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, Cu2+ 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 Cu2+. We have investigated the relative contributions of the N- and C-terminal (C-TDαB-crystallin) domains of αB-crystallin to its Cu2+-binding and redox-attenuation properties and mapped the Cu2+-binding regions. C-TDαB-crystallin binds Cu2+ with slightly less affinity and inhibits Cu2+-catalyzed, ascorbate-mediated generation of ROS to a lesser extent than αB-crystallin. [Cu2+]/[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 Cu2+-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 Cu2+-NTA column chromatography identified four additional Cu2+-binding peptides located in the C-terminal domain. Thus, Cu2+-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 Cu2+ binding. Our study indicates that an oligomer of αB-crystallin can sequester a large number (~ 150) of Cu2+ ions. It acts like a “Cu2+ sponge,” exhibits redox attenuation of Cu2+, and has potential roles in Cu2+ homeostasis and in preventing oxidative stress.