E. coli sabotages the in vivo production of O-linked ß-N-acetylglucosamine-modified proteins.

The O-linked ß-N-acetylglucosamine (O-GlcNAc) post-translational modification is an important, regulatory modification of cytosolic and nuclear enzymes. To date, no 3-dimensional structures of O-GlcNAc-modified proteins exist due to difficulties in producing sufficient quantities with either in vitro or in vivo techniques. Recombinant co-expression of substrate protein and O-GlcNAc transferase in Escherichia coli was used to produce O-GlcNAc-modified domains of human cAMP responsive element-binding protein (CREB1) and Abelson tyrosine-kinase 2 (ABL2). Recombinant expression in E. coli is an advantageous approach, but only small quantities of insoluble O-GlcNAc-modified protein were produced. Adding ß-N-acetylglucosaminidase inhibitor, O-(2-acetamido-2-dexoy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), to the culture media provided the first evidence that an E. coli enzyme cleaves O-GlcNAc from proteins in vivo. With the inhibitor present, the yields of O-GlcNAc-modified protein increased. The E. coli ß-N-acetylglucosaminidase was isolated and shown to cleave O-GlcNAc from a synthetic O-GlcNAc-peptide in vitro. The identity of the interfering ß-N-acetylglucosaminidase was confirmed by testing a nagZ knockout strain. In E. coli, NagZ natively cleaves the GlcNAc-ß1,4-N-acetylmuramic acid linkage to recycle peptidoglycan in the cytoplasm and cleaves the GlcNAc-ß-O-linkage of foreign O-GlcNAc-modified proteins in vivo, sabotaging the recombinant co-expression system.

X-ray crystallographic structure of an artificial beta-sheet dimer.

This paper describes the X-ray crystallographic structure of a designed cyclic beta-sheet peptide that forms a well-defined hydrogen-bonded dimer that mimics beta-sheet dimers formed by proteins. The 54-membered ring macrocyclic peptide (1a) contains molecular template and turn units that induce beta-sheet structure in a heptapeptide strand that forms the dimerization interface. The X-ray crystallographic structure reveals the structures of the two "Hao" amino acids that help template the beta-sheet structure and the two delta-linked ornithine turn units that link the Hao-containing template to the heptapeptide beta-strand. The Hao amino acids adopt a conformation that resembles a tripeptide in a beta-strand conformation, with one edge of the Hao unit presenting an alternating array of hydrogen-bond donor and acceptor groups in the same pattern as that of a tripeptide beta-strand. The delta-linked ornithines adopt a conformation that resembles a hydrogen-bonded beta-turn, in which the ornithine takes the place of the i+1 and i+2 residues. The dimers formed by macrocyclic beta-sheet 1a resemble the dimers of many proteins, such as defensin HNP-3, the lambda-Cro repressor, interleukin 8, and the ribonuclease H domain of HIV-1 reverse transcriptase. The dimers of 1a self-assemble in the solid state into a barrel-shaped trimer of dimers in which the three dimers are arranged in a triangular fashion. Molecular modeling in which one of the three dimers is removed and the remaining two dimers are aligned face-to-face provides a model of the dimers of dimers of closely related macrocyclic beta-sheet peptides that were observed in solution.