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-
The preparation of high quality samples is a critical challenge for the structural characterization of helical integral membrane proteins. Solving the structures of this diverse class of proteins by solution nuclear magnetic resonance spectroscopy (NMR) requires that well-resolved 2D 1H/15N chemical shift correlation spectra be obtained. Acquiring these spectra demands the production of samples with high levels of purity and excellent homogeneity throughout the sample. In addition, high yields of isotopically enriched protein and efficient purification protocols are required. We describe two robust sample preparation methods for preparing high quality, homogeneous samples of helical integral membrane proteins. These sample preparation protocols have been combined with screens for detergents and sample conditions leading to the efficient production of samples suitable for solution NMR spectroscopy. We have examined 18 helical integral membrane proteins, ranging in size from approximately 9 kDa to 29 kDa with 1-4 transmembrane helices, originating from a number of bacterial and viral genomes. 2D 1H/15N chemical shift correlation spectra acquired for each protein demonstrate well-resolved resonances, and >90% detection of the predicted resonances. These results indicate that with proper sample preparation, high quality solution NMR spectra of helical integral membrane proteins can be obtained greatly enhancing the probability for structural characterization of these important proteins.
Dominant mutations in the tetraspan membrane protein peripheral myelin protein 22 (PMP22) are known to result in peripheral neuropathies such as Charcot-Marie-Tooth Type 1A (CMT1A) disease via mechanisms that appear to be closely linked to misfolding of PMP22 in the membrane of the endoplasmic reticulum (ER). To characterize the molecular defects in PMP22, we examined the structure and folding stability of two human disease mutant forms of PMP22 that are also the basis for mouse models of peripheral neuropathies: G150D (Trembler phenotype), and L16P (Trembler-J phenotype). Circular dichroism and NMR spectroscopic studies indicated that, when folded, the 3-D structures of these disease-linked mutants are similar to the folded wild type protein. However, the folded forms of the mutants were observed to be destabilized relative to the wild type protein, with the L16P mutant being particularly unstable. The rate of refolding from an unfolded state was observed to be very slow for the wild type protein, and no refolding was observed for either mutant. These results lead to the hypothesis that ER quality control recognizes the G150D and L16P mutant forms of PMP22 as defective through mechanisms closely related to their conformational instability and/or slow folding. It was also seen that wild type PMP22 binds Zn(II) and Cu(II) with micromolar affinity, a property that may be important to the stability and function of this protein. Zn(II) was able to rescue the stability defect of the Tr mutant.Peripheral myelin protein 22 (PMP22) is a 160 residue integral membrane protein with four putative transmembrane spans. PMP22 is a major protein of the peripheral nervous system myelin(1;2), where it is known to play important roles in regulating Schwann cell proliferation and in myelin formation and maintenance(3;4). A high resolution structure is yet to be determined for PMP22, although some of its general structural and topological features have been established(5;6). PMP22 represents the PMP22/EMP/MP20/Claudin superfamily (pfam00822 in NCIB (7)), which share both sequence homology and their predicted tetraspan topology. PMP22 and some of these other proteins are found at specialized membrane junctions found in myelin and in epithelia(8-11).PMP22 is highly expressed in Schwann cells and represents 2-5% of the total protein content of the myelin membrane. Changes in gene dosage or dominant missense mutations in the gene encoding PMP22 result in several inherited peripheral neuropathies(12), including hereditary neuropathy with liability to pressure palsies (HNPP), Dejerine-Sottas syndrome (DSS), and
Gene duplications, deletions, and point mutations in peripheral myelin protein 22 (PMP22) are linked to several inherited peripheral neuropathies. However, the structural and biochemical properties of this very hydrophobic putative tetraspan integral membrane protein have received little attention, in part because of difficulties in obtaining milligram quantities of wild type and disease-linked mutant forms of the protein. In this study a fusion protein was constructed consisting of a fragment of lambda repressor, a decahistidine tag, an intervening TEV protease cleavage site, a Strep tag, and the human PMP22 sequence. This fusion protein was expressed in Escherichia coli at a level of 10-20 mg/L of protein. Following TEV cleavage of the fusion partner, PMP22 was purified and its structural properties were examined in several different types of detergent micelles using cross-linking, near and far-UV circular dichroism, and nuclear magnetic resonance (NMR) spectroscopy. PMP22 is highly helical and, in certain detergents, shows evidence of stable tertiary structure. The protein exhibits a strong tendency to dimerize. The 1H-15N TROSY NMR spectrum is well dispersed and contains signals from all regions of the protein. It appears that detergent-solubilized PMP22 is amenable to detailed structural characterization via crystallography or NMR. This work sets the stage for more detailed studies of the structure, folding, and misfolding of wild type and disease-linked mutants in order to unravel the molecular defects underlying peripheral neuropathies.
The reaction between Mn(6)L(12) and Mg(6)L(12) (L = N,N-diethylcarbamate) results in isolation of heteronuclear complexes Mn(n)Mg(6)(-)(n)L(12). A series was prepared with different doping factors n by varying the Mn/Mg ratio in the crystallization solutions. Single-crystal X-ray diffraction shows that MnMg(5)L(12) is isostructural with Mn(6)L(12) and Mg(6)L(12). Magnetic susceptibility data on the series Mn(n)Mg(6)(-)(n)L(12) (n = 1-6) are consistent with antiferromagnetic Mn.Mn interactions. At low n, the magnetic data demonstrate the formation of magnetically isolated Mn(2+) centers. This was confirmed by measurement of the EPR spectrum at a doping factor n = 0.06 in solution, as a powder, and as single crystals. These show hyperfine interactions consistent with isolated Mn(2+). The EPR spectrum of Mn(0.06)Mg(5.94)L(12) exhibits a dominant signal at g(eff) = 4, and a wide series of less intense signals spanning 200-6000 G in the X-band regime. This unusual behavior in a weak-field Mn(2+) complex is attributed to the substantial distortions from cubic ligand field geometry in this system. The g(eff) = 4 signals are attributed to a C(2)-symmetric hexacoordinate Mn(2+) ion with D > 0.3 cm(-)(1) and E/D = 0.33. The wide series is assigned to an axial C(4)(v) pentacoordinate Mn(2+) site with D = 0.05 cm(-)(1). Comparison of the g(eff) = 4 signals to the g = 4.1 signals exhibited by the tetramanganese complex in photosystem II belies the fact that they almost certainly arise from different spin systems. In addition, the similarity of the spectrum of Mn(n)Mg(6)(-)(n)L(12) to mononuclear Mn(4+) complexes suggests that considerable care must be exercised in the use of EPR as a fingerprint for the manganese oxidation state, particularly in manganese proteins where molecular composition may not be precisely established.
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