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Convergent chemical synthesis of histone H2B protein for the site-specific ubiquitination at Lys34.

Posttranslational modifications (PTMs) play a vital role in regulating the structure and dynamics of chromatin as well as DNA-driven cellular processes by covalently modifying the primary structures of the histone proteins (H2B, H2A, H3, H4) with various chemical entities. One such example is the modification of the histone H2B by ubiquitin (Ub) at multiple lysine residues. Of these, ubiquitination at Lys120 (H2K120Ub) is best studied. This PTM was suggested to modulate gene expression, trigger H3 methylation, and activate DNA damage response pathways. In contrast, little is known about the effects and functions of the other sites of ubiquitination in H2B, K34, K46, K108, and K116. Notably, the preparation of any of these ubiquitinated histone proteins for biochemical analyses has been a challenge in the field and has thus hampered a variety of studies aiming to understand the effect of ubiquitination on chromatin. Only recently, novel nonenzymatic approaches were introduced and enabled the preparation of homogeneously native H2K120Ub on a milligram scale for structural and functional analyses. These approaches proved to be successful for the incorporation of this single modification or any other modification within the short C-terminal synthetic peptide. In order to investigate the role of ubiquitination at other sites and to study the interplay of multiple modifications, a new synthetic strategy is needed. For example, it has been reported recently that ubiquitination of H2B at Lys34 directly regulates H3K4 and K79 methylation through trans-tail crosstalk both in vitro and in cells. Hence, the preparation of such an analogue will enable studies aiming to understand how this modification regulates H3 methylation and potentially affects the structure of chromatin, as well as help to delineate mechanisms of K34 ubiquitination and deubiquitination. Here, we report our endeavors toward the total chemical synthesis of the H2B protein, and the successful sitespecific ubiquitination at Lys34 (H2K34Ub) for initial functional characterization. If one considers the preparation of H2K34Ub semisynthetically through the expression of a large C-terminal H2B fragment that bears an N-terminal Cys or through total chemical synthesis by applying a ligation approach with three fragments, the 57mer H2B(1–57), which bears the K34 modification, has to be prepared synthetically. Because H2B lacks any Cys residue, an Ala to Cys mutation for native chemical ligation (NCL) combined with desulfurization should be performed at Ala58 (Scheme 1). Even if the semisynthetic method were applicable, it would not allow the insertion of different PTMs along the sequence but only at the N-terminal fragment. Our initial studies with a threefragment approach revealed that the synthesis of the 57mer peptide via Fmoc-SPPS was very difficult. We thus concluded that a total chemical synthesis of the H2B protein from four fragments is inevitable. Hence, we divided the H2B sequence, taking into consideration the position of Ala residues, to include H2B(1–20) labeled with an HA tag, H2B(21–57), H2B(58–96), and H2B(97–125). In such an approach, ubiquitination at K34 can be achieved by incorporating dmercaptolysine (K*) in the fragment H2B(21–57), thus enabling isopeptide native chemical ligation (ICL). We also envisioned a convergent chemical synthesis approach via a peptide hydrazide intermediate for the final assembly, first by ligating fragments H2B(1–20) and H2B(21–57), which bears a Ub, followed by ligation with H2B(58–125), which is prepared through ligation of fragments H2B(58–96) and H2B(97–125). Convergent protein synthesis offers several advantages over linear approaches and several promising strategies have been reported recently. Initially, we examined the synthesis of unmodified H2B to set the ground for the preparation of H2B that is modified at any desired site. Peptides H2B(1–20), H2B(21–57), and H2B(58–96), which bear activated C-termini, were prepared using the N-acylurea method (Supporting Information). The N-terminal Ala in H2B(21–57) and H2B(97–125) were substituted with Cys to give peptides 3 and 1, respectively, and in H2B(58–96) with a 1,3-thiazolidine-4-carboxo (Thz) moiety to give peptide 2. To enable a convergent synthesis, the N-acylurea moiety in the H2B(21–57) fragment was substituted with hydrazine to form the stable C-terminal acyl hydrazide 3 (Scheme 1). Direct synthesis of this peptide on hydrazine-functionalized resin failed to give the desired product directly. Overall, the syntheses of all fragments were straightforward with good yields (30–50 %; Supporting Information). [*] P. Siman, Dr. S. V. Karthikeyan, Prof. A. Brik Department of Chemistry Ben Gurion University of the Negev Beer Sheva 84105 (Israel) E-mail: abrik@bgu.ac.il Homepage: http://www.bgu.ac.il/~abrik