Inhibition of Cu2+-mediated generation of reactive oxygen species by the small heat shock protein αB-crystallin: the relative contributions of the N- and C-terminal domains.

Oxidative stress, Cu(2+) homeostasis, and small heat shock proteins (sHsp's) have important implications in several neurodegenerative diseases. The ubiquitous sHsp αB-crystallin is an oligomeric protein that binds Cu(2+). We have investigated the relative contributions of the N- and C-terminal (C-TDαB-crystallin) domains of αB-crystallin to its Cu(2+)-binding and redox-attenuation properties and mapped the Cu(2+)-binding regions. C-TDαB-crystallin binds Cu(2+) with slightly less affinity and inhibits Cu(2+)-catalyzed, ascorbate-mediated generation of ROS to a lesser extent than αB-crystallin. [Cu(2+)]/[subunit] stoichiometries for redox attenuation by αB-crystallin and C-TDαB-crystallin are 5 and 2, respectively. Both αB-crystallin and C-TDαB-crystallin also inhibit the Fenton reaction of hydroxyl radical formation. Trypsinization of αB-crystallin bound to a Cu(2+)-NTA column and MALDI-TOF analysis of column-bound peptides yielded three peptides located in the N-terminal domain, and in-solution trypsinization of αB-crystallin followed by Cu(2+)-NTA column chromatography identified four additional Cu(2+)-binding peptides located in the C-terminal domain. Thus, Cu(2+)-binding regions are distributed in the N- and C-terminal domains. Small-angle X-ray scattering and sedimentation-velocity measurements indicate quaternary structural changes in αB-crystallin upon Cu(2+) binding. Our study indicates that an oligomer of αB-crystallin can sequester a large number (~150) of Cu(2+) ions. It acts like a "Cu(2+) sponge," exhibits redox attenuation of Cu(2+), and has potential roles in Cu(2+) homeostasis and in preventing oxidative stress.