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Metal allergens nickel and cobalt facilitate TLR4 homodimerization independently of MD2.

ABSTRACT: Development of contact allergy requires cooperation of adaptive and innate immunity. Ni(2+) stimulates innate immunity via TLR4/MD2, the bacterial LPS receptor. This likely involves receptor dimerization, but direct proof is pending and it is unclear if related haptens share this mechanism. We reveal Co(2+) as second metal stimulating TLR4 and confirm necessity of H456/H458 therein. Experiments with a new TLR4 dimerization mutant established dimerization as a mechanism of metal- and LPS-induced TLR4 activation. Yet, in interaction studies only LPS- but not metal-induced dimerization required MD2. Consistently, soluble TLR4 expressed without MD2 inhibited metal- but not LPS-induced responses, opening new therapeutic perspectives.

SUBMITTER: Raghavan B

PROVIDER: S-EPMC3512400 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Metal allergens nickel and cobalt facilitate TLR4 homodimerization independently of MD2.

Raghavan Badrinarayanan B Martin Stefan F SF Esser Philipp R PR Goebeler Matthias M Schmidt Marc M

EMBO reports 20121012 12

Development of contact allergy requires cooperation of adaptive and innate immunity. Ni(2+) stimulates innate immunity via TLR4/MD2, the bacterial LPS receptor. This likely involves receptor dimerization, but direct proof is pending and it is unclear if related haptens share this mechanism. We reveal Co(2+) as second metal stimulating TLR4 and confirm necessity of H456/H458 therein. Experiments with a new TLR4 dimerization mutant established dimerization as a mechanism of metal- and LPS-induced ...[more]

PMID: 23059983

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Project description:A novel ArsR-SmtB family transcriptional repressor, KmtR, has been characterised from mycobacteria. Mutants of M. tuberculosis lacking kmtR show elevated expression of Rv2025c encoding a deduced CDF-family metal-exporter. KmtR-dependent repression of the cdf and kmtR operator-promoters was alleviated by nickel and cobalt in minimal medium. Electrophoretic mobility shift assays (EMSA) and fluorescence anisotropy (FA) show binding of purified KmtR to nucleotide sequences containing a region of dyad symmetry from the cdf and kmtR operator-promoters. A relatively large deltar(obs) in FA implies formation of high order apo-KmtR(n)-DNA and multiple complexes were detected by EMSA. Incubation of KmtR with cobalt inhibits DNA-complex assembly and metal-protein binding was confirmed by competition against 4-(2-pyridylazo)-resorcinol. KmtR is the second, to NmtR, characterised ArsR-SmtB sensor of nickel and cobalt from M. tuberculosis suggesting special significance for these ions in this pathogen. KmtR-dependent expression is elevated in complete medium with no increase in response to metals, while NmtR retains a response to nickel and cobalt under these conditions. Mixing equimolar apo-KmtR and apo-NmtR with 0.8 equivalents of nickel or cobalt gave nickel- and cobalt-dependent difference emission spectra similar to nickel(0.8)-KmtR and cobalt(0.8)-KmtR, respectively. Thus, KmtR has tighter affinities for nickel and cobalt than NmtR consistent with basal levels of these metals being sensed by KmtR but not NmtR in complete medium. More than a thousand genes encoding ArsR-SmtB related proteins are listed in databases and a proportion can be predicted to detect metals through known allosteric sites. KmtR has none of the previously defined sites. Substitution of His(88), Glu(101), His(102), His(110) or His(111) with Gln generated KmtR-variants that repress the cdf and kmtR operator-promoters even in elevated nickel and cobalt, revealing a new sensory site. Importantly, ArsR-SmtB sequence groupings do not correspond with the different sensory-motifs revealing that only the latter should be used to predict metal-sensing. Data is also available from http://bugs.sgul.ac.uk/E-BUGS-49

Dataset Information

Metal allergens nickel and cobalt facilitate TLR4 homodimerization independently of MD2.

Publications

Metal allergens nickel and cobalt facilitate TLR4 homodimerization independently of MD2.

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