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Lipid‐binding properties and/or involvement with host defense are often found in allergen proteins, implying that these intrinsic biological functions likely contribute to the allergenicity of allergens. The group 2 major mite allergens, Der f 2 and Der p 2, show structural homology with MD‐2, the lipopolysaccharide (LPS)‐binding component of the Toll‐like receptor (TLR) 4 signalling complex. Elucidation of the ligand‐binding properties of group 2 mite allergens and identification of interaction sites by structural studies are important to explore the relationship between allergenicity and biological function. Here, we report a ligand‐fishing approach in which His‐tagged Der f 2 was incubated with sonicated stable isotope‐labelled Escherichia coli as a potential ligand source, followed by isolation of Der f 2‐bound material by a HisTrap column and NMR analysis. We found that Der f 2 binds to LPS with a nanomolar affinity and, using fluorescence and gel filtration assays that LPS binds to Der f 2 in a molar ratio of 1 : 1. We mapped the LPS‐binding interface of Der f 2 by NMR perturbation studies, which suggested that LPS binds Der f 2 between the two large β‐sheets, similar to its binding to MD‐2, the LPS‐binding component of the innate immunity receptor TLR4.

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Arginyl-tRNA synthetase (ArgRS) recognizes two major identity elements of tRNA Arg : A20, located at the outside corner of the L-shaped tRNA, and C35, the second letter of the anticodon. Only a few exceptional organisms, such as the yeast Saccharomyces cerevisiae, lack A20 in tRNA Arg . In the present study, we solved the crystal structure of a typical A20-recognizing ArgRS from Thermus thermophilus at 2.3 Å resolution. The structure of the T. thermophilus ArgRS was found to be similar to that of the previously reported

The Raman spectra of L-histidine, L-histidine-13C (labelled at C2 of the imidazole ring) and related compounds, namely, imidazole, 4-methylimidazole, N-acetyl-L-histidine methylamide, were observed in H 2 0 and D,O solutions at various pH and pD values. A strong Raman band at around 1410cm-' was found for these compounds in acidic D,O solutions, and it was assigned to a mode characteristic of the N-deuterated imidazolium ring ( I m ' ) in which the NI-C2-NJ symmetric stretching and N-D bending vibrations are mixed. It is concluded that this band would be useful as a probe for studying the ionization states of histidine residues in proteins.

Interaction of an iodide ion with lactoperoxidase was studied by the use of 1H NMR, 127I NMR, and optical difference spectrum techniques. 1H NMR spectra demonstrated that a major broad hyperfine-shifted signal at about 60 ppm, which is ascribed to the heme peripheral methyl protons, was shifted toward high field by adding KI, indicating the binding of iodide to the active site of the enzyme; the dissociation constant was estimated to be 38 mM at pH 6.1. The binding was further detected by 127I NMR, showing no competition with cyanide. Both 1H NMR and 127I NMR revealed that the binding of iodide to the enzyme is facilitated by the protonation of an ionizable group with a pKa value of 6.0-6.8, which is presumably the distal histidyl residue. Optical difference spectra showed that the binding of an aromatic donor molecule to the enzyme is slightly but distinctly affected by adding KI. On the basis of these results, it was suggested that an iodide ion binds to lactoperoxidase outside the heme crevice but at the position close enough to interact with the distal histidyl residue which possibly mediates electron transport in the iodide oxidation reaction.

The X-ray crystal structure of the complex of salicylhydroxamic acid (SHA) with Arthromyces ramosus peroxidase (ARP) has been determined at 1.9 A resolution. The position of SHA in the active site of ARP is similar to that of the complex of benzhydroxamic acid (BHA) with ARP [Itakura, H., et al. (1997) FEBS Lett. 412, 107-110]. The aromatic ring of SHA binds to a hydrophobic region at the opening of the distal pocket, and the hydroxamic acid moiety forms hydrogen bonds with the His56, Arg52, and Pro154 residues but is not asscoiated with the heme iron. X-ray analyses of ARP-resorcinol and ARP-p-cresol complexes failed to identify the aromatic donor molecules, most likely due to the very low affinities of these aromatic donors for ARP. Therefore, we examined the locations of these and other aromatic donors on ARP by the molecular dynamics method and found that the benzene rings are trapped similarly by hydrophobic interactions with the Ala92, Pro156, Leu192, and Phe230 residues at the entrance of the heme pocket, but the dihedral angles between the benzene rings and the heme plane vary from donor to donor. The distances between the heme iron and protons of SHA and resorcinol are similar to those obtained by NMR relaxation. Although SHA and BHA are usually considered potent inhibitors for peroxidase, they were found to reduce compound I and compound II of ARP and horseradish peroxidase C in the same manner as p-cresol and resorcinol. The aforementioned spatial relationships of these aromatic donors to the heme iron in ARP are discussed with respect to the quantum chemical mechanism of electron transfer in peroxidase reactions.