LAUSR

Structural characterization of small molecule bind-ing site hotspots within the global proteome is uniquely enabled by photo-affinity labeling (PAL) coupled with chemical enrichment and unbiased analysis by mass spectrometry (MS). MS-based binding site maps provide structural resolution of interaction sites in conjunction with identification of target proteins. However, binding site hotspot mapping has been confined to relatively simple small molecules to date; extension to more com-plex compounds would enable the structural defi-nition of new binding modes in the proteome. Here, we extend PAL and MS methods to derive a binding site hotspot map for the immunosuppres-sant rapamycin, a complex macrocyclic natural product that forms a ternary complex with the pro-teins FKBP12 and FRB. Photo-rapamycin was de-veloped as a diazirine-based PAL probe for ra-pamycin, and the FKBP12-photo-rapamycin-FRB ternary complex formed readily in vitro. Photo-irradiation, digestion, and MS analysis of the ter-nary complex revealed a McLafferty rearrangement product of photo-rapamycin conjugated to specific surfaces on FKBP12 and FRB. Molecular modeling based on the binding site map revealed two distinct conformations of complex-bound photo-rapamycin, providing a 5.0 Å distance constraint between the conjugated residues and the diazirine carbon and a 9.0 Å labeling radius for the diazirine upon photo-activation. These measurements may be broadly useful in the interpretation of binding site measurements from PAL. Thus, in characteriz-ing the ternary complex of photo-rapamycin by MS, we applied binding site hotspot mapping to a macrocyclic natural product and extracted precise structural measurements for interpretation of PAL products that may enable the discovery of new binding sites in the "undruggable" proteome.