*2.5. MDM2 Exhibits a sMall Conformational Change upon Binding*

There is a small conformational rearrangement of the backbone (1.9 Å RMSD) between the apo (unbound) and holo (bound) crystal structures (1z1m [29] and 1ycr [30], respec-

tively), which opens up the cavity for binding. Sidechain rearrangement of the surface residues happens on a faster timescale, changing the surface accessibility to the binding cavity. In MELD×MD binding, the conformational freedom of MDM2 by using flat-bottom harmomic restraints on the *C<sup>α</sup>* around the holo structure to prevent unfolding in the replica exchange ladder (see methods). At high temperatures, we find that the protein is sampling conformations between 2–3.5 Åbackbone RMSD from the holo structure and a similar range (2.5–4.0 Å) with respect to the apo MDM2 structure (see Figure A9). At low temperatures, the thermal ensemble is narrower, with an RMSD in between 1 to 2 Åfrom the holo structure and 2–4 Å from the apo structure. The presence of the peptide binding for a significant amount of time to the active site further shifts the RMSD to lower values (see *pdiq* and *ATSP*-7041 in Figure A9). There are no restraints on the sidechains, in which fluctuations determine the open/closed state of the cavity. Reorientation of these sidechains is fast and adapts to the presence of the peptide near the active site.
