Affinity-ligand purification of native human low-abundance multi-protein complexes for structure determination

In human cells, large multi-protein transcription co-activators, such as chromatin remodelers or histone acetyltransferases, play critical roles in gene-expression regulation and are often implicated in disease. However, interrogating their structures or analyzing their properties and interactions in different organs or in medically relevant cell-types is hindered by the difficulty in purifying them. We overcome this difficulty by applying an affinity-ligand composed of a small molecule that specifically recognizes a particular domain in a given co-activator multiprotein complex. This molecule is coupled to a desthiobiotin moiety, which allows binding to streptavidin beads and can be eluted using biotin. To demonstrate the universal utility of this idea to practically any co-activator complex from any cell source we synthesized a compound conjugating desthiobiotin to GSK4027, a molecule that targets the bromodomain in the GCN5/PCAF catalytic subunit of SAGA and ATAC acetyltransferase complexes. Employing this heterobifunctional affinity ligand and a novel purification scheme adapted to low-abundance complexes, we isolated the 1.6 MDa SAGA complex from two cancer cell lines to high degree of purity and activity. We then solved the structure of the isolated 20-subunit SAGA complex to high-resolution (2.3-3 Å) by cryo-EM, elucidating for the first time the molecular details of how its enigmatic splicing module anchors into SAGA. Analyzing these interactions raises the possibility that SAGA serves to relay this module to the splicing machinery. Our approach will be instrumental for characterizing many other multi-protein complexes from medically important sources.