Destabilization of Peptide Binding and Interdomain Communication by an E543K Mutation in the Bovine 70-kDa Heat Shock Cognate Protein, a Molecular Chaperone

Jeung-Hoi Ha,Ulf Hellman,Eric R Johnson,Lushen Li,David B Mckay,Marcelo C Sousa,Shigeki Takeda,Christer Wernstedt,Sigurd M Wilbanks

doi:10.1074/jbc.272.44.27796

Abstract

We have compared 70-kDa heat shock cognate protein (Hsc70) isolated from bovine brain with recombinant wild type protein and mutant E543K protein (previously studied as wild type in our laboratory). Wild type bovine and recombinant protein differ by posttranslational modification of lysine 561 but interact similarly with a short peptide (fluorescein-labeled FYQLALT) and with denatured staphylococcal nuclease-(Delta135-149). Mutation E543K results in 4. 5-fold faster release of peptide and lower stability of complexes with staphylococcal nuclease-(Delta135-149). ATP hydrolysis rates of the wild type proteins are enhanced 6-10-fold by the addition of peptide. The E543K mutant has a peptide-stimulated hydrolytic rate similar to that of wild type protein but a higher unstimulated rate, yielding a mere 2-fold enhancement. All three versions of Hsc70 possess similar ATP-dependent conformational shifts, and all show potassium ion dependence. These data support the following model: (i) in the presence of K+, Mg2+, and ATP, the peptide binding domain inhibits the ATPase; (ii) binding of peptide relieves this inhibition; and (iii) the E543K mutation significantly attenuates the inhibition by the peptide binding domain and destabilizes Hsc70-peptide complexes.

Highlights

The 70-kDa heat shock-related proteins (Hsp70s)1 comprise a family of molecular chaperones that bind and release unstructured, hydrophobic segments of polypeptide in an ATP-dependent manner, thereby presumably suppressing intermolec
Purification and Peptide Mapping of brain Hsc70 (bHsc70) and Our Original Recombinant Hsc70 —Both bovine brain and recombinant Hsc70 proteins were purified on anion exchange, ATP-agarose, Mono P, and gel filtration columns sequentially. bHsc70 behaves in a manner similar to recombinant protein on anion exchange and ATP-agarose columns
BHsc70 behaved differently on gel filtration; approximately half of the protein eluted from Superdex 75 as monomer, and the remainder eluted as dimer and higher order oligomers

Summary

Introduction

The 70-kDa heat shock-related proteins (Hsp70s) comprise a family of molecular chaperones that bind and release unstructured, hydrophobic segments of polypeptide in an ATP-dependent manner, thereby presumably suppressing intermolec-. Self-aggregation can be reversed by the addition of ATP or the addition of competing peptides and is thought to be similar to, and competitive with, the heterologous peptide binding activity of the proteins [8]. The structure of a peptide binding fragment of the Escherichia coli DnaK protein complexed to peptide has been solved, revealing a subdomain of eight antiparallel ␤strands, which form a peptide binding pocket, and a second, ␣-helical subdomain, one helix of which lies over the peptide binding pocket [29]. The level of amino acid similarity within the Hsp protein family (e.g. 47% sequence identity between E. coli DnaK and bovine Hsc70) is such that the tertiary fold and many of the intramolecular interactions seen in a threedimensional structure of one member of the family can be extrapolated safely to other Hsp70s

Methods

Results

Conclusion