Chemically Synthesized 58‐mer LysM Domain Binds Lipochitin Oligosaccharide

Abstract

Recognition of carbohydrates by proteins is a ubiquitous biochemical process. In legume-rhizobium symbiosis, lipochitin oligosaccharides, also referred to as nodulation (nod) factors, function as primary rhizobial signal molecules to trigger root nodule development. Perception of these signal molecules is receptor mediated, and nod factor receptor 5 (NFR5) from the model legume Lotus japonicus is predicted to contain three LysM domain binding sites. Here we studied the interactions between nod factor and each of the three NFR5 LysM domains, which were chemically synthesized. LysM domain variants (up to 58 amino acids) designed to optimize solubility were chemically assembled by solid-phase peptide synthesis (SPPS) with microwave heating. Their interaction with nod factors and chitin oligosaccharides was studied by isothermal titration calorimetry and circular dichroism (CD) spectroscopy. LysM2 showed a change in folding upon nod factor binding, thus providing direct evidence that the LysM domain of NFR5 recognizes lipochitin oligosaccharides. These results clearly show that the L. japonicus LysM2 domain binds to the nod factor from Mesorhizobium loti, thereby causing a conformational change in the LysM2 domain. The preferential affinity for nod factors over chitin oligosaccharides was demonstrated by a newly developed glycan microarray. Besides the biological implications, our approach shows that carbohydrate binding to a small protein domain can be detected by CD spectroscopy.

Structural Insight Into the Role of Mutual Polymorphism and Conservatism in the Contact Zone of the NFR5–K1 Heterodimer With the Nod Factor

“…The key step in the formation of the rhizobia-legume symbiosis is the dialogue between partners through Nod factors (NFs, signaling molecules) from the rhizobia side and the system of receptors from the plant side that specifically recognizes the NF decorations. In spite of the fact that putative NF receptors were discovered more than 10 years ago, the direct interaction of NFs with any of the receptors was demonstrated in only a few studies (Broghammer et al, 2012 ; Sørensen et al, 2014 ). We supposed that the reason that blocks the experimental detection of this direct interaction concerns the hypothesis of a heterodimer receptor for NF.…”

High‐Performance Reversed‐Phase Flash Chromatography Purification of Peptides and Chemically Modified Insulins

“…The 54‐amino acid peptide, derived from Lys M1 domain from nfr5, Lotus japonicus , [39] was synthesized using microwave assisted SPPS but without optimization and was obtained with a crude purity of 20 % as determined by LC–MS. The Lys M1 peptide was challenging to purify due to its inherent low solubility.…”

Microarray Strategies for Exploring Bacterial Surface Glycans and Their Interactions With Glycan-Binding Proteins

“…Analysis of the binding to the array of P60, an autolysin of Listeria monocytogenes that hydrolyzes the cell wall peptidoglycan and is essential for bacterial virulence, revealed recognition of chitin oligosaccharides of ≥ 5 GlcNAc units and selective binding to some Nod factors, in particular those containing a C18:1 lipid chain. Interestingly, a chemically synthesized LysM domain of the Nod factor receptor 5 from the legume Lotus japonicas was also found to show preference for Nod factors with C18:1 lipid chains (Sorensen et al, 2014). Thus, the bacterial and plant LysM domains appeared to exhibit a similar dependence on the lipid structure, hinting at a possible role of the lipid moiety in the binding of LysM domains to Nod factors.…”