Quasi-Racemic X-ray Structures of K27-Linked Ubiquitin Chains Prepared by Total Chemical Synthesis

Abstract

Quasi-racemic crystallography has been used to determine the X-ray structures of K27-linked ubiquitin (Ub) chains prepared through total chemical synthesis. Crystal structures of K27-linked di- and tri-ubiquitins reveal that the isopeptide linkages are confined in a unique buried conformation, which provides the molecular basis for the distinctive function of K27 linkage compared to the other seven Ub chains. K27-linked di- and triUb were found to adopt different structural conformations in the crystals, one being symmetric whereas the other triangular. Furthermore, bioactivity experiments showed that the ovarian tumor family de-ubiquitinase 2 significantly favors K27-linked triUb than K27-linked diUb. K27-linked triUb represents the so-far largest chemically synthesized protein (228 amino acids) that has been crystallized to afford a high-resolution X-ray structure.

A Highly Efficient Synthesis of Polyubiquitin Chains

“…For the synthesis of K48‐diUb 10 and K63‐diUb 11 , two equivalents of segment 1 were ligated with one equivalent of either segment 2 (for K48) or 3 (for K63) in a single hydrazide‐based native chemical ligation64, 65, 66, 67, 68, 69, 70 step followed by a single desulfurization procedure 42, 71, 72. One representative ligation process between 1 and 3 was complete within 12 h, and the subsequent desulfurization step afforded full‐length K63‐diUb 11 on an 80 mg scale in an overall yield of 32%, as confirmed by HPLC and ESI‐MS ( Figure 3 A–C and Figure S7 (Supporting Information)).…”

A molecular engineering toolbox for the structural biologist

“…Baculovirus vectors that can deliver a large cargo of genetic material (>30 kb) to a variety of mammalian cells have also been developed for more efficient UAA incorporation (Chatterjee et al . 2013c; Zheng et al . 2016).…”

“…D-enantiomers of proteins can be made using solid-phase peptide synthesis and NCL, with synthesis efficiencies that have allowed the structure determination of constructs in the 200-amino acid range (Mandal et al . 2012; Pan et al . 2016).…”

Chemical Protein Synthesis Enabled Mechanistic Studies on the Molecular Recognition of K27‐linked Ubiquitin Chains

“…[12,13] Recently,c hemical protein synthesis was used to prepare K27 diUb and triUb, and their X-ray crystal structures were solved. [14] Fushman et al generated K27 diUb using the genetically encoded orthogonal protection and activated ligation (GOPAL) method and reported its NMR structure. [15] An interesting finding of both studies was that the K27 isopeptide bond is rigidly buried, such that the inter-domain mobility of K27 Ubs is restricted.…”

“…To this end, smFRET technology [17] was used and FRET-compatible fluorescent donor and acceptor (Cy3/ Cy5) were attached separately onto the two Ub units of K27 diUb (probes 1 to 4,F igure 1A). [14,22] Taking the synthesis of probe 1 as example,Ubmutant 1a' ' was expressed in E. coli bearing aC-terminal Cys (obtained in ay ield of 80 mg L À1 of E. coli cell culture). [14,22] Taking the synthesis of probe 1 as example,Ubmutant 1a' ' was expressed in E. coli bearing aC-terminal Cys (obtained in ay ield of 80 mg L À1 of E. coli cell culture).…”