ABSTRACT
The CorA family of divalent cation transporters utilizes Mg2+ and Co2+ as primary substrates. The molecular mechanism of its function, including ion selectivity and gating, has not been fully characterized. Recently we reported a new structure of a CorA homologue from Methanocaldococcus jannaschii, which provided novel structural details that offered the conception of a unique gating mechanism involving conversion of an open hydrophilic gate into a closed hydrophobic one. In the present study we report functional evidence for this novel gating mechanism in the Thermotoga maritima CorA together with an improved crystal structure of this CorA to 2.7 Å (1 Å=0.1 nm) resolution. The latter reveals the organization of the selectivity filter to be similar to that of M. jannaschii CorA and also the previously unknown organization of the second signature motif of the CorA family. The proposed gating is achieved by a helical rotation upon the binding of a metal ion substrate to the regulatory binding sites. Additionally, our data suggest that the preference of this CorA for Co2+ over Mg2+ is controlled by the presence of threonine side chains in the channel. Finally, the roles of the intracellular metal-binding sites have been assigned to increased thermostability and regulation of the gating. These mechanisms most likely apply to the entire CorA family as they are regulated by the highly conserved amino acids.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Cation Transport Proteins/chemistry , Cation Transport Proteins/metabolism , Cobalt/chemistry , Magnesium/chemistry , Thermotoga maritima/metabolism , Amino Acid Motifs , Bacterial Proteins/genetics , Binding Sites , Biological Transport , Cation Transport Proteins/genetics , Cations, Divalent , Cobalt/metabolism , Crystallography, X-Ray , Escherichia coli/genetics , Hydrophobic and Hydrophilic Interactions , Ion Channel Gating , Kinetics , Magnesium/metabolism , Models, Molecular , Molecular Sequence Data , Mutation , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Thermotoga maritima/geneticsABSTRACT
The yeast Malassezia furfur belongs to the normal cutaneous flora, but is also a triggering allergen that can contribute to atopic dermatitis. To illuminate the effect of circulating allergen-specific T cells in atopic dermatitis, the peripheral mononuclear cell response was correlated with the in vivo skin prick test and atopy patch test reactivity to M. furfur. None of 16 healthy controls showed any positive in vivo reaction. The 40 atopic dermatitis patients, of whom 18 had serum IgE reactivity to M. furfur, were subdivided according to their in vivo reaction to M. furfur extract into three groups: skin prick test positive/atopy patch test positive (n = 12), skin prick test positive/atopy patch test negative (n = 12), and skin prick test negative/atopy patch test negative (n = 16). The skin prick test positive/atopy patch test positive and the skin prick test positive/atopy patch test negative groups had a significantly higher peripheral mononuclear cell stimulation index than the healthy controls. Interestingly, the stimulation index values in the skin prick test positive/atopy patch test positive group were significantly higher than in the skin prick test positive/atopy patch test negative group. In the M. furfur skin prick test positive atopic dermatitis patients (n = 24) a correlation was found between stimulation index and the M. furfur atopy patch test reactions, but not between stimulation index and M. furfur-specific serum IgE levels. Skin prick test positive and/or atopy patch test positive reactions to the recombinant M. furfur allergens rMal f 1, rMal f 5, and rMal f 6 were observed in 7, 14, and 16 of the 40 atopic dermatitis patients, respectively. Further, there was a correlation between production of the T helper 2-related cytokines interleukins 4, 5, and 13 and stimulation index to M. furfur extract, but not between the T helper 1-related interferon-gamma and stimulation index to M. furfur extract. Our data strongly suggest a relationship between circulating specific T cells with a T helper 2-like cytokine profile and positive atopy patch test reactions.
