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The three-dimensional structure of human β-endorphin amyloid fibrils

Nature Structural & Molecular Biology volume 27, pages 1178–1184 (2020)Cite this article

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Abstract

In the pituitary gland, hormones are stored in a functional amyloid state within acidic secretory granules before they are released into the blood. To gain a detailed understanding of the structure–function relationship of amyloids in hormone secretion, the three-dimensional (3D) structure of the amyloid fibril of the human hormone β-endorphin was determined by solid-state NMR. We find that β-endorphin fibrils are in a β-solenoid conformation with a protonated glutamate residue in their fibrillar core. During exocytosis of the hormone amyloid the pH increases from acidic in the secretory granule to neutral level in the blood, thus it is suggested—and supported with mutagenesis data—that the pH change in the cellular milieu acts through the deprotonation of glutamate 8 to release the hormone from the amyloid. For amyloid disassembly in the blood, it is proposed that the pH change acts together with a buffer composition change and hormone dilution. In the pituitary gland, peptide hormones can be stored as amyloid fibrils within acidic secretory granules before release into the blood stream. Here, we use solid-state NMR to determine the 3D structure of the amyloid fiber formed by the human hormone β-endorphin. We find that β-endorphin fibrils are in a β-solenoid conformation that is generally reminiscent of other functional amyloids. In the β-endorphin amyloid, every layer of the β-solenoid is composed of a single peptide and protonated Glu8 is located in the fibrillar core. The secretory granule has an acidic pH but, on exocytosis, the β-endorphin fibril would encounter neutral pH conditions (pH 7.4) in the blood; this pH change would result in deprotonation of Glu8 to release the hormone peptide from the amyloid. Analyses of β-endorphin variants carrying mutations in Glu8 support the role of the protonation state of this residue in fibril disassembly, among other environmental changes.

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Fig. 1: Initial structural characterization of β-endorphin amyloid fibrils.
Fig. 2: 3D structure of a single peptide layer of β-endorphin amyloid protofibrils.
Fig. 3: 3D structure of β-endorphin amyloid protofibrils.
Fig. 4: Wild-type β-endorphin fibrils disassemble faster with increasing pH than the variants E8L and E8Q.

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Data availability

Structural coordinates and experimental restraints have been deposited in the wwPBD under accession code PDB 6TUB. NMR chemical shifts have been deposited in the BMRB under entry 26715.

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Acknowledgements

We thank ETH and the Swiss National Science Foundation for financial support.

Author information

Author notes

  1. These authors contributed equally: Carolin Seuring, Joeri Verasdonck.

Authors and Affiliations

  1. Laboratory of Physical Chemistry, ETH Zürich, Zürich, Switzerland

    Carolin Seuring, Joeri Verasdonck, Julia Gath, Dhimam Ghosh, Nadezhda Nespovitaya, Marielle Aulikki Wälti, Riccardo Cadalbert, Peter Güntert, Beat H. Meier & Roland Riek

  2. Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron, Hamburg, Germany

    Carolin Seuring

  3. Department of Biosciences and Bioengineering, IIT Bombay, Powai, Mumbai, India

    Dhimam Ghosh & Samir K. Maji

  4. Institute of Biophysical Chemistry, Center for Biomolecular Magnetic Resonance, Goethe University Frankfurt am Main, Frankfurt am Main, Germany

    Peter Güntert

  5. Graduate School of Science, Tokyo Metropolitan University, Tokyo, Japan

    Peter Güntert

  6. Structural Biology Laboratory, The Salk Institute, La Jolla, CA, USA

    Roland Riek

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Contributions

C.S., N.N., M.A.W., D.G. and R.C. prepared samples. C.S., J.V., D.G., S.K.M., B.H.M. and R.R. planned the research. C.S., J.V., J.G. and D.G. collected and analyzed data. C.S., J.V., J.G., B.H.M., R.R. and P.G. analyzed NMR data and performed structure calculations. C.S., J.V., P.G., S.K.M., B.H.M. and R.R. wrote the paper.

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Correspondence to Beat H. Meier or Roland Riek.

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Peer review information Inês Chen was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 NMR spectrum for distance restraint collection: 400 ms PDSD spectrum recorded on uniformly 15N,13C-labelled fibrils.

a, Spectrally unambiguous contacts are labeled in red for intermolecular contacts and in blue for contacts that are ambiguous with respect to being intra- or intermolecular. Some structurally relevant restraints defining crucial contacts in the 3D fold of the protein (Supplementary Table 2, PDSD) were assigned manually and are shown in b as zoomed-in regions: E8 sidechain to L14, E8 to T16; F4 sidechain to A21 and I23. Intramolecular contacts are framed in grey, intermolecular contacts in orange. Contacts that are ambiguous with respect to being intra- or intermolecular are framed in black.

