Molecular Dynamics Simulations of the Hypoxia-Inducible Factor PAS-B Domain Confirm That Internally Bound Water...

Hypoxia-inducible factors (HIFs) are heterodimeric transcription factors that are induced in many cancer types, promoting tumor-forming pathways. The HIF-2α PAS-B domain of the human HIF-2 complex contains a large buried hydrophilic cavity that is filled with eight ordered water molecules, making the domain a promising drug target. Recent work has led to the hypothesis that these buried water molecules act as an internal scaffold that stabilizes the buried cavity, holding it open to ease ligand binding. Here we show that extensive molecular dynamics simulations of the HIF-2α PAS-B domain strongly support this hypothesis. If water is prevented from entering the buried cavity, several amino acid side chains lining the cavity undergo a major conformational change that shrinks the cavity volume by an average of ∼45% relative to the hydrated cavity. The structural change within the cavity is due mainly to side chain rearrangements; the absence of bound water has only minor effects on the protein backbone conformation. Moreover, when the cavity lacks internally bound water, the side chain of Tyr307 rotates such that it blocks a major route for entry of the ligand into the cavity, greatly raising the free energy barrier for binding of an artificial ligand compared to when the cavity contains water. Finally, there is no difference in global protein stability of PAS-B when the cavity lacks water, as indicated by the same degree of protein flexibility and identical predicted melting temperatures. These findings help to explain further how water stabilizes large internal cavities, a relatively underexamined feature of proteins.