The regulation of the copper homeostasis pathway in Enterococcus hirae is conducted through activity of the zinc metalloprotein Zn(II)CopY, which is a Cu(I)-responsive dimeric repressor (Cobine et al., Biochemistry 41:5822-5829, 2002). Its dimerization domain contains a C-terminal cysteine-rich metal-binding motif used for Cu(I) sensing adjacent to an aliphatic-rich repeating sequence, but it is unclear as to which regions contribute most to the interaction. To accomplish this, a synthetically produced CopY construct (CDG) was fused with solubility enhancement tags so the key components of the elements of the aliphatic repeat and metal-binding site could be probed for their dimerization activity. The resultant fusion constructs were tested using two independent methods. Isothermal titration calorimetry, an in vitro technique, was employed to determine dimer affinity thermodynamically. Protein fragment complementation, an in vivo technique, made it possible to rapidly screen homodimeric and heterodimeric complexes within live cells. The combination of in vivo and in vitro studies enabled the identification of CDG sequences that dimerize and sequences that do not, in addition to deciphering relative dimer affinity between all constructs screened. The in vivo technique allowed the formation of heterodimers to be tested for their ability to form specific complexes between dissimilar CDG analogs.
[Show abstract][Hide abstract] ABSTRACT: Interleukin-8 has been shown by X-ray crystallography and NMR to be a homodimer, suggesting that this is the form which binds to its receptor. Here we measure, for the first time, the monomer-dimer equilibrium of interleukin-8 using analytical ultracentrifugation and titration microcalorimetry and find that it dissociates readily to monomers with an equilibrium dissociation constant of 18 +/- 6 microM at 37 degrees C. The present findings suggest that the monomer is the form which binds to the receptor. Comparison of experimental and structure-based calculated thermodynamics of interleukin-8 dimerization argues for limited subunit conformational changes upon dissociation to monomer.
[Show abstract][Hide abstract] ABSTRACT: alphaA- and alphaB-crystallins are abundantly present in the eye lens, belong to the small heat shock protein family, and exhibit molecular chaperone activity. They are also known to interact with metal ions such as Cu(2+), and their metal-binding modulates the structure and chaperone function. Unlike other point mutations in alphaA-crystallin that cause congenital cataracts, the G98R mutation causes pre-senile cataract. We have investigated the effect of Cu(2+) on the structure and function of G98R alphaA-crystallin.
Fluorescence spectroscopy and isothermal titration calorimetry were used to study Cu(2+) binding to alphaA- and G98R alphaA-crystallin. Circular dichroism spectroscopy was used to study secondary and tertiary structures, and dynamic light scattering was used to determine the hydrodynamic radii of the proteins. Chaperone activity and self-aggregation of the wild type and the mutant protein in the absence and the presence of the metal ions was monitored using light scattering.
Our fluorescence quenching and isothermal titration calorimetric studies show that like alphaA-crystallin, G98R alphaA-crystallin binds Cu(2+) with picomolar range affinity. Further, both wild type and mutant alphaA-crystallin inhibit Cu(2+)-induced generation of reactive oxygen species with similar efficiency. However, G98R alphaA-crystallin undergoes pronounced self-aggregation above a certain concentration of Cu(2+) (above subunit to Cu(2+) molar ratio of 1:3 in HEPES-NaOH buffer, pH 7.4). At concentrations of Cu(2+) below this ratio, G98R alphaA-crystallin is more susceptible to Cu(2+)-induced tertiary and quaternary structural changes than alphaA-crystallin. Interestingly, Cu(2+) binding increases the chaperone-like activity of alphaA-crystallin toward the aggregation of citrate synthase at 43 degrees C while it decreases the chaperone-like activity of G98R alphaA-crystallin. Mixed oligomer formation between the wild type and the mutant subunits modulates the Cu(2+)-induced effect on the self-aggregation propensity. Other heavy metal ions, namely Cd(2+) and Zn(2+) but not Ca(2+), also promote the self-aggregation of G98R alphaA-crystallin and decrease its chaperone-like activity.
Our study demonstrates that unlike wild type alphaA-crystallin, G98R alphaA-crystallin and its mixed oligomers with wild type protein are vulnerable to heavy metal ions. Our study provides insight into aspects of how environmental factors could augment phenotype(s) in certain genetically predisposed conditions.
[Show abstract][Hide abstract] ABSTRACT: A hallmark of a group of neurodegenerative diseases such as Alzheimer disease is the formation of neurofibrillary tangles, which are principally composed of bundles of filaments formed by microtubule-associated protein Tau. Clarifying how natively unstructured Tau protein forms abnormal aggregates is of central importance for elucidating the etiology of these diseases. There is considerable evidence showing that zinc, as an essential element that is highly concentrated in brain, is linked to the development or progression of these diseases. Herein, by using recombinant human Tau fragment Tau(244-372) and its mutants, we have investigated the effect of zinc on the aggregation of Tau. Low micromolar concentrations of Zn(2+) dramatically accelerate fibril formation of wild-type Tau(244-372) under reducing conditions, compared with no Zn(2+). Higher concentrations of Zn(2+), however, induce wild-type Tau(244-372) to form granular aggregates in reducing conditions. Moreover, these non-fibrillar aggregates assemble into mature Tau filaments when Zn(2+) has been chelated by EDTA. Unlike wild-type Tau(244-372), low micromolar concentrations of Zn(2+) have no obvious effects on fibrillization kinetics of single mutants C291A and C322A and double mutant C291A/C322A under reducing conditions. The results from isothermal titration calorimetry show that one Zn(2+) binds to one Tau molecule via tetrahedral coordination to Cys-291 and Cys-322 as well as two histidines, with moderate, micromolar affinity. Our data demonstrate that low micromolar zinc accelerates the fibrillization of human Tau protein via bridging Cys-291 and Cys-322 in physiological reducing conditions, providing clues to understanding the relationship between zinc dyshomeostasis and the etiology of neurodegenerative diseases.
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