Extended Data Fig. 2 Spectral distinction between inter and intra-molecular contacts.

Selected traces through the indirect dimension of the individual spectra (as indicated on the right) of a fully uniformly 13C,15N-labeled sample in black and a sample diluted four-fold with unlabeled peptide in red at the resonance frequencies of 14LeuCD2 (23.62 ppm, three top panels) or 16ThrCA (62.70 ppm, three bottom panels) as indicated. The dashed lines indicate the peaks that were used for scaling. Red labels are used for intermolecular contacts, green labels for intramolecular contacts, and blue labels for contacts that fall in between. In short, if the relative cross peak intensity in the spectra of the diluted sample is similar in intensity as the one in the spectrum of the fully-labeled sample (see green-labeled cross peak 8GluCG) the cross peak originates from an intra-molecular contact, while an inter-molecular contact would give rise to a 5-fold less intense cross peak attributed to the dilution (see, for instance, the red-labeled cross peak 6ThrCA). Signal intensities in the two spectra to be compared (uniformly labelled versus diluted) were determined by summing over the peak regions in a 0.5 by 0.5 ppm square centered at the peak positions. The noise on these signal intensities was estimated by the standard deviation of a set of 1000 signal intensities in an empty region of the spectrum. For each slice j, a scaling factor \(S_j = \left( {\mathop {\sum }\limits_i w_iU_i/D_i} \right)/\left( {\mathop {\sum }\limits_i w_i} \right)\) was determined, where the sums run over all peaks in slice j, Ui and Di are the intensities of a given peak \(i\) in the uniformly labelled and diluted spectrum, respectively, and \(w_i = 1/\sigma _i^2\) with σi equal to the error of the intensity ratio obtained by error propagation from the estimated noise of the intensities. For each peak i in slice j that spans more than 2 residues, the scaled intensity ratio Ri = Sj Di/Ui and its error \(\sigma _{R_i}\), obtained by error propagation, were computed. Restraints were classified as intramolecular if \(R_i + \sigma _{R_i} > 0.8\) and \(R_i - \sigma _{R_i} > 0.7\), or as intermolecular if \(R_i + \sigma _{R_i} < 0.4\) and \(R_i - \sigma _{R_i} < 0.3\). Ratios that do not meet either of these criteria are classified as ambiguous with respect to being an intra- or intermolecular restraint.

Extended Data Fig. 3 The identification of the staggering extent (A) and of the relative orientation of the β-sheets (B).

The weighted mean ratio between relative cross peak intensities from spectra obtained of a fully uniformly 15N,13C-labeled sample and a 80% (1:4) diluted sample (shown in part in Extended Data Fig. 2 and Supplementary Fig. 4) are shown per residue pair (as indicated on the x-axis). The weighted mean ratios were obtained by averaging over all cross peaks observed between the two residues in the various spectra, weighing the contribution of the i-th cross peak by \(1/\sigma _i^2\), where σi is the error of the peak intensity ratio for cross peak i. Error bars show the estimated standard error of the weighted mean, determined by performing a bootstrap with a sample size of 10,000. If the cross peaks between a residue pair is due to an intra-molecular interaction, the value should be 1, if it is of entire inter-molecular origin, the value should be 0.2. In a staggered structure, individual contacts across the fibrillar core can have an intra- or intermolecular nature. However, the weighted average of all contacts for a cross fibrillar-core contacts should tend towards a value in between the intra- and intermolecular regime. Here, these ratios are all substantially lower than 1 and mostly higher than 0.2, indicating that the structure is indeed staggered. Once the in-register parallel β-sheet conformation has been established, the relative orientation of the β-sheets, that is the direction of the hydrogen bonds along the fibril axis (N-H…O=C, denoted here as A, or C=O…H-N, denoted here as B), was determined. Four unique possibilities for the relative orientation of the three β-strands β1, β2, β3 exist (Supplementary Fig. 4). These are labeled with AAA, AAB, ABA, and ABB. (The BBB orientation is equivalent to AAA, BAB to ABA, BBA to AAB, and BAA to ABB.) CYANA structure calculations of β-endorphin fibrils were performed with all four orientations of the β-sheets using in addition to the experimental restraints for each of the possible orientations their corresponding intermolecular hydrogen-bond restraints. The plot shows the CYANA target function value, which is a weighted sum of all squared restraint violations. Only the ABA (and equivalent BAB) orientation yielded a low target function value and thus a well fulfillment of the experimental restraints and therefore the ABA orientation was used in the subsequent final structure calculations. ABA is the one arrangement for which the hydrogen bonds of β-sheet β2 have the opposite orientations along the fibril axis to the β -sheets β1 and β3 strands.

