Abstract
Although Hsp90-family chaperones have been extensively targeted with ATP-competitive inhibitors, it is unknown whether high affinity is achieved from a few highly stabilizing contacts or from many weaker contacts within the ATP-binding pocket. A large-scale analysis of Hsp90α:inhibitor structures shows that inhibitor hydrogen-bonding to a conserved aspartate (D93 in Hsp90α) stands out as most universal among Hsp90 inhibitors. Here we show that the D93 region makes a dominant energetic contribution to inhibitor binding for both cytosolic and organelle-specific Hsp90 paralogs. For inhibitors in the resorcinol family, the D93:inhibitor hydrogen-bond is pH-dependent because the associated inhibitor hydroxyl group is titratable, rationalizing a linked-protonation event previously observed by the Matulis group. The inhibitor hydroxyl group pK
associated with the D93 hydrogen-bond is therefore critical for optimizing the affinity of resorcinol derivatives, and we demonstrate that spectrophotometric measurements can determine this pK
value. Quantifying the energetic contribution of the D93 hotspot is best achieved with the mitochondrial Hsp90 paralog, yielding 3-6 kcal/mol of stabilization (35-60% of the total binding energy) for a diverse set of inhibitors. The Hsp90 Asp93➔Asn substitution has long been known to abolish nucleotide binding, yet puzzlingly, native sequences of structurally similar ATPases, such as Topoisomerasese II, have an asparagine at this same crucial site. While aspartate and asparagine sidechains can both act as hydrogen bond acceptors, we show that a steric clash prevents the Hsp90 Asp93➔Asn sidechain from adopting the necessary rotamer, whereas this steric restriction is absent in Topoisomerasese II.
