Total synthesis of Ser68-phosphorylated CENP-A

Abstract

As a conserved marker of the centromere, CENP-A is crucial for controlling the accurate segregation of chromatins in mitosis and meiosis. Phosphorylation at Ser68 is suggested to be crucial to CENP-A deposition at the centromere, but it is still under debate. To verify the importance of Ser68 phosphorylation, CENP-A with phosphorylated Ser68 was totally synthesized. Total chemical synthesis is a powerful tool to obtain proteins with unnatural or modified amino acids. Traditional recombinant protein expression, which can only generate wild-type proteins, is not suitable for special or complicated proteins bearing post-translational modifications. In chemical protein synthesis, peptide fragments are built manually step by step, so that special amino acid coupling can be controlled at the atomic level. During total synthesis of phosphorylated CENP-A at Ser68, we applied N-Fmoc-protected solid-phase peptide synthesis (SPPS) to produce peptides, and native chemical ligation (NCL) to connect them together by forming a native amide bond at the ligation site. Standard SPPS is efficient for synthesizing short peptides of approximately 50 amino acids. This restriction is overcome by chemo-selective amide bond forming ligation reaction for which NCL is employed frequently. Generally, two fragments of non-protected polypeptides are required, one with a C-terminal thioester and another with cysteinyl peptide. In our project, CENP-A was spilt into five fragments that ligated convergently: CENP-A-1: GPRRRSRKPEAPRRRSPSPTPTPGPSRRGPSLG-NHNH2; CENP-A-2: CSSHQH SRRRQGWLKE IRKLQKSTHL LIRKLPFS(P)RL-Dbz; CENP-A-3: Thz-REICVKFTR GVDFNWQAQA LLALQEA-Nbz-R-R; CENP-A-4: Thz-EAF LVHLFEDAYL LTLH-Nbz-R; and CENP-A-5: Thz-GRVT LFPKDVQLAR RIRGLEEGLG-HMBA-R-G-Nbz. Three types of thioester precursors—hydrazide, benzotriazole, and Nbz—were employed, based on convenience and efficiency, to produce thioester in situ. The cysteine at the C-terminal of CENP-A-3, CENP-A-4, and CENP-A-5 was protected by thiazolidine in case of ligation with itself. Solution-phase and solid-phase approaches were combined in the designed synthetic route. The CENP-3-4-5 fragment was prepared using solid phase because purification of the product subsequent to ligation was challenging in solution-phase methodology. With final desulfurization, we obtained proteins of interest with qualified purity. This study demonstrates the synthesis of phosphorylated CENP-A in detail. Importantly, several reaction conditions, including the Dbz protecting protocol and conversion yield from Dbz to Nbz, were largely improved compared to previous studies.