Details

Autor(en) / Beteiligte

• Piemontese, Emanuele
• Herfort, Alina
• Perevedentseva, Yulia
• Möller, Heiko M.
• Seitz, Oliver

Titel

Multiphosphorylation-Dependent Recognition of Anti-pS2 Antibodies against RNA Polymerase II C‑Terminal Domain Revealed by Chemical Synthesis

Ist Teil von

• Journal of the American Chemical Society, 2024-05, Vol.146 (17), p.12074-12086

Ort / Verlag

United States: American Chemical Society

Erscheinungsjahr

2024

Link zum Volltext

Quelle

MEDLINE

Beschreibungen/Notizen

• Phosphorylation is a major constituent of the CTD code, which describes the set of post-translational modifications on 52 repeats of a YSPTSPS consensus heptad that orchestrates the binding of regulatory proteins to the C-terminal domain (CTD) of RNA polymerase II. Phospho-specific antibodies are used to detect CTD phosphorylation patterns. However, their recognition repertoire is underexplored due to limitations in the synthesis of long multiphosphorylated peptides. Herein, we describe the development of a synthesis strategy that provides access to multiphosphorylated CTD peptides in high purity without HPLC purification for immobilization onto microtiter plates. Native chemical ligation was used to assemble 12 heptad repeats in various phosphoforms. The synthesis of >60 CTD peptides, 48–90 amino acids in length and containing up to 6 phosphosites, enabled a detailed and rapid analysis of the binding characteristics of different anti-pSer2 antibodies. The three antibodies tested showed positional selectivity with marked differences in the affinity of the antibodies for pSer2-containing peptides. Furthermore, the length of the phosphopeptides allowed a systematic analysis of the multivalent chelate-type interactions. The absence of multivalency-induced binding enhancements is probably due to the high flexibility of the CTD scaffold. The effect of clustered phosphorylation proved to be more complex. Recognition of pSer2 by anti-pSer2-antibodies can be prevented and, perhaps surprisingly, enhanced by the phosphorylation of “bystander” amino acids in the vicinity. The results have relevance for functional analysis of the CTD in cell biological experiments.