Conformational Changes of an Hsp70 Molecular Chaperone Induced by Nucleotides, Polypeptides, and N-Ethylmaleimide
详细信息 查看全文
- 作者：William J. Chirico ; Micheline L. Markey ; and Anthony L. Fink
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：September 29, 1998
- 年：1998
- 卷：37
- 期：39
- 页码：13862 - 13870
- 全文大小：141K
- 年卷期：v.37,no.39(September 29, 1998)
- ISSN：1520-4995
文摘
Hsp70 molecular chaperones are highly conserved ATPases that guide the folding and assemblyof proteins in many cellular pathways. They use the energy of ATP binding and hydrolysis to regulatetheir interactions with hydrophobic regions of unfolded proteins. The activities and the conformations ofthe N-terminal nucleotide- and C-terminal polypeptide-binding domains of Hsp70s are coupled. We recentlyreported that the sulfhydryl-modifying reagent N-ethylmaleimide (NEM) inactivates the yeast Hsp70 Ssa1pby reacting with its three cysteine residues which are located in the nucleotide-binding domain. To furthercharacterize conformational changes associated with interdomain coupling and to determine whether NEMalters Ssa1p's conformation, the structures of Ssa1p and NEM-modified Ssa1p (NEM-Ssa1p) were comparedusing a variety of biophysical techniques. Size exclusion chromatography revealed that NEM-Ssa1p ismore oligomeric and more resistant to nucleotide- or polypeptide-dependent depolymerization than Ssa1p.Measurement of the thermal stability indicated that NEM modification has an effect very similar to thatof binding of nucleotides to the unmodified protein. Circular dichroism demonstrated small differencesin the secondary structure of Ssa1p and NEM-Ssa1p, and in their complexes with nucleotides. NEMmodification increased the ANS fluorescence of Ssa1p and exposed numerous trypsin-sensitive sites inits nucleotide-binding domain. The intrinsic fluorescence of Ssa1p's only tryptophan residue, which islocated in a C-terminal 
-helical region adjacent to the polypeptide-binding cleft, was quenched in thepresence of ATP, but not ADP. NEM modification altered nucleotide-dependent changes in the intrinsicfluorescence of Ssa1p. Together, these results demonstrate that NEM alters the conformation of Ssa1pand disrupts, but does not eliminate, interdomain communication. Furthermore, the results provide evidencefor a model in which the polypeptide-binding cleft of Hsp70s is covered by an -helical lid that is openin the presence of ATP, but closed in the presence of ADP.
-helical region adjacent to the polypeptide-binding cleft, was quenched in thepresence of ATP, but not ADP. NEM modification altered nucleotide-dependent changes in the intrinsicfluorescence of Ssa1p. Together, these results demonstrate that NEM alters the conformation of Ssa1pand disrupts, but does not eliminate, interdomain communication. Furthermore, the results provide evidencefor a model in which the polypeptide-binding cleft of Hsp70s is covered by an -helical lid that is openin the presence of ATP, but closed in the presence of ADP.