HspB2/myotonic dystrophy protein kinase binding protein (MKBP) as a novel molecular chaperone: structural and functional aspects.

The small heat shock protein, human HspB2, also known as Myotonic Dystrophy Kinase Binding Protein (MKBP), specifically associates with and activates Myotonic Dystrophy Protein Kinase (DMPK), a serine/threonine protein kinase that plays an important role in maintaining muscle structure and function. The structure and function of HspB2 are not well understood. We have cloned and expressed the protein in E.coli and purified it to homogeneity. Far-UV circular dichroic spectrum of the recombinant HspB2 shows a ß-sheet structure. Fluorescence spectroscopic studies show that the sole tryptophan residue at the 130(th) position is almost completely solvent-exposed. Bis-ANS binding shows that though HspB2 exhibits accessible hydrophobic surfaces, it is significantly less than that exhibited by another well characterized small HSP, αB-crystallin. Sedimentation velocity measurements show that the protein exhibits concentration-dependent oligomerization. Fluorescence resonance energy transfer study shows that HspB2 oligomers exchange subunits. Interestingly, HspB2 exhibits target protein-dependent chaperone-like activity: it exhibits significant chaperone-like activity towards dithiothreitol (DTT)-induced aggregation of insulin and heat-induced aggregation of alcohol dehydrogenase, but only partially prevents the heat-induced aggregation of citrate synthase, co-precipitating with the target protein. It also significantly prevents the ordered amyloid fibril formation of α-synuclein. Thus, our study, for the first time, provides biophysical characterization on the structural aspects of HspB2, and shows that it exhibits target protein-dependent chaperone-like activity.

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.