We report a structural and functional analysis of the prophage Ba02 endolysin (PlyL) encoded by the Bacillus anthracis genome. We show that PlyL comprises two autonomously folded domains, an N-terminal catalytic domain and a C-terminal cell wall-binding domain. We determined the crystal structure of the catalytic domain; its three-dimensional fold is related to that of the cell wall amidase, T7 lysozyme, and contains a conserved zinc coordination site and other components of the catalytic machinery. We demonstrate that PlyL is an N-acetylmuramoyl-L-alanine amidase that cleaves the cell wall of several Bacillus species when applied exogenously. We show, unexpectedly, that the catalytic domain of PlyL cleaves more efficiently than the full-length protein, except in the case of Bacillus cereus, and using GFP-tagged cell wall-binding domain, we detected strong binding of the cell wall-binding domain to B. cereus but not to other species tested. We further show that a related endolysin (Ply21) from the B. cereus phage, TP21, shows a similar pattern of behavior. To explain these data, and the species specificity of PlyL, we propose that the C-terminal domain inhibits the activity of the catalytic domain through intramolecular interactions that are relieved upon binding of the C-terminal domain to the cell wall. Furthermore, our data show that (when applied exogenously) targeting of the enzyme to the cell wall is not a prerequisite of its lytic activity, which is inherently high. These results may have broad implications for the design of endolysins as therapeutic agents.
Background: Lytic enzymes (lysins) from bacteriophages are potential bactericidal agents. Results: Positive net charge on lysin catalytic domains is a major factor in determining lytic activity and host range. Conclusion: Lysins can be engineered to modify their dependence on their cell wall targeting domains. Significance: This work suggests a number of approaches for fine-tuning lysins as novel antibiotics.
Plants use a highly evolved immune system to exhibit defense response against microbial infections. The plant TIR domain, together with the nucleotide-binding (NB) domain and/or a LRR region, forms a type of molecule, named resistance (R) proteins, that interact with microbial effector proteins and elicit hypersensitive responses against infection. Here, we report the first crystal structure of a plant TIR domain from Arabidopsis thaliana (AtTIR) solved at a resolution of 2.0 Å . The structure consists of five b-strands forming a parallel b-sheet at the core of the protein. The b-strands are connected by a series of a-helices and the overall fold mimics closely that of other mammalian and bacterial TIR domains. However, the region of the aD-helix reveals significant differences when compared with other TIR structures, especially the aD3-helix that corresponds to an insertion only present in plant TIR domains. Available mutagenesis data suggest that several conserved and exposed residues in this region are involved in the plant TIR signaling function.
Macrophages detect pathogen infection via the activation of their plasma membrane-bound Toll-like receptor proteins (TLRs). The heterotypic interaction between the Toll/interleukin-1 receptor (TIR) domains of TLRs and adaptor proteins, likeMyeloid differentiation primary response gene 88 (MyD88), is the first intracellular step in the signaling pathway of the mammalian innate immune response. The hetero-oligomerization of the TIRs of the receptor and adaptor brings about the activation of the transcription factor NF-B, which regulates the synthesis of pro-inflammatory cytokines. Here, we report the first crystal structure of a bacterial TIR domain solved at 2.5 Å resolution. The three-dimensional fold of Paracoccus denitrificans TIR is identical to that observed for the TIR of human TLRs and MyD88 proteins. The structure shows a unique dimerization interface involving the DD-loop and EE-loop residues, whereas leaving the BB-loop highly exposed. Peptide amide hydrogendeuterium exchange mass spectrometry also reveals that the same region is used for dimerization in solution and in the context of the full-length protein. These results, together with a functional interaction between P. denitrificans TIR and MyD88 visualized in a co-immunoprecipitation assay, further substantiate the model that bacterial TIR proteins adopt structural mimicry of the host active receptor TIR domains to interfere with the signaling of TLRs and their adaptors to decrease the inflammatory response. Toll/interleukin-1 receptor (TIR)2 domain is a key mediator in the Toll-like receptor (TLR) signaling. TLRs are involved in early detection of pathogen-associated molecular patterns to enable quick responses to infection by triggering innate immune reactions through activation of the gene program regulated by the transcription factor nuclear factor-B (NF-B) and the recruitment of macrophages to the infection sites (1). The signaling of TLRs requires the homo-or heterodimerization of their extracellular leucine-rich repeats region mediated by the microbial pathogen-associated molecular patterns, leading to the dimerization of the receptor cytoplasmic TIR domains (2). Only in this active conformation are the receptor TIR domains capable of a functionally productive interaction with TIR domains of adaptor molecules, such as Myeloid differentiation primary response gene 88 (MyD88), MyD88 adaptor-like (Mal, also known as Toll/IL-1 receptor domain-containing adaptor protein (TIRAP)), TIR domain-containing adapter-inducing interferon- (TRIF), or TRIF-related adaptor molecule (TRAM), to initiate the signaling cascade (3).Structures of TIR domains from human TLR1(4), TLR2(5), TLR10(6), IL-1RAPL(7), and MyD88 (Protein Data Bank (PDB) IDs: 2JS7; 2Z5V) have been determined, and they all showed a flavodoxin-like fold consisting of three layers ␣//␣ with fivestranded parallel -sheet ordered 2,1,3,4,5 surrounded by ␣-helices on each side. However, neither the homotypic interactions of TIR domains nor the heterotypic ones between the TIRs of receptors and a...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.