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Intron-Encoded Domain of Herstatin, An Autoinhibitor of Human Epidermal Growth Factor Receptors, Is Intrinsically Disordered - PubMed

  • ️Sat Jan 01 2022

Intron-Encoded Domain of Herstatin, An Autoinhibitor of Human Epidermal Growth Factor Receptors, Is Intrinsically Disordered

Daisuke Tashiro et al. Front Mol Biosci. 2022.

Abstract

Human epidermal growth factor receptors (HER/ERBB) form dimers that promote cell proliferation, migration, and differentiation, but overexpression of HER proteins results in cancer. Consequently, inhibitors of HER dimerization may function as effective antitumor drugs. An alternatively spliced variant of HER2, called herstatin, is an autoinhibitor of HER proteins, and the intron 8-encoded 79-residue domain of herstatin, called Int8, binds HER family receptors even in isolation. However, the structure of Int8 remains poorly understood. Here, we revealed by circular dichroism, NMR, small-angle X-ray scattering, and structure prediction that isolated Int8 is largely disordered but has a residual helical structure. The radius of gyration of Int8 was almost the same as that of fully unfolded states, although the conformational ensemble of Int8 was less flexible than random coils. These results demonstrate that Int8 is intrinsically disordered. Thus, Int8 is an interesting example of an intrinsically disordered region with tumor-suppressive activity encoded by an intron. Furthermore, we show that the R371I mutant of Int8, which is defective in binding to HER2, is prone to aggregation, providing a rationale for the loss of function.

Keywords: herstatin; human epidermal growth factor receptor; intrinsically disordered protein; intron-encoded protein; pre-molten globule state; small-angle X-ray scattering.

Copyright © 2022 Tashiro, Suetaka, Sato, Ooka, Kunihara, Kudo, Inatomi, Hayashi and Arai.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1

Structures of HER2 and herstatin. (A) Structure of HER2. The extracellular domain is composed of four subdomains, ECD I, ECD II, ECD III, and ECD IV. Seven glycosylation sites are shown by asterisks [N68, N124, N187, N259, N530, N571, and N629 according to the UniProt database (Accession number P04626) (UniProt Consortium, 2021)]. (B) Structure of herstatin. ECD I and ECD II are identical to those in HER2. An intron-8 encoded 79-residue domain, named Int8 (or ECD IIIa), is retained at the C-terminal region of herstatin. Four glycosylation sites identical to those in HER2 are shown by asterisks. Note that Int8 does not have a glycosylation site.

FIGURE 2
FIGURE 2

CD measurements. (A) Far-UV CD spectra of the wild type (red) and R371I mutant (blue) of Int8 in the absence (continuous lines) and presence (broken lines) of 4 M GdnHCl. The MRE values are shown. The spectra in the presence of 4 M GdnHCl were measured up to 211 nm due to large absorption by GdnHCl at lower wavelengths. (B) Difference CD spectra calculated by subtracting the CD spectrum in the presence of 4 M GdnHCl from that measured in the absence of GdnHCl.

FIGURE 3
FIGURE 3

NMR measurements. (A,C) One-dimensional NMR spectra of the wild type (A) and R371I mutant (C) of Int8. Arrows show the peaks used for the analysis of pulsed-field gradient (PFG) NMR measurement. The DSS peak is at 0 ppm. (B,D) Peak intensity decay curves obtained by the PFG NMR measurement of the wild type (B) and R371I mutant (D). (E) Two-dimensional 1H−15N heteronuclear single quantum coherence spectrum of the wild-type Int8.

FIGURE 4
FIGURE 4

SAXS analysis of wild-type Int8. (A) The ln I(Q) versus Q plot. The continuous line was obtained by the EOM fit. The intensity is shown in an arbitrary unit. (B) Kratky plot. (C) A pair-distance distribution function, P(r). (D) Guinier plot. The continuous line was obtained by Guinier approximation. (E) The I(Q)−1 versus Q 2.206 plot. The continuous line was obtained by fitting to the Debye function for a random coil. (F) Scaling relationship for the native (green), intermediate (blue), and unfolded state (purple). The red circle shows the R g of Int8 obtained by fitting to the Debye function.

FIGURE 5
FIGURE 5

EOM analysis of the SAXS data. (A,B) The distribution of R g (A) and D max (B) for the completely random pool (black) and the ensemble of Int8 conformations (red). (C) Five representative conformations of the wild-type Int8 involved in the ensemble that was best fitted to the scattering curve of Int8 (Figure 4A). The R g, D max, and fraction (%) of the conformations are shown at the bottom.

FIGURE 6
FIGURE 6

Secondary structure and disorder predictions of wild-type Int8. (A) Secondary structure prediction by PSIPRED. Pred indicates the predicted secondary structure (H, α-helix; E, β-sheet; and C, coil). Conf shows the confidence level of the prediction. Regions predicted to form α-helices and β-sheets are shown by red and yellow boxes, respectively. (B) Disorder prediction by nine different prediction servers. Black thick line shows the average of the predictions. Regions with a score larger than 0.5 are predicted to be disordered.

FIGURE 7
FIGURE 7

SEC measurements. (A,B) Elution profiles of the wild type (red) and R371I mutant (blue) of Int8 measured at low (∼100 μM) (A) and high (∼500 μM) protein concentrations (B).

FIGURE 8
FIGURE 8

Structure modeling of Int8. (A,B) Overall structures of the wild type (A) and R371I mutant (B) of Int8 predicted by AlphaFold2. (C,D) Expanded views of the helical regions of the wild type (C) and R371I mutant (D). R371 and I371 are shown by red and blue balls, respectively. The Figures were drawn using the PyMOL Molecular Graphics System, Version 2.4.0 Schrödinger, LLC.

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