ATP and the Core “α-Crystallin” Domain of the Small Heat-shock Protein αB-crystallin

Abstract

Electrospray ionization mass spectrometry (ESI-LC/ MS) of tryptic digests of human ␣B-crystallin in the presence and absence of ATP identified four residues located within the core "␣-crystallin" domain, Lys 82 , Lys 103 , Arg 116 , and Arg 123 , that were shielded from the action of trypsin in the presence of ATP. In control experiments, chymotrypsin was used in place of trypsin. The chymotryptic fragments of human ␣B-crystallin produced in the presence and absence of ATP were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS , located near or within the core ␣-crystallin domain, were shielded from the action of chymotrypsin in the presence of ATP. Chemically similar analogs of ATP were less protective than ATP against proteolysis by trypsin or chymotrypsin. ATP had no effect on the enzymatic activity of trypsin and the K m for trypsin was 0.031 mM in the presence of ATP and 0.029 mM in the absence of ATP. The results demonstrated an ATP-dependent structural modification in the core ␣-crystallin domain conserved in nearly all identified small heatshock proteins that act as molecular chaperones.␣-Crystallin is a protein that is expressed at high levels in all vertebrate eye lenses and is composed of two M r 20,000 subunits, ␣A and ␣B, which associate to form high molecular mass complexes with an average molecular mass of 800 kDa (1-3). In 1992 it was demonstrated that ␣A-and ␣B-crystallin function as molecular chaperones by suppressing the aggregation of other polypeptides in vitro (4). A large body of experimental evidence confirmed that ␣B-crystallin is a prototype for the small heat-shock protein (sHSP) 1 superfamily, which is characterized by the presence of a conserved core "␣-crystallin" domain that spans 80 -100 amino acids in the C-terminal domain (2, 5, 6). sHSPs are expressed ubiquitously in nature (5, 6), are up-regulated in several neurodegenerative diseases that include multiple sclerosis (7), Alzheimer's (8, 9), and Creutzfeldt-Jacob disease (10), and are mutated in inherited diseases including a desmin-related myopathy and cataract (11,12). Initially thought to be lens-specific structural proteins, both ␣A-and ␣B-crystallin have been identified in a variety of non-lens tissues (13,14). While ␣-crystallins and sHSPs function as molecular chaperones both in vitro and in vivo (15-35) a function for the conserved core ␣-crystallin domain has yet to be demonstrated. In this report trypsin proteolysis identified regions in the core domain that may be involved with the effect of ATP on chaperone activity of sHSPs.In contrast to the large heat-shock protein families HSP60, HSP70, HSP90, and HSP100, the effect of ATP on the molecular chaperone functions of sHSPs has only been reported recently (31). Several publications have supported a structural and functional relationship between ATP and the sHSPs. Equilibrium binding studies, intrinsic tryptophan fluorescence, and 31 P nuclear magnetic resonance spectroscopy demonstrated an interaction between ATP and total bovine ␣-crystallin (36...

α-Crystallin-Type Heat Shock Proteins: Socializing Minichaperones in the Context of a Multichaperone Network

“…Nonhydrolyzable ATP analogs had no stimulatory effect. Tryptophan fluorescence studies and mass spectrometry of tryptic digests of the chaperone demonstrated ATP-dependent structural modifications in the ␣-crystallin domain, which might influence its functionality (216,217). ATP-enhanced chaperone activity of ␣B-crystallin was independently confirmed in experiments with the bovine protein (297).…”

Role of ATP on the Interaction of α-Crystallin with Its Substrates and Its Implications for the Molecular Chaperone Function

“…Limited tryptic digestion studies of ␣-crystallin reported by us (38) as well as by others (31) showed that cleavage sites located at the C-and N-terminal regions are relatively easily accessible by trypsin, but those on the ␣-crystallin domain are less accessible. We have checked whether the addition of ATP leads to any differences in the accessibility of these sites of ␣A-crystallin by trypsin.…”

Molecular Characterization ofhsp20, Encoding a Small Heat Shock Protein ofBifidobacterium breveUCC2003

“…In the case of bacteria, sHSPs may be absent in some species, e.g., Mycoplasma genitalium, while in other species only a small number of sHSP-encoding genes are present, such as in the case of Escherichia coli, which specifies just two types of sHSPs (13). In contrast, rhizobia possess a large set of genes specifying sHSPs (12).…”