Universität zu Köln

Hasegawa, Keiichi ORCID: 0009-0004-4251-877X (2024). Activation mechanism of mixed-lineage-kinase-like proteins in plants. PhD thesis, Universität zu Köln.

PDF Phd thesis_Keiichi_Hasegawa Final.pdf - Accepted Version Download (11MB)

Abstract

Plants and animals have similarities in their immune system. A new protein family was reported to be conserved across seed plants that structurally resembles animal mixed lineage kinase domain-like (MLKL), a mediator of necroptotic cell death. The Arabidopsis MLKLs (AtMLKLs) play a role in disease resistance mediated by TIR-type nucleotide binding–leucine-rich repeats (NLRs). Cryo-EM structures of Arabidopsis MLKLs (AtMLKLs) reveal a tetrameric configuration in contrast with the monomeric structure of animal MLKL. To understand the biological meaning of the AtMLKL tetramer, I performed a structure-guided study. Together with a newly established disease resistance assay in N. benthamiana, the data imply that the AtMLKL tetramers represent an auto-repressed conformation of plant MLKLs and the exposure of the N-terminal HeLo-domain from the tetramer is an important activation step, which could be mediated by a phosphorylation in the activation loop of the pseudokinase domain. To elucidate the genetic relationship between helper NLRs and plant MLKLs, the disease resistance activities of AtMLKL1 variants were examined in helper NLR null background and <I>eds1 pad4 sag101</I> null background. The plant MLKL-mediated immunity was partially retained in the helperless mutant background but no longer retained in the <I>eds1 pad4 sag101</I> mutant in <I>N. benthamiana</I>. It was found that the AtMLKL1 HeLo domain elicits Ca2+ influx in human cell line and in N. benthamiana. These data demonstrate that plant and animal MLKLs commonly utilize the N-terminal HeLo-domain as a signaling domain for immune responses. Furthermore, to study the activation step, I purified N-terminal HeLo domain of AtMLKL1 and a gain-of-function variant of AtMLKL1 carrying a phosphomimetic mutation in the activation loop of the pseudokinase domain. The purified recombinant N-terminal HeLo domain of AtMLKL1 autonomously formed a higher-order oligomer with a 10 nm ring-like structure. Additionally, the purified recombinant phosphomimetic variant of AtMLKL1 retained a tetrameric configuration similar as purified recombinant AtMLKL1 wild-type, implying that AtMLKL does not change it conformation via phosphorylation in the pseudokinase domain upon activation unlike animal MLKL.

Item Type:	Thesis (PhD thesis)
Creators:	Creators Email ORCID ORCID Put Code Hasegawa, Keiichi hasegawa.k.999@gmail.com orcid.org/0009-0004-4251-877X UNSPECIFIED
URN:	urn:nbn:de:hbz:38-725656
Date:	2024
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Mathematics and Natural Sciences > Department of Chemistry > Institute of Biochemistry
Subjects:	Life sciences
Uncontrolled Keywords:	Keywords Language Mixed lineage kinase domain-like (MLKL) proteins English NOD-like receptor (NLR) English TNL-triggered immunity English Cell death and immunity English Plant immunity English
Date of oral exam:	11 January 2024
Referee:	Name Academic Title Chai, Jijie Prof. Dr. Thomma, Bart Prof. Dr.
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/72565