Mapping Purinosome Protein‐Protein Interactions Using In Vivo Photocrosslinking

Abstract

Efficient de novo purine biosynthesis in higher eukaryotes is hypothesized to depend on the existence of a multienzyme complex — the purinosome. However, there is as yet no compelling identification of specific protein‐protein interactions between purine biosynthetic enzymes. Similarly, functionally important interactions between purine biosynthetic enzymes and other proteins have yet to be elucidated. Inspired by the highly symmetric oligomeric structure of PAICS, a de novo purine biosynthetic enzyme, we identified several putative interaction surfaces. Using genetically encoded unnatural amino acid incorporation, we inserted photocrosslinking amino acid analogs into these surfaces at specific residues. Upon UV irradiation, we were able to detect multiple crosslinked products between PAICS and other proteins, formed in their native context inside living cells. Distinct sites of photocrosslinker incorporation yielded distinct products; this suggests they are the result of specific protein‐protein interactions. Using mass spectrometry, we are in the process of identifying PAICS’s crosslinked partners, as well as their interaction interface with PAICS. We will also extend this approach to study other pathway enzymes as we seek to identify functionally important protein‐protein interactions in de novo purine biosynthesis.Support or Funding InformationThe National Institutes of Health (GM024129 to S.J.B.)