A Photo-clickable ATP-Mimetic Reveals Nucleotide Interactors in the Membrane Proteome

ATP is an important energy metabolite and allosteric signal in health and disease. ATP-interacting proteins, such as P2 receptors, control inflammation, cell death, migration, and wound healing. However, identification of allosteric ATP sites remains challenging, and our current inventory of ATP-controlled pathways is likely incomplete. Here, we develop and verify mipATP as a minimally invasive photoaffinity probe for ATP-interacting proteins. Its N6 functionalization allows target enrichment by UV crosslinking and conjugation to reporter tags by “click” chemistry. The additions are compact, allowing mipATP to completely retain the calcium signaling responses of native ATP in vitro and in vivo. mipATP specifically enriched for known nucleotide binders in A549 cell lysates and membrane fractions. In addition, it retrieved unannotated ATP interactors, such as the FAS receptor, CD44, and various SLC transporters. Thus, mipATP is a promising tool to identify allosteric ATP sites in the proteome. © 2020 Elsevier Ltd Jelcic et al. developed and verified a minimally invasive photoaffinity ATP (mipATP) probe that retains the signaling functions of native ATP in vivo and in vitro and provide proof-of-principle that mipATP can be used to map ATP-protein interaction space using proteomic screens. © 2020 Elsevier Ltd