title: Molecular mechanism of the chaperone function of mini-α-crystallin, a 19-residue peptide of human α-crystallin.
abstract: α-Crystallin is the archetypical chaperone of the small heat-shock protein family, all members of which contain the so-called "α-crystallin domain" (ACD). This domain and the N- and C-terminal extensions are considered the main functional units in its chaperone function. Previous studies have shown that a 19-residue fragment of the ACD of human αA-crystallin called mini-αA-crystallin (MAC) shows chaperone properties similar to those of the parent protein. Subsequent studies have confirmed the function of this peptide, but no studies have addressed the mechanistic basis for the chaperone function of MAC. Using human γD-crystallin (HGD), a key substrate protein for parent α-crystallin in the ocular lens, we show here that MAC not only protects HGD from aggregation during thermal and chemical unfolding but also binds weakly and reversibly to HGD (Kd ≈ 200-700 μM) even when HGD is in the native state. However, at temperatures favoring the unfolding of HGD, MAC forms a stable complex with HGD similar to parent α-crystallin. Using nuclear magnetic resonance spectroscopy, we identify the residues in HGD that are involved in these two modes of binding and show that MAC protects HGD from aggregation by binding to Phe 56 and Val 132 at the domain interface of the target protein, and residues Val 164 to Leu 167 in the core of the C-terminal domain. Furthermore, we suggest that the low-affinity, reversible binding of MAC on the surface of HGD in the native state is involved in facilitating its binding to both the domain interface and core regions during the early stages of the unfolding of HGD. This work highlights some structural features of MAC and MAC-like peptides that affect their chaperone activity and can potentially be manipulated for translational studies.
authors.affiliation: Department of Chemistry, Life Sciences, University at Albany, State University of New York , Albany, New York 12222, United States.
authors.forename: Priya R
authors.initials: PR
authors.lastname: Banerjee
authors.forename: Ajay
authors.initials: A
authors.lastname: Pande
authors.forename: Alexander
authors.initials: A
authors.lastname: Shekhtman
authors.forename: Jayanti
authors.initials: J
authors.lastname: Pande
mesh_terms: D000595:Amino Acid Sequence; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D066329:Protein Aggregates; D011485:Protein Binding; D055550:Protein Stability; D058767:Protein Unfolding; D038202:alpha-Crystallin A Chain / Q000737:chemistry / Q000378:metabolism*; D038222:gamma-Crystallins / Q000737:chemistry / Q000378:metabolism*
chemical_list: C504903:CRYGD protein, human; D010446:Peptide Fragments; D066329:Protein Aggregates; D038202:alpha-Crystallin A Chain; D038222:gamma-Crystallins