Towards understanding of HMGA1a protein post-translational modifications (PTMs) via protein chemical synthesis approach

Abstract

Post-translational modifications (PTMs) on proteins not only dramatically increase the proteome diversity, but also affect protein structure, activity, stability, localization, interaction and other functions in cellular activities. As a typical member of intrinsically disordered proteins (IDPs), High-mobility group (HMG) A1a protein bears many sites for PTMs. The function of these PTMs on this protein remains largely elusive. One of the reasons that impeded the systemic study of how PTMs on HMGA1a affect its function is the lack of homogenous HMGA1a with site-specific modification(s). In this study, we mainly focus on 1) using protein chemical synthesis to address the accessibility problem of homogenous proteins with site-specific PTM(s), 2) using Hmb-assisted Ser/Thr ligation to synthesize site-specifically modified HMGA1a analogs for deciphering the function of HMGA1a PTMs such as phosphorylation at the C-terminal tail and acetylation on the protein binding domain. 3) exploring a novel prior-capture Ser/Thr ligation strategy for peptide ligation at high dilution conditions.

To systemically study the function of triple phosphorylations (Ser98/101/102) at the HMGA1a C-terminal tail domain, nine HMGA1a proteins with site-specific phosphorylation have been synthesized via Hmb-assisted Ser/Thr ligation according to their endogenously existing forms. By employing in vitro kinase assay, nuclear magnetic resonance (NMR), and pull-down assay, we showed that the triple phosphorylation at the HMGA1a C-terminal tail domain could alter HMGA1a conformation from a disordered conformation to a more compacted and stable form by the interaction between the phosphorylated C-terminal tail and the certain region of HMGA1a (01-91). Furthermore, the influence of the C-terminal phosphorylation on protein-protein interactions (PPIs) between HMGA1a and other proteins was studied. Pull-down assay was used to investigate the influence of HMGA1a C-terminal phosphorylation on the interaction between HMGA1a and P53. Besides, the importance of C-terminal phosphorylation on modulating HMGA1a PPIs was demonstrated using mass spectrometry-based chemical proteomics. Moreover, biotinylated HMGA1a protein with four Lys in the protein binding domain (Lys64/66/70/73) being acetylated was synthesized for investigating the influence of HMGA1a acetylation on the interaction between HMGA1a and P53. Taken together, an integrated chemical biology approach has been set up to study PTMs’ affect on HMGA1a PPIs, which provides a good example to unveil how PTMs affect IDPs' conformation and the resulting influence on their function such as PPIs. On the other hand, this work showcased that the protein chemical synthesis serves as a good tool for the studies of protein PTMs.

With an aim to further improve the efficiency of chemical ligation in the synthesis of complex proteins, we explored the idea of a prior-capture Ser/Thr ligation, which involved bringing two ligating peptide fragments of Ser/Thr ligation together via a disulfide formation prior to ligation. Model studies showed that such a prior-capture via disulfide formation was fully compatible with Ser/Thr ligation and could expedite the ligation process at highly diluted conditions, which laid the foundation for further exploration of prior-capture Ser/Thr ligation in the complex protein chemical synthesis.