Extended Data Fig. 4 Embedment of the 3D structure of the proto-fibril into the fibril.

The width of 3.2 +/− 0.2 nm of proto-fibrils within fibrils extracted from (a) negative-stain transmission electron microscopy data from Seuring et al. 2017 (ref. 19) fit well (b) the dimension of the determined 3D solid state NMR structure. In the depicted micrographs, β-endorphin forms straight, striated fibril ribbons of variable widths in buffer A. The full width at half-maximum (FWHM) of approximately 3 nm (9 pixels) can be retrieved from the corresponding intensity profiles.

Extended Data Fig. 5 Negative-stain transmission electron micrographs and NMR spectra show that the two variants E8L and E8Q form also amyloid fibrils with similar morphologies and 3D structure as wild type β-endorphin.

Negatively stained transmission electron micrographs of wild-type β-endorphin and the two variants E8L and E8Q are shown (all prepared in buffer B). Scale bars correspond to 200 nm. Below, a superposition of the 20 ms [13C,13C]-DARR spectra of wild type β-endorphin (blue) and the variant E8Q (red) is shown. The close resemblance between the two spectra indicates the formation of the same fold. Only residues in spatial proximity to residue 8, that is Leu14, Val15, Thr16, and, to a lesser extent, Leu17 show significant chemical shift changes. The cross peaks for which the chemical shift perturbation due to the mutation exceeds the line width, are labeled for both the wild-type and the variant E8Q amyloid fibrils with one letter amino acid residue codes. The line widths are overall comparable between the two spectra, indicating homogeneous samples for both endorphin forms. The spectrum of wild type β-endorphin fibrils was recorded at 20.0 T static magnetic field and 17 kHz MAS. The spectrum of the mutant E8Q was recorded at 14.1 T static magnetic field and 13 kHz MAS.

Extended Data Fig. 6 Fibril disassembly of wild type and mutant β-endorphin fibrils in absence (A) and presence (B) of sonication in the acetate buffer A.

a, b, Bar diagram representation of the relative disassembly for (a) sonicated and (b) not-sonicated wild type β-endorphin (WT) fibrils and in (a) also its variants E8L (8L) and E8Q (8Q) measured at different pH values after 50 h disassembly time. The release was measured by UV absorbance at 280 nm outside the dialysis membrane and normalized such that 100% corresponds to the entire peptide material. The data show that with increasing pH, wild type β-endorphin fibrils disassemble more than those of the mutants E8Q and E8L. In addition, the disassembly is more pronounced in the sonicated wild type fibrils than the non sonicated ones indicating disassembly also from the end of the fibrils.

Extended Data Fig. 7 Glu8 is protonated at pH 5.5–7.4 as evidenced by solid state NMR.

20 ms DARR spectra of wild type β-endorphin amyloids at pH 5.5 (blue) and 7.4 (green) are shown. The arrow highlights the Glu8 Cδ/Cβ cross-peak, which is present in both spectra. The Cδ chemical shift indicates the protonated state of the Glu8 carboxyl group.

Extended Data Fig. 8 Presence of natural abundance heparin in the β-endorphin NMR sample as measured by solid state NMR.

A weak diagonal signal from natural abundance (1%) 13C heparin indicated by the grey rectangle is observed in the 400 ms [13C,13C]-PDSD spectrum in addition to the strong signals from 15N,13C-labeled β-endorphin amyloids.

Extended Data Fig. 9 Sequence alignment of β-endorphins.

The 37 sequences shown in this alignment are the non-redundant output of a PSI-BLAST search with the β-endorphin sequence comprising residues 1–31 (carried to convergence at an E-value threshold of 0.005). The query sequence is shown as the first sequence of the alignment. Of the other sequences displayed, 31 correspond to β-endorphin sequences of the precursor POMC. The six “only”-β-endorphin sequences are marked by black dots on the left. The color code represents hydrophilic, hydrophobic, negative and positive charged residues in green, white, blue and red, respectively. The consensus and sequence conservations are displayed below. The high conservation of the five N-terminal residues is attributed to their role in receptor activation.

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Seuring, C., Verasdonck, J., Gath, J. et al. The three-dimensional structure of human β-endorphin amyloid fibrils. Nat Struct Mol Biol 27, 1178–1184 (2020). https://doi.org/10.1038/s41594-020-00515-z

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