In Vitro Dissociation of BiP-Peptide Complexes Requires a Conformational Change in BiP after ATP Binding but Does Not Require ATP Hydrolysis

Abstract

In the present study, we produced single point mutations in the ATP binding site of hamster BiP, isolated recombinant proteins, and characterized them in terms of their affinity for ATP and ADP, their ability to undergo a conformational change upon nucleotide binding, and their rate of ATP hydrolysis. These analyses allowed us to classify the mutants into three groups: ATP hydrolysis (T229G), ATP binding (G226D, G227D), and ATP-induced conformation (T37G) mutants, and to test the role of these activities in the in vitro ATP-mediated release of proteins from BiP. All three classes of mutants were still able to bind peptide demonstrating that nucleotide is not involved in this function. Addition of ATP to either wild-type BiP or the T229G mutant caused the in vitro release of bound peptide, confirming that ATP hydrolysis is not required for protein release. ATP did not dissociate G226D, G227D, or T37G mutant BiP-peptide complexes, suggesting that ATP binding to BiP is not sufficient for the release of bound peptides, but that an ATP-induced conformational change in BiP is necessary. The identification of BiP mutants that are defective in each of these steps of ATP hydrolysis will allow the in vivo dissection of the role of nucleotide in BiP's activity.The heat shock protein 70 (HSP70) 1 family of chaperones are components of the cellular machinery for folding, assembly, and degradation of proteins (1). These proteins are thought to undergo cycles of nucleotide-mediated binding and release to unfolded polypeptides. The binding of peptides to HSP70 proteins stimulates their ATPase activity (2), and bound peptides or proteins are released with ATP but not with non-hydrolyzable analogues (2-5). These observations have led to the conclusion that ATP hydrolysis is required for HSP70 activity and that there are functional interactions between the ATP binding and protein binding domains. All HSP70 proteins bind ATP tightly but the ATP hydrolysis rates of purified proteins are so low under physiological conditions, that other co-factors may be required to enhance the rate of ATP hydrolysis. In bacteria two co-factors, dnaJ and grpE, have been identified that together increase the ATPase activity of dnaK (bacterial HSP70 homologue) up to 50-fold (6). Although dnaJ homologues have been identified in several organelles in various organisms (7), the only eukaryotic grpE homologues found thus far are mitrochondrial (8, 9). Alternatively, it is possible that ATP binding rather than ATP hydrolysis is essential for HSP70 function. In support of this hypothesis, investigators have recently demonstrated that peptides are released from both dnaK and hsc70 (mammalian cytosolic homologue) after ATP binding but before ATP hydrolysis occurs (10). Thus, it is presently unclear whether ATP binding, ATP hydrolysis, or both are important for HSP70 function in vivo.The ATP binding domain of all HSP70 members resides within a highly conserved NH 2 -terminal 44-kDa fragment that can be generated by proteolysis (11,12). The structure of the ATP ...

BAP, a Mammalian BiP-associated Protein, Is a Nucleotide Exchange Factor That Regulates the ATPase Activity of BiP

“…The BiPT229G binds ATP but cannot hydrolyze it (30,31). We isolated four independent positive clones that carried target genes encoding putative BiP-interacting proteins.…”

“…DNA encoding the ATPase domain of the hamster BiPT229G mutant without the ER targeting signal sequence (30,31) was cloned into the NdeI and BamHI sites of the pAS1 vector. The resulting construct, pAS1(T229G) was transformed into the yeast HF7c strain.…”

“…We have identified a number of BiP mutants that are impaired in nucleotide binding, in the ATP-dependent conformational change, or in ATP hydrolysis (30,31). Transfected cells were metabolically labeled with [ 35 S]methionine and [ 35 S]cysteine and then treated with the membrane-permeable cross-linking agent DSP to stabilize BAP-BiP complexes within the cell.…”

Hsc66 and Hsc20, a new heat shock cognate molecular chaperone system from Escherichia coli

“…Figure 8 shows the variation in rates of ATP hydrolysis observed for Hsc66 and DnaK over the range 10-55". Hsc66 exhibits greater activity than DnaK (0.6 min" vs. 0.1 min" at pH 7.5, 37"C), and the Hsc66 ATPase rates are also somewhat higher than those typical of other hsp70 proteins (McCarty & Walker, 1991;Palleros et al, 1993a;Kamath-Loeb et al, 1995;O'Brien & McKay, 1995;Wei et al, 1995;Ziegelhoffer et al, 1995). The increased activity of Hsc66 at high temperatures (approximately two-fold greater at 44°C than at 37°C) suggests that it is active in the cell under conditions of heat stress; however, activity declined at temperatures greater than 50°C suggesting that Hsc66 is less thermostable than DnaK.…”

Radicicol-sensitive Peptide Binding to the N-terminal Portion of GRP94

“…As shown here, the recombinant N355 protein exists predominantly as monomers and binds peptides as monomers, although dimerization (and higher order oligomerization) neither augments nor interferes with peptide binding. In this respect, GRP94 is different from BiP/GRP78, which is converted to monomers upon peptide binding (47)(48)(49).…”