High pressure promotes binding of the allosteric inhibitor Zn2+‐cyclen in crystals of activated H‐Ras

In this work, we experimentally investigate the potency of high pressure to drive a protein toward an excited state where an inhibitor targeted for this state can bind. Ras proteins are small GTPases cycling between active GTP‐bound and inactive GDP‐bound states. Various states of GTP‐bound Ras in active conformation coexist in solution, amongst them, state 2 which binds to effectors, and state 1, weakly populated at ambient conditions, which has a low affinity for effectors. Zn2+‐cyclen is an allosteric inhibitor of Ras protein, designed to bind specifically to the state 1. In H‐Ras(wt).Mg2+.GppNHp crystals soaked with Zn2+‐cyclen, no binding could be observed, as expected in the state 2 conformation which is the dominant state at ambient pressure. Interestingly, Zn2+‐cyclen binding is observed at 500 MPa pressure, close to the nucleotide, in Ras protein that is driven by pressure to a state 1 conformer. The unknown binding mode of Zn2+‐cyclen to H‐Ras can thus be fully characterized in atomic details. As a more general conjunction from our study, high pressure x‐ray crystallography turns out to be a powerful method to induce transitions allowing drug binding in proteins that are in low‐populated conformations at ambient conditions, enabling the design of specific inhibitors.