green at 200 MPa, cyan at 500 MPa, pink at 650 MPa and orange at 90 MPa.

PRINCIPAL PUBLICATION AND AUTHORS

Equilibria between conformational states of the Ras oncogene protein revealed by high pressure crystallography, E. Girard (a), P. Lopes (b), M. Spoerner (b), A.C. Dhaussy (c), T. Prangé (d), H.R. Kalbitzer (b), N. Colloc h (e), Chem. Sci. 13, 2001-2010 (2022); https:/doi.org/10.1039/d1sc05488k (a) University Grenoble Alpes, CEA, CNRS, IBS, Grenoble (France) (b) Institute of Biophysics and Physical Biochemistry, University of Regensburg (Germany) (c) Normandie University, Ensicaen, CNRS, CRISTMAT UMR 6508, Caen (France) (d) CitCOM UMR 8038, CNRS, Paris-Cité University, Paris (France) (e) ISTCT UMR 6030, CNRS, University Caen-Normandie, Caen (France)

only at 900 MPa, a very mobile segment (A59-R68) that adopts variable intermediate conformations, and a long segment (D69-V109) that is similar to state 1(T) conformation at 500 MPa, to 2(T)*-RBD conformation at 650 MPa and to state 1(0) conformation at 900 MPa (Figure 10). As a consequence, residue-by-residue modifications have been precisely observed, revealing the structural plasticity involved in allosteric equilibria between conformers. In particular, the role of the residue tyrosine 71 as a sensor has been revealed, facing the core of the protein at 200 MPa and 650 MPa (Tyr in ), and oriented toward the bulk at 0.1 MPa, 500 MPa and 900 MPa (Tyr out ) (Figure 11).

Crystallography under high hydrostatic pressure has thus made it possible to analyse in detail, without any mutations nor addition of exogenous compounds, the conformational states adopted by Ras when it interacts with regulators of its activity, and to identify step-by-step the protein segments that drive the transition between states. This work opens the way to designing specific inhibitors of these states.