LysM Domains from Pteris ryukyuensis Chitinase-A

Abstract

The LysM domain probably binds peptidoglycans, but how it does so has yet to be described. For this report, we measured the thermal stabilities of recombinant LysM domains derived from Pteris ryukyuensis chitinase-A (PrChi-A) and monitored their binding to N-acetylglucosamine oligomers ((GlcNAc) n ) using differential scanning calorimetry, isothermal titration calorimetry, and NMR spectroscopy. We thereby characterized certain of the domains' functional and structural features. We observed that the domains are very resistant to thermal denaturation and that this resistance depends on the presence of disulfide bonds. We also show that the stoichiometry of (GlcNAc) n /LysM domain binding is 1:1. (GlcNAc) 5 titration experiments, monitored by NMR spectroscopy, allowed us to identify the domain residues that are critical for (GlcNAc) 5 binding. The binding site is a shallow groove formed by the N-terminal part of helix 1, the loop between strand 1 and helix 1, the C-terminal part of helix 2, and the loop between helix 2 and strand 2. Furthermore, mutagenesis experiments reiterate the critical involvement of Tyr 72 in (GlcNAc) n /LysM domain binding. Ours is the first report describing the physical structure of a LysM oligosaccharide-binding site based on experimental data.Carbohydrates are often the signals that initiate biochemical processes. For example, as a response to pathogenic attacks, plant defense proteins first recognize and then attack the carbohydrate components of the pathogens' cell walls (1). As these defense proteins bind carbohydrates (2), via their noncatalytic carbohydrate-binding modules, their binding mechanisms are physiologically interesting. Many carbohydrate-binding modules have been structurally and/or functionally characterized (3) and, based on these characteristics, are currently grouped into 49 families in the Carbohydrate-Active Enzyme (CAZy) data base (available on the World Wide Web).The LysM domain, which probably binds chitin, a ␤-(134)-linked N-acetylglucosamine oligosaccharide ((GlcNAc) n ), was originally identified as a component of bacterial lysins. This domain is found in many of the enzymes involved in cell wall degradation and is also present in other proteins that are associated with bacterial cell walls (4 -8). Basal level resistance by plants against certain pathogens also appears to involve the recognition of chitin oligosaccharides and related compounds. Recently, a chitin oligosaccharide elicitor receptor was purified, and it contains a LysM domain within its extracellular domain (9). Additionally, certain symbiotic relationships between leguminous plants and rhizobial bacteria appear to be mediated by LysM domain/chitin oligosaccharide interactions. Recent studies have identified a class of proteins that recognize Nod factors, which are the lipochitin oligosaccharide signal molecules secreted by symbiotic bacteria (10 -12). These plant proteins are members of a serine/threonine receptor kinase family and contain extracellular LysM domains. After reviewing the availab...

Molecular basis for bacterial peptidoglycan recognition by LysM domains

“…Interestingly, our results suggest that unlike the eukaryotic LysM domains from plants and fungi studied so far25263132 tandem bacterial LysM modules do not form any stable quaternary structure, and contribute to binding in an additive manner. Using peptidoglycan fragments and commercially available polysaccharides, we show that LysM recognizes both the GlcNAc moiety of polysaccharides and to a lesser extent the peptide stems.…”

“…Despite a wealth of knowledge as to the role of LysM proteins, only scarce information is available concerning the mechanism underpinning LysM–carbohydrate interaction242526. The recent elucidation of the structure of Arabidopsis thaliana AtCERK1 LysM domain in complex with a chitooligosaccharide revealed the molecular basis for chitin recognition25.…”

The Lysin Motif Receptor-like Kinase (LysM-RLK) CERK1 Is a Major Chitin-binding Protein in Arabidopsis thaliana and Subject to Chitin-induced Phosphorylation

“…The requirement of several LysM domains for optimum substrate binding has been demonstrated for bacterial peptidoglycan hydrolases (39). Isothermal titration calorimetry of LysM domains from a fern chitinase suggests that each LysM domain can bind one chitin oligomer (41). Consequently, binding to individual oligomer molecules might be brought about by single LysM domains, whereas several LysM motifs may act cooperatively in binding to polymeric chitin.…”

“…ryukyuensis (41,42). Also, legume LysM-RLKs, which are closely related to CERK1 and implicated in Nod-factor perception, contain more than one LysM motif (10,11).…”

The Cpx Envelope Stress Response Modifies Peptidoglycan Cross-Linking via the l,d-Transpeptidase LdtD and the Novel Protein YgaU

“…YgaU is localized to the inner membrane (our unpublished data), likely by virtue of its BON domain (16), while its LysM domain is predicted to bind the NAG moiety of PG (44,45). How, then, could YgaU bind to the NAG moiety of PG and regulate 3-3 cross-linking imposed by LdtD from the cytoplasm?…”