The Notch intracellular domain (NICD) forms a transcriptional activation complex 
with the DNA-binding factor CSL and a transcriptional co-activator of the 
Mastermind family (MAML). The "RAM" region of NICD recruits Notch to CSL, 
facilitating the binding of MAML at the interface between the ankyrin (ANK) 
repeat domain of NICD and CSL. Here, we report the X-ray structure of a human 
MAML1/RAM/ANK/CSL/DNA complex, and probe changes in component dynamics upon 
stepwise assembly of a MAML1/NICD/CSL complex using HX-MS. Association of CSL 
with NICD exerts remarkably little effect on the exchange kinetics of the ANK 
domain, whereas MAML1 binding greatly retards the exchange kinetics of ANK 
repeats 2-3. These exchange patterns identify critical features contributing to 
the cooperative assembly of Notch transcription complexes (NTCs), highlight the 
importance of MAML recruitment in rigidifying the ANK domain and stabilizing its 
interface with CSL, and rationalize the requirement for MAML1 in driving 
cooperative dimerization of NTCs on paired-site DNA.