© 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Background: Elucidating the mechanochemistry of ABC transporters is essential for understanding several human disease states. Results: We show a novel intermolecular binding step that governs CFTR channel activity. Conclusion: CFTR gating and ATP hydrolysis require a properly configured transmembrane domain interface. Significance: These findings indicate a new approach for discovering therapeutic targets within ABC proteins that modulate function.