Structural and Biochemical Studies of LysM Proteins

Lysin motivet (LysM) er et velkarakteriseret carbohydrat-bindende modul hvilken normalt findes som gentagende enheder i proteiner fra alle organismer, med undtagelse af Archaea. Den funktionelle betydning af denne multiplicitet blev undersøgt med udgangspunkt i to homologe endopeptidaser, Nlp/P60. Disse endopeptidasaer er involveret i peptidoglycan hydrolyse; Cell Wall Lytic Enzyme associated with cell Separation (CwlS) fra Bacillus subtilis og P60_Tth fra Thermus thermophilus. Biokemiske studier, foretaget med heterolog udtrykt CwlS, viste at flere LysM domæner opererer kooperativt for at binde N-acetylglukosamin (NAG) polymerer. Denne øgede affinitet korrelerede positivt med den katalytiske aktivitet af enzymet. Den kooperative ligand binding af LysM domænerne var yderligere demonstreret ved strukturelle studier på P60_2LysM, en variant af P60_Tth bestående af to LysM domæner og derved mangler det katalytiske domæne. Ligand induceret intermolekylær dimerisering blev observeret i co‑krystalstrukturen af P60_2LysM og NAG6. Indtil videre er dette det eneste strukturelle bevis der illustrerer intermolekylær dimerisering af LysM proteiner.Intermolekylær dimerisering af plante LysM receptor kinaser (RK) er blevet foreslået som initierings mekanismen foregåede down-stream signalering. Flere LysM-RK par er endog vist impliceret i symbiose- eller forsvarssignalering. Den ene partner i et LysM-RK par mangler normalt et aktivt kinase domæne og rekrutterer sandsynligvis en aktiv LysM-RK som co-receptor for at mediere down-stream signalering, eksempelvis nodfaktor receptorne 1 og 5 (NFR1 og NFR5) fra Lotus japonicus. Ved binding og genkendelse af rhizobiale nodfaktorer vil NFR1 og NFR5 initiere symbiosesignalering, hvilket fører til udviklingen af rodknolde og symbiotisk nitrogenfiksering. De fleste signaleringskomponenter i nodfaktor pathwayen er blevet identificeret via genetiske metoder. Den aktuelle signaleringsmodel mangler dog komponenter der kan koble genkendelse af nodfaktore ved plasmamembranen til down-stream responser som calcium influks og perinukleær calcium spiking, begge nødvendige for formationen af infektionstråde og rodknolde. Ved brug af nye proteomiske fremgangsmåder er ni receptor kinaser og fire receptor-lignede cytoplasmiske kinaser fundet i pull-down eksperimenter med NFR5 som receptor. Disse formodede interaktører bliver i øjeblikket valideret som komponenter af NFR5 signaleringskomplekset og deres funktionelle roller bliver karakteriseret. The Lysin Motif (LysM) is a well characterised carbohydrate-binding module that is present, usually as repeated entities, in proteins from all organisms except archaea. The functional significance of the multiplicity of the LysM module was investigated using two homologous NlpC/P60 endopeptidases involved in peptidoglycan hydrolysis; the Cell Wall Lytic enzyme associated with cell Separation (CwlS) from Bacillus subtilis, and P60_Tth from Thermus thermopiles. Biochemical studies conducted on purified CwlS showed that multiple LysM modules function cooperatively to bind N-acetylglucosamine (NAG) polymers, and that the increased affinity was positively correlated to the catalytic activity of the enzyme. The cooperativity of LysM domains was further demonstrated in structural studies on P60_2LysM, a variant of P60_Tth that contains only two LysM domains and lacks the catalytic domain. Ligand-induced intermolecular dimerization was observed in the co-crystal structure of P60_2LysM and NAG6. Until today, this is the only structural evidence illustrating intermolecular dimerization of LysM proteins.Intermolecular dimerization of plant LysM receptor kinases (RK) has been proposed as a mechanism for the initiation of downstream signalling. Indeed, several LysM-RK pairs have been reported to be implicated in symbiosis and defence signalling. One partner of the LysM-RK pair usually lacks an active kinase domain and most probably recruits the other active LysM-RK as a co-receptor to mediate downstream signalling. An example of such a LysM-RK pair is the Nod factor receptors 1 and 5 (NFR1 and NFR5) from Lotus japonicus. Upon recognition of rhizobial Nod factors, NFR1 and NFR5 initiate symbiosis signalling, subsequently leading to the development of root nodules and symbiotic nitrogen fixation. Most of the signalling components in the Nod factor signalling pathway have been identified through genetic approaches. The current symbiosis signalling model, however, lacks components that could link Nod factor perception at the plasma membrane to downstream responses, such as calcium influx and perinuclear calcium spiking events that are required for the formation of infection threads and nodules. Using a novel proteomics approach, nine receptor kinases and four receptor-like cytoplasmic kinases were found in pull-down experiments using an NFR5 bait. These putative interactors are currently being validated as components of the NFR5 signalling complex and their functional roles are being characterised.