Application of Luminescence Resonance Energy Transfer (LRET) for Study of Substrate Peptide Binding to PglB from N-Linked Glycosylation Pathway in C. Jejuni and C. Lari

Abstract

PglB, a monomeric membrane-bound protein, is the oligosacchryltransferase (OTase) of the N-linked glycosylation system from the gram-negative bacterium Campylobacter jejuni (C. jejuni). PglB is a homologue of the Stt3p subunit of the multimeric eukaryotic OTase and represents a simplified model of this highly conserved enzymatic process. OTases transfer glycans from polyisoprenyl diphosphate-linked carriers to selected Asn residues of target proteins. A recently published 3.4A-resolution crystal structure of PglB from C. lari (Lizak C. et al., 2011 Nature 474, 350–355) provides important insight into the binding determinants of the peptide, although some of the critical periplasmic loops are not well-ordered and it has not yet been possible to experimentally determine the glycan substrate binding site. To gain insight into the potential conformational changes involved in PglB substrate binding and catalysis we have established a Luminescence Resonance Energy Transfer (LRET) approach for studying the system. The strategy devised includes expression of PglB with an N-terminal genetically encoded lanthanide-binding tag (LBT). The LBT is an encodable peptide sequence comprising 24 amino acids that binds and sensitizes Tb3+ as a luminescence donor in LRET experiments. The LBT enables intermolecular LRET measurements between the LBT-Tb3+ complex and a Bodipy-TMR maleimide acceptor attached to a cysteine-containing peptide substrates. Here, we report the establishment of a system to study structural changes associated with substrate peptide binding to PglB from C. jejuni and C. lari using LRET technique. LRET is measured through the span of the membrane with A accuracy, since the Tb3+ donor emission is unpolarized. Additionally, the long-lived luminescence lifetime of the bound Tb3+ is exploited by including a short (50 μs) time delay to eliminate background emission of the acceptor.