Processing of amyloid precursor protein (APP) by ␥-secretase is the last step in the formation of the A␤ peptides associated Alzheimer's disease. Solid-state NMR spectroscopy is used to establish the structural features of the transmembrane (TM) and juxtamembrane (JM) domains of APP that facilitate proteolysis. Using peptides corresponding to the APP TM and JM regions (residues 618 -660), we show that the TM domain forms an ␣-helical homodimer mediated by consecutive GxxxG motifs. We find that the APP TM helix is disrupted at the intracellular membrane boundary near the -cleavage site. This helix-to-coil transition is required for ␥-secretase processing; mutations that extend the TM ␣-helix inhibit cleavage, leading to a low production of A␤ peptides and an accumulation of the ␣-and ␤-C-terminal fragments. Our data support a progressive cleavage mechanism for APP proteolysis that depends on the helix-to-coil transition at the TM-JM boundary and unraveling of the TM ␣-helix. The most unusual feature of APP proteolysis is the intramembraneous cleavage by the ␥-secretase complex. The mechanism of proteolysis is of considerable interest because of its role in (i) generating the A␤ peptides associated with Alzheimer's disease and (ii) releasing the AICD involved in APP-dependent gene transcription. Several cleavage sites have been identified that generate different length A␤ peptides. The ␥-cleavage site cuts the APP sequence in the middle of the TM domain to predominantly produce the A␤40 peptide, and to a lesser extent the A␤42 peptide. However, A␤42 has a higher propensity to form aggregates than the shorter isoforms and is the most toxic peptide generated by ␥ cleavage (3). There is another cleavage site (4), referred to as the -cleavage site, a few residues downstream between Leu-645 and Val-646 that has been identified by N-terminal sequencing of the AICD peptide (4). An open question has been whether the same enzyme activity is responsible for both the ␥-and -cleavage sites.The ␥-secretase complex has a diverse set of type I membrane protein substrates. Notch, Cd44, ErbB4, and E-cadherin are cleaved by the ␥-secretase in vivo. For each of these substrates, truncation of the extracellular domain to just a few amino acids is required to bind to the ␥-secretase complex. These substrates are all cleaved near the intracellular TM-JM boundary. However, like APP, Notch, and Cd44 are also cleaved in the middle of the TM domain, although their sequences are not conserved (see SI).To address the mechanism of intramembrane proteolysis, we focus on the structure of the TM domain of APP in membrane bilayers. Proteolysis requires local unraveling of the helical secondary structure of the TM domain to expose backbone carbonyl carbons for nucleophilic attack by polarized water in the enzyme active site. This requirement raises the question of whether there are sequence motifs in the TM domain of APP that destabilize the helical structure in cell membranes in a fashion similar to that proposed for the conserved Asn-Pro sequenc...