Figure 6. Concerted action of exosites and active site in substrate binding and cleavage.

• A
  Schematic representation of the substrate‐binding chase experiment assaying whether C99 crosslinked at exosites (Exo) can be cleaved during chase incubation. Note that crosslink product of substrates with Bpa placed in the Aβ region cannot be recovered by Ni‐NTA affinity pulldown after γ‐secretase cleavage in the 37°C chase.
• B
  Substrates bound to exosites in the PS1 NTF at 4°C can be cleaved in the 37°C chase incubation in a L‐685,458‐dependent manner. Note that although many of these substrates crosslinked only weakly at the exosites, their cleavage in the 37°C chase proved that these reflected functional substrate‐binding states.
• C
  Substrates crosslinking in the exosites to NCT and PEN‐2 cannot be cleaved.
• D, E
  Substrates can be cleaved when crosslinked to PS1 NTF, but not when crosslinked to NCT (D) or PEN‐2 (E).
• F
  Excess amounts of Notch1 substrate N102‐FmH compete with exosite binding of C99. See Fig EV4 for competition of C99 binding in the active site region.