Pyk2 is a non‐receptor tyrosine kinase enriched at the post‐synaptic density of glutamatergic synapses. Activated by Ca2+influx, Pyk2 participates in the translation of synaptic activity into signaling cascades that tune synaptic strength. Pyk2 is a multi‐domain protein kinase with a regulatory FERM domain. Structures have been reported for isolated Pyk2 domains, yet the architectures of the auto‐inhibited Pyk2 and Pyk2 activation complex remain unclear. We employed a cysteine cross‐linking strategy to define the inter‐domain interactions responsible for kinase regulation. Cysteine residues were engineered at putative interfaces in both kinase and FERM domains. Rates of cysteine disulfide bond formation were measured using mass spectrometry (MS). Ultimately, cross‐linking results were integrated with hydrogen/deuterium exchange MS to develop a model of the auto‐inhibited architecture.Support or Funding InformationNSF MCB grant 1715411; Linder Family Undergraduate Research FellowshipThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.