Endolysins of Bacillus anthracis bacteriophages recognize unique carbohydrate epitopes of vegetative cell wall polysaccharides with high affinity and selectivity.

Bacteriophages express endolysins which are the enzymes that hydrolyze peptidoglycan resulting in cell lysis and release of bacteriophages. Endolysins have acquired stringent substrate specificities, which have been attributed to cell wall binding domains (CBD). Although it has been realized that CBDs of bacteriophages that infect Gram-positive bacteria target cell wall carbohydrate structures, molecular mechanisms that confer selectivity are not understood. A range of oligosaccharides, derived from the secondary cell wall polysaccharides of Bacillus anthracis, has been chemically synthesized. The compounds contain an α-d-GlcNAc-(1→4)-ß-d-ManNAc-(1→4)-ß-d-GlcNAc backbone that is modified by various patterns of α-d-Gal and ß-d-Gal branching points. The library of compounds could readily be prepared by employing a core trisaccharide modified by the orthogonal protecting groups N(α)-9-fluorenylmethyloxycarbonate (Fmoc), 2-methylnaphthyl ether (Nap), levulinoyl ester (Lev) and dimethylthexylsilyl ether (TDS) at key branching points. Dissociation constants for the binding the cell wall binding domains of the endolysins PlyL and PlyG were determined by surface plasmon resonance (SPR). It was found that the pattern of galactosylation greatly influenced binding affinities, and in particular a compound having a galactosyl moiety at C-4 of the nonreducing GlcNAc moiety bound in the low micromolar range. It is known that secondary cell wall polysaccharides of various bacilli may have both common and variable structural features and in particular differences in the pattern of galactosylation have been noted. Therefore, it is proposed that specificity of endolysins for specific bacilli is achieved by selective binding to a uniquely galactosylated core structure.

Role of net charge on catalytic domain and influence of cell wall binding domain on bactericidal activity, specificity, and host range of phage lysins.

The recombinant lysins of lytic phages, when applied externally to Gram-positive bacteria, can be efficient bactericidal agents, typically retaining high specificity. Their development as novel antibacterial agents offers many potential advantages over conventional antibiotics. Protein engineering could exploit this potential further by generating novel lysins fit for distinct target populations and environments. However, access to the peptidoglycan layer is controlled by a variety of secondary cell wall polymers, chemical modifications, and (in some cases) S-layers and capsules. Classical lysins require a cell wall-binding domain (CBD) that targets the catalytic domain to the peptidoglycan layer via binding to a secondary cell wall polymer component. The cell walls of Gram-positive bacteria generally have a negative charge, and we noticed a correlation between (positive) charge on the catalytic domain and bacteriolytic activity in the absence of the CBD (nonclassical behavior). We investigated a physical basis for this correlation by comparing the structures and activities of pairs of lysins where the lytic activity of one of each pair was CBD-independent. We found that by engineering a reversal of sign of the net charge of the catalytic domain, we could either eliminate or create CBD dependence. We also provide evidence that the S-layer of Bacillus anthracis acts as a molecular sieve that is chiefly size-dependent, favoring catalytic domains over full-length lysins. Our work suggests a number of facile approaches for fine-tuning lysin activity, either to enhance or reduce specificity/host range and/or bactericidal potential, as required.

The crystal structure of a TIR domain from Arabidopsis thaliana reveals a conserved helical region unique to plants.

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 A. The structure consists of five beta-strands forming a parallel beta-sheet at the core of the protein. The beta-strands are connected by a series of alpha-helices and the overall fold mimics closely that of other mammalian and bacterial TIR domains. However, the region of the alphaD-helix reveals significant differences when compared with other TIR structures, especially the alphaD3-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.

Molecular mimicry in innate immunity: crystal structure of a bacterial TIR domain.

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, like Myeloid 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-kappaB, which regulates the synthesis of pro-inflammatory cytokines. Here, we report the first crystal structure of a bacterial TIR domain solved at 2.5 A 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 hydrogen-deuterium 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.

Characterization of a TIR-like protein from Paracoccus denitrificans.

