CONFORMATIONAL-CHANGES OF AN HSP70 MOLECULAR CHAPERONE INDUCED BY NUCLEOTIDES, POLYPEPTIDES, AND N-ETHYLMALEIMIDE

Hsp70 molecular chaperones are highly conserved ATPases that guide the folding and assembly of proteins in many cellular pathways. They use the energy of ATP binding and hydrolysis to regulate their interaction s with hydrophobic regions of unfolded proteins. The activities and th e conformations of the N-terminal nucleotide- and C-terminal polypepti de-binding domains of Hsp70s are coupled. We recently reported that th e sulfhydryl-modifying reagent N-ethylmaleimide (NEM) inactivates the yeast Hsp70 Ssa1p by reacting with its three cysteine residues which a re located in the nucleotide-binding domain. To further characterize c onformational changes associated with interdomain coupling and to dete rmine whether NEM alters Ssa1p's conformation, the structures of Ssa1p and NEM-modified Ssa1p (NEM-Ssa1p) were compared using a variety of b iophysical techniques. Size exclusion chromatography revealed that NEM -Ssa1p is more oligomeric and more resistant to nucleotide- or polypep tide-dependent depolymerization than Ssa1p. Measurement of the thermal stability indicated that NEM modification has an effect very similar to that of binding of nucleotides to the unmodified protein. Circular dichroism demonstrated small differences in the secondary structure of Ssa1p and NEM-Ssa1p, and in their complexes with nucleotides, NEM mod ification increased the ANS fluