C99 is the transmembrane carboxyl-terminal domain of the amyloid precursor protein that is cleaved by γ-secretase to release the amyloid-β polypeptides, which are associated with Alzheimer’s disease. Nuclear magnetic resonance and electron paramagnetic resonance spectroscopy show that the extracellular amino terminus of C99 includes a surface-embedded “N-helix” followed by a short “N-loop” connecting to the transmembrane domain (TMD). The TMD is a flexibly curved α helix, making it well suited for processive cleavage by γ-secretase. Titration of C99 reveals a binding site for cholesterol, providing mechanistic insight into how cholesterol promotes amyloidogenesis. Membrane-buried GXXXG motifs (G, Gly; X, any amino acid), which have an established role in oligomerization, were also shown to play a key role in cholesterol binding. The structure and cholesterol binding properties of C99 may aid in the design of Alzheimer’s therapeutics.

The amyloid precursor protein (APP) is subject to alternative pathways of proteolytic processing, leading either to production of the amyloid-β (Aβ) peptides or to non-amyloidogenic fragments. Here, we report the first structural study of C99, the 99-residue transmembrane C-terminal domain of APP liberated by β-secretase cleavage. We also show that cholesterol, an agent that promotes the amyloidogenic pathway, specifically binds to this protein. C99 was purified into model membranes where it was observed to homodimerize. NMR data show that the transmembrane domain of C99 is an α-helix that is flanked on both sides by mostly disordered extramembrane domains, with two exceptions. First, there is a short extracellular surface-associated helix located just after the site of α-secretase cleavage that helps to organize the connecting loop to the transmembrane domain, which is known to be essential for Aβ production. Second, there is a surface-associated helix located at the cytosolic C-terminus, adjacent to the YENPTY motif that plays critical roles in APP trafficking and protein-protein interactions. Cholesterol was seen to participate in saturable interactions with C99 that are centered at the critical loop connecting the extracellular helix to the transmembrane domain. Binding of cholesterol to C99 and, most likely, to APP may be critical for the trafficking of these proteins to cholesterol-rich membrane domains, which leads to cleavage by β-and γ-secretase and resulting amyloid-β production. It is proposed that APP may serve as a cellular cholesterol sensor that is linked to mechanisms for suppressing cellular cholesterol uptake.The human amyloid precursor protein (APP) 1 is a single-span membrane protein that is alternatively processed by either α-or β-secretase to release its large ectodomain from the cell surface, a process referred to as "shedding". β-Secretase (β-site APP cleaving enzyme 1, BACE1) cleaves APP after Met671, leading to production of the C-terminal 99-residue domain of APP, C99, a single-span membrane protein. Subsequent cleavage of C99 at membranedisposed sites by γ-secretase leads to release of both the amyloid-β (Aβ) peptides and the water-

Escherichia coli diacylglycerol kinase (DAGK) represents a family of integral membrane enzymes that is unrelated to all other phosphotransferases. We have determined the three-dimensional structure of the DAGK homotrimer using solution NMR. The third transmembrane helix from each subunit is domain-swapped with the first and second transmembrane segments from an adjacent subunit. Each of DAGK's three active sites resembles a portico. The cornice of the portico appears to be the determinant of DAGK's lipid substrate specificity and overhangs the site of phosphoryl transfer near the water-membrane interface. Mutations to cysteine that caused severe misfolding were located in or near the active site, indicating a high degree of overlap between sites responsible for folding and for catalysis.

Long QT syndrome–associated mutations in KCNQ1 most often destabilize the protein, leading to mistrafficking and degradation.

Summary Mutations in peripheral myelin protein 22 (PMP22) can result in the common peripheral neuropathy, Charcot-Marie-Tooth disease (CMTD). The Leu16Pro mutation in PMP22 results in misassembly of the protein, which causes the Trembler-J (TrJ) disease phenotype. Here we elucidate the structural defects present in a partially folded state of TrJ PMP22 that are decisive in promoting CMTD-causing misfolding. In this state transmembrane helices 2-4 (TM2-4) form a molten-globular bundle while transmembrane helix 1 (TM1) is dissociated from this bundle. The TrJ mutation was seen to profoundly disrupt the TM1 helix, resulting in increased backbone dynamics and changes in the tertiary interactions of TM1 with the PMP22 TM2-4 core in the folded state. Consequently, TM1 undergoes enhanced dissociation from the other transmembrane segments in TrJ PMP22, becoming available for recognition and sequestration by protein folding quality control, leading to loss of function and toxic accumulation of aggregates that results in CMTD.