Based on protein sequence homology searches, we found a conserved open reading frame within the genome of several human pathogenic bacteria showing a resemblance to the mammalian TIR domain. We cloned, expressed, and characterized the corresponding gene product from Paracoccus denitrificans using several biophysical techniques. The protein consists of two independently folded domains. As predicted from the amino acid sequence and experimentally confirmed here, the N-terminal domain consists of a alpha-helical coiled-coil. The NMR data indicates that the C-terminal TIR-like domain folds into a compact protein. Finally, using GST pull-down experiments, we show that the bacteria TIR-like domain binds to the mammalian receptor (TLR4) and adaptor (MyD88) TIR domains. We postulate that prokaryotic pathogens utilize the TIR-like proteins to interfere with the innate immune response of the mammalian host so that the bacterial infection can progress undetected.

Structure and lytic activity of a Bacillus anthracis prophage endolysin.

We report a structural and functional analysis of the lambda 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.

The zinc-dependent redox switch domain of the chaperone Hsp33 has a novel fold.

The Escherichia coli chaperone Hsp33 contains a C-terminal zinc-binding domain that modulates activity by a so-called "redox switch". The oxidized form in the absence of zinc is active, while the reduced form in the presence of zinc is inactive. X-ray crystal structures of Hsp33 invariably omit details of the C-terminal domain, which is truncated in protein constructs that are capable of forming crystals. We report the solution structure of a recombinant 61-residue protein containing the zinc-binding domain (residues 227-287) of Hsp33, in the presence of stoichiometric amounts of Zn2+. The zinc-bound protein is well folded, and forms a novel structure unlike other published zinc-binding domains. The structure consists of two helices at right-angles to each other, a two-stranded B-hairpin and a third helix at the C terminus. The zinc site comprises the side-chains of the conserved cysteine residues 232, 234, 262 and 265, and connects a short sequence before the first helix with the tight turn in the middle of the B-hairpin. The structure of the C-terminal zinc-binding domain suggests a mechanism for the operation of the redox switch: loss of the bound zinc ion disrupts the folded structure, allowing the ligand cysteine residues to be oxidized, probably to disulfide bonds. The observation that the C-terminal domain is poorly structured in the active oxidized form suggests that the loss of zinc and unfolding of the domain precedes the oxidation of the thiolate groups of the cysteine residues, since the formation of disulfides between distant parts of the domain sequence would presumably promote the formation of stable three-dimensional structure in the oxidized form.Hsp33 provides an example of a redox signaling system that utilizes protein folding and unfolding together with chemical modification for transduction of external stimuli, in this case oxidative stress, to activate the machinery of the cell that is designed to deal with that stress.

Metal-dependent folding and stability of nuclear hormone receptor DNA-binding domains.

The nuclear/hormone receptors are an extensive family of ligand-activated transcription factors that recognise DNA targets through a highly conserved, structurally autonomous DNA-binding domain. The compact structure of the DNA-binding domain is supported by two zinc ions, each of which is co-ordinated by the tetrahedral arrangement of thiol groups from four cysteine residues. Metal binding is expected to be linked with deprotonation of the co-ordinating thiol groups and folding of the polypeptide. Using a variety of biophysical approaches, we characterise these linked equilibria for the isolated DNA-binding domains (DBD) of the receptors for estrogen and glucocorticoid. Mass spectrometry and equilibrium denaturation indicate that, near neutral pH, approximately four of the eight co-ordinating thiol groups release protons with zinc uptake, in agreement with the expected pK(a) change for the -SH group in the presence of the metal. Mass spectrometry reveals that the protein charge distribution changes with the uptake of zinc and that metal binding is co-operative. The co-operativity is consistent with observations from equilibrium denaturation, which indicate that the folding event is a two-state process. A crucial residue that stabilises the equilibrium structure of the DBD fold itself is a cysteine residue situated in the hydrophobic core of all known nuclear hormone receptors (but not involved in metal binding): it appears to be conserved absolutely for its unique combination of size and hydrophobicity. Stabilisation of the DBDs could be achieved by truncating the flexible, basic termini, suggesting that like-charge clusters may have deleterious effects on protein folds. While the metal-free apo protein and the chemically denatured state have little defined secondary structure, these states were expanded only partially in comparison with the native structure, according to data from small-angle X-ray scattering. The comparatively compact shapes of the denatured and apo forms may explain, in part, the marginal stability of the native fold.