Research ArticleBIOCHEMISTRY

Structural and biochemical differences between the Notch and the amyloid precursor protein transmembrane domains

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Science Advances  12 Apr 2017:
Vol. 3, no. 4, e1602794
DOI: 10.1126/sciadv.1602794
  • Fig. 1 Domain organization of full-length Notch 1.

    The ligand-binding N-terminal extracellular domain includes the repeat EGF domain followed by three Lin-12/Notch repeats (LNR) and the heterodimerization domain (HD). The LNR segments and the HD comprise the NRR. The transmembrane segment follows. The cytosolic domain contains the RAM domain, followed by ankyrin (ANK) repeats, the transcriptional activation domain (TAD), and the C-terminal terminal proline-, glutamic acid–, serine-, and threonine-rich (PEST) domain. The construct used in this study (the Notch-TMD) is the transmembrane segment plus the immediate juxtamembrane regions, highlighted on the right. Major γ-secretase cut sites are labeled red. The S2 metalloprotease cut site precedes Val1721.

  • Fig. 2 Structures of the Notch-TMD.

    (A) Representative low-energy NMR-determined XPLOR-NIH structure. The approximate location of the membrane span is shown by the gray bars. The residue represented in green van der Waals mode is Trp1768 of the LWF motif. (B) Representative frame from one of the 10 × 60–ns restrained MD trajectories in a hydrated DMPC bilayer. The blue and red balls indicate the positions of the DMPC head group phosphodiester and tertiary amino groups, respectively. (C) Representative frames from the other nine restrained MD trajectories, illustrating variations in TMD tilt, conformational disorder in the JMDs, and the presence of a membrane reentrant segment (LWF) in the cytosolic C-terminal domain.

  • Fig. 3 Water and lipid contacts for the Notch-TMD.

    Analysis of the observed frequency of lipid side-chain (A) and water (B) contacts with Notch-TMD side chains in rMD simulations of the Notch-TMD in DMPC bilayers (compilation of results for the final 40 ns of all 10 simulations).

  • Fig. 4 Comparison of site-specific amide backbone 15N NMR chemical shifts for the Notch-TMD as a function of the lipid used to form the bicelles in each of the three samples.

    This plot shows the differences between the observed chemical shifts for each site minus the random coil chemical shift value for that amino acid. DMPC has C14 chains. ESM has one C16 chain as part of ceramide and a second usually C16 chain at the fatty amide position. MSM has the ceramide C16 and a usually C22-to-C24 chain at the fatty amide position.

  • Fig. 5 Access of the Notch-TMD backbone amide protons to water-soluble (Gd-DTPA) and lipophilic (16-DSA) paramagnetic probes in bicelles containing lipids of different types and chain lengths.

    Short chain, DMPC; medium chain, ESM; long chain, MSM. Iexp/Iref is the ratio of the NMR peak intensities for each site in the presence of the probe versus the intensity for that same site under matched diamagnetic conditions. The solid vertical lines indicate the length of the α helix that spans the TMD. The DMPC accessibility plots represent the average of two matched trials, one of which was previously reported (40).

  • Fig. 6 Superimposed 900-MHz TROSY spectra from a cholesterol titration of the Notch-TMD demonstrate an absence of specific association of cholesterol with the Notch-TMD.

    NMR samples were prepared with increasing concentrations of cholesterol (0, 2.5, 5, 10, and 15 mol %). Samples contained ~0.35 mM protein in 15% (w/v) DMPC/DHPC bicelles. There are no significant chemical shift perturbations that suggest direct cholesterol binding [compare to results for C99 (30)]. Moreover, even the modest shifts seen for some residues vary linearly with cholesterol concentration rather than showing any sign of saturation of binding.

  • Fig. 7 Summary of differences between C99 and the Notch-TMD.

    ECD and ICD are the large Notch extracellular and intracellular domains, respectively (see Fig. 1). The general locations of the key residues in the cholesterol binding site of C99 are indicated in purple. Not illustrated here is the propensity of C99, but not the Notch-TMD, to dimerize. Also not illustrated here is the fact that, if the membrane shown in this figure were thinned, the Notch-TMD is predicted by the results of this work to adjust by tilting with respect to the bilayer normal, whereas C99 would remain untilted, with the N-terminal end of its TMD jutting out into the ectoplasm.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/4/e1602794/DC1

    Supplementary Methods

    fig. S1. Top 20 NMR-determined XPLOR-NIH structures of the Notch-TMD.

    table S1. Statistics for Notch-TMD structure determination and structure quality for the 10 final XPLOR-NIH NMR structures and for the 10 representative restrained MD structures.

    References (9496)

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Methods
    • fig. S1. Top 20 NMR-determined XPLOR-NIH structures of the Notch-TMD.
    • table S1. Statistics for Notch-TMD structure determination and structure quality for the 10 final XPLOR-NIH NMR structures and for the 10 representative restrained MD structures.
    • References (94–96)

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