Effect of Phosphorylation on the Structural Behaviour of Peptides Derived from the Intrinsically Disordered C-Terminal Domain of Histone H1.0

Abstract

To investigate the structural impact of phosphorylation on the human histone H1.0 C-terminal domain, we performed NMR structural studies of model peptides containing a single phosphorylation site: T¹¹⁸ -H1.0 (T¹¹⁸ PKK motif) and T¹⁴⁰ -H1.0 (T¹⁴⁰ PVK motif). Both model peptides are mainly disordered in aqueous solution in their non-phosphorylated and phosphorylated forms, but become structured in the presence of trifluoroethanol. The peptides T¹¹⁸ -H1.0 and pT¹¹⁸ -H1.0 contain two helical regions, a long amphipathic α helix spanning residues 104-115 and a short α/3₁₀ helix (residues 119-123), that are almost perpendicular in T¹¹⁸ -H1.0 but have a poorly defined orientation in pT¹¹⁸ -H1.0. Peptides T¹⁴⁰ -H1.0 and pT¹⁴⁰ -H1.0 form very similar α helices between residues 141-147. The TPKK and TPVK motifs show the same backbone conformation, but differ in their side-chain contacts; the Thr and pThr side chains interact with the i+2 Lys side chain in the TPKK motif, and with the i+3 Lys side chain in the TPVK motif. The pT phosphate group in pT¹¹⁸ -H1.0 and pT¹⁴⁰ -H1.0 has pK_a values below the intrinsic values, which can be explained by non-specific charge-charge interactions with nearby Lys. The non-polar Val in the TPVK motif accounts for the pT¹⁴⁰ pK_a being closer to the intrinsic pK_a value than the pT¹¹⁸ pK_a . Altogether, these results validate that minimalist strategies using model peptides can provide structural details difficult to obtain in short-lived intrinsically disordered proteins and domains.

Keywords: NMR spectroscopy; peptides; phosphorylation; protein models; protein structures; structure elucidation.