A CON-based NMR assignment strategy for pro-rich intrinsically disordered proteins with low signal dispersion: the C-terminal domain of histone H1.0 as a case study

Abstract

The C-terminal domain of histone H1.0 (C-H1.0) is involved in DNA binding and is a main determinant of the chromatin condensing properties of histone H1.0. Phosphorylation at the (S/T)-P-X-(K/R) motifs affects DNA binding and is crucial for regulation of C-H1.0 function. Since C-H1.0 is an intrinsically disordered domain, solution NMR is an excellent approach to characterize the effect of phosphorylation on the structural and dynamic properties of C-H1.0. However, its very repetitive, lowamino acid-diverse and Pro-rich sequence, together with the low signal dispersion observed at the 1 H-15 N HSQC spectra of both non-and tri-phosphorylated C-H1.0 preclude the use of standard 1 H-detected assignment strategies. We have achieved an essentially complete assignment of the heavy backbone atoms (15 N, 13 C' and 13 Cα), as well as 1 H N and 13 Cβ nuclei, of non-and tri-phosphorylated C-H1.0 by applying a novel 13 C-detected CON-based strategy. No C-H1.0 region with a clear secondary structure tendency was detected by chemical shift analyses, confirming at residue level that C-H1.0 is disordered in aqueous solution. Phosphorylation only affected the chemical shifts of phosphorylated Thr's, and their adjacent residues. Heteronuclear { 1 H}-15 N NOEs were also essentially equal in the non-and tri-phosphorylated states. Hence, structural tendencies and dynamic properties of C-H1.0 free in aqueous solution are unmodified by phosphorylation. We propose that the assignment strategy used for C-H1.0, which is based on the acquisition of only a few 3D spectra, is an excellent choice for short-lived intrinsically disordered proteins with repetitive sequences.

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

“…These results are consistent with data from a non-phosphorylated peptide, which encompasses residues 99-121 of C-H1.0 (Figure 1) and contains the T 118 PKK phosphorylation site, [16] as well as from the full-length C-H1.0, [3,10] which were shown to be disordered in aqueous solution. Furthermore, we compared the 13 Cα and 13 Cβ chemical shifts in the non-phosphorylated peptides with those in the nonphosphorylated C-H1.0 domain, [10] and the equivalent values in phosphorylated peptides with those in the tri-phosphorylated pT-C-H1.0.…”

“…[8] [9] Based on the fact that dephosphorylation of two different H1 subtypes is dependent on the cis-trans prolyl-isomerase activity Pin1 in vivo, [8] it was proposed that cis-trans proline isomerization might play a role in the observed phosphorylation-induced conformational change in DNAbound H1 C-terminal domain. [7] More recently we performed a NMR characterisation of the C-terminal domain of the H1.0 subtype (C-H1.0; [10] ) in aqueous solution. However this construct presents some experimental problems caused by its short sample life, due to degradation and aggregation.…”

“…We compare the structural behaviour between non-phosphorylated (T 118 -H1.0 and T 140 -H1.0) and phosphorylated (pT 118 -H1.0 and pT 140 -H1.0) peptides, and with the corresponding regions in C-H1.0/pT-C-H1.0 domain. [10] In addition, we determined the pKa of the phospho-threonine in the two phosphorylated peptides, and analysed the impact of the ionization state on the structure of these peptides.…”

Exclusively heteronuclear NMR experiments for the investigation of intrinsically disordered proteins: focusing on proline residues

“…These observations clearly show the importance of experimental atomic resolution information on the structural and dynamic properties of proline residues to understand their role in modulating protein function. While abundant information about proline residues in globular protein folds is available either through NMR or X-ray studies (MacArthur and Thornton, 1991), including several examples of cis-trans isomerization of peptide bonds involving proline nitrogen as molecular switches (Lu et al, 2007), their characterization in highly flexible, disordered polypeptides is available only in a handful of cases (Chaves-Arquero et al, 2018;Gibbs et al, 2017;Haba et al, 2013;Hošek et al, 2016;Knoblich et al, 2009;Pérez et al, 2009;Piai et al, 2016) and actually early studies on IDPs/IDRs routinely reported assignment statistics only considering all other amino acids ("excluding prolines").…”

HACANCOi: a new Hα-detected experiment for backbone resonance assignment of intrinsically disordered proteins

“…The base level of iHA(CA)N(CO) and HA(CA) N(CO)i experiments at each temperature is identical i.e. the peak intensities are directly comparable between the two experiments experiments they often apply the 'CON' strategy for connecting neighboring residues with highly degenerate chemical shifts (Pantoja-Uceda and Santoro 2014; Sahu et al 2014;Brutscher et al 2015;Chaves-Arquero et al 2018). Another salient feature of the 13 C-detected strategy is the absence of the disturbing residual water signal, i.e.…”