Phosphate‐Rich Biomimetic Peptides Shed Light on High‐Affinity Hyperphosphorylated Uranyl Binding Sites in Phosphoproteins

Abstract

Some phosphoproteins such as osteopontin (OPN) have been identified as high‐affinity uranyl targets. However, the binding sites required for interaction with uranyl and therefore involved in its toxicity have not been identified in the whole protein. The biomimetic approach proposed here aimed to decipher the nature of these sites and should help to understand the role of the multiple phosphorylations in UO22+ binding. Two hyperphosphorylated cyclic peptides, pS168 and pS1368 containing up to four phosphoserine (pSer) residues over the ten amino acids present in the sequences, were synthesized with all reactions performed in the solid phase, including post‐phosphorylation. These β‐sheet‐structured peptides present four coordinating residues from four amino acid side chains pointing to the metal ion, either three pSer and one glutamate in pS168 or four pSer in pS1368. Significantly, increasing the number of pSer residues up to four in the cyclodecapeptide scaffolds produced molecules with an affinity constant for UO22+ that is as large as that reported for osteopontin at physiological pH. The phosphate‐rich pS1368 can thus be considered a relevant model of UO22+ coordination in this intrinsically disordered protein, which wraps around the metal ion to gather four phosphate groups in the UO22+ coordination sphere. These model hyperphosphorylated peptides are highly selective for UO22+ with respect to endogenous Ca2+, which makes them good starting structures for selective UO22+ complexation.

Uranyl Binding to Proteins and Structural-Functional Impacts

“…Both acidic residues (Asp/Glu) and phosphoserines (pSer) were systematically introduced in the key positions (X1, X3, X6, and X8) of the peptide [32][33][34]. It showed that the stability of UO 2 2+ -peptide complex increased successively by replacing the 4 Glu residues (logK = 8.2) in these positions with 1-4 pSer residues, i.e., 3 Glu/1 pSer, logK = 9.2; 2 Glu/2 pSer, logK = 10.1; 1 Glu/3 pSer, logK = 10.7; and 4 pSer, logK = 11.3.…”

“…It showed that the stability of UO 2 2+ -peptide complex increased successively by replacing the 4 Glu residues (logK = 8.2) in these positions with 1-4 pSer residues, i.e., 3 Glu/1 pSer, logK = 9.2; 2 Glu/2 pSer, logK = 10.1; 1 Glu/3 pSer, logK = 10.7; and 4 pSer, logK = 11.3. Moreover, both 1 Glu/3 pSer and 4 pSer peptides can trap two UO 2 2+ ions and form bimetallic complexes, suggesting a decisive role of the phosphate groups [34]. In an application research, Hu, Wang and co-workers designed a fluorescent sensor for detection of uranyl ions based on the cyclic peptide [35].…”

Hydrophobic core formation and secondary structure elements in uranyl(vi)-binding peptides

“…19 Higher affinity was achieved by phosphorylation of the binding sites. [20][21][22][23] However, we found that high affinity can be achieved not only through phosphorylation but also by selecting an appropriate size of peptides. For example, previously studied peptide A1 (c[EREPGEWEPG]) was found to have a binding energy of À43.6 kcal mol À1 with UO 2…”

A Simple Fluorescence Affinity Assay to Decipher Uranyl‐Binding to Native Proteins

“…four coordinating glutamate and/or phosphoserine (pSer) residues. [11,[17][18][19] These peptides were named pSn, n being the number of pSer and (4À n) being accordingly the number of coordinating glutamates in the sequence. Sequences of the peptides used in this study are described in the Supporting Information (Table S1).…”

“…As a consequence, analysis of the Trp fluorescence data had been performed making assumptions on the emitting or non-emitting character of these metal species. [17] It was thus necessary to confirm the obtained stability constants prior to building an affinity scale. NaphP fluorescence being independent from the fluorescence of the UO 2 pSn complexes, it was possible to re-assess these constants by observing Naph fluorescence (λ ex = 344 nm, λ em = 380 nm).…”