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PRINCIPAL PUBLICATION AND AUTHORS

Amyloid and hydrogel formation of a peptide sequence from a coronavirus spike protein, V. Castelletto (a), I. Hamley (a), ACS Nano 16, 1887-1867 (2022); https:/doi.org/10.1021/acsnano.1c10658 (a) Department of Chemistry, University of Reading (UK)

REFERENCES

[1] B. Robson, Computers Biol. Med. 121, 103749 (2020). [2] I.W. Hamley, Small-Angle Scattering: Theory, Instrumentation, Data and Applications. Wiley, Chichester (2021). [3] S. Nyström & P. Hammarström, J. Am. Chem. Soc. 144, 8945- 8950 (2022).

Fig. 36: a) Measured SAXS data (open symbols) with fitted form factor profiles (red lines) for 1 wt% RSAIEDLLFDKV samples at the pH values indicated (data shifted for convenience, data at pH 3 measured on a different beamline). The limiting slope of I(q)~q-2 shown confirms the presence of planar structures, here nanotapes at pH 4 - 6. b) One possible proposed model for nanotape structures.

Rapid determination of the crystal structure of human mitochondrial NADK2

X-ray crystallography studies of human NADK2 reveals a dimeric organisation and active site occlusion by lysine acetylation. These findings reveal important molecular insights into the structure and regulation of a vital enzyme in mitochondrial NADPH and proline metabolism.

NAD kinases (NADKs) form an original family of enzymes that were identified about a decade ago through an extensive sequence comparison [1]. NADKs are ubiquitously conserved in the various kingdoms of life, and phosphorylate the cofactor NAD to produce NADP,

a limiting precursor for the generation of NADPH cellular reducing power. NAD(H) and NADP(H) supply reductive power in numerous oxidoreductive enzymatic reactions in living organisms. These two molecules possess the same moiety (a nicotinamide) that is responsible for electron and proton transfer, but they markedly differ by their biological roles. Indeed, reduced NAD (NADH) is mainly consumed during respiration (to produce water from dioxygen) and in cellular catabolism, while reduced NADP (NADPH) is mostly dedicated to oxidative stress response and anabolic metabolism, including the biosynthesis of nucleotides, amino acids and lipids. These distinct biological roles are not due to distinct oxidoreductive properties but to specific recognition by their respective enzymes thanks to the earmark (a phosphate group) added by NADKs. Accordingly, NADKs are now recognised as attractive therapeutic targets.

(SAXS), transmission electron microscopy (TEM) and spectroscopic methods. Synchrotron SAXS experiments on solutions were performed using the BioSAXS robots on beamline BM29 and at Diamond Light Source, UK. Under most conditions of pH, SAXS form factor measurements on RSAIEDLLFDKV are consistent with the presence of unaggregated monomers (Figure 36a). Amyloid formation by peptides can be induced by pH adjustment, changing the net charge on peptide molecules and hence enabling aggregation into β-sheet structures, especially near the isoelectric point. Indeed, SAXS data at pH 4 - 6 shows a very different shape to the intensity profiles at low and high pH. The data show a limiting slope I(q) ~ q-2 at low q, which is characteristic of planar structures such as nanotapes [2], consistent with TEM images. The SAXS data at these pH values was fitted to a form factor of bilayers. The electron density profiles indicate the presence of a dense core 40 Å in extent, with diffuse outer layers. This is a little larger than the calculated molecular length, so the molecules must be interdigitated. The hydrophobic core includes the LLF

residues, while the charged residues are present on each side of the bilayer structure (Figure 36b). Nanotape amyloid form factors were observed in SAXS data in the concentration range 0.2 1 wt% at pH 4. Very precise pH adjustment to pH 4.4 led to the observation of hydrogels at sufficiently high concentration, which were characterised by SAXS and other methods.

In summary, the coronavirus spike protein fragment peptide RSAIEDLLFDKV forms amyloid structures at pH values close to the isoelectric point. This is unexpected because very closely related sequences lie in α-helical regions of coronavirus spike protein S2 domain, and also because this sequence is predicted to have no β-sheet aggregation propensity at pH 4. Subsequent to this work, amyloid formation of several SARS-CoV-2 spike peptides was demonstrated and suggested to be a possible factor in the formation of blood clots because the spike peptides were shown to be able to induce amyloid formation of fibrinogen [3]. Such blood clot formation is characteristic of severe and long COVID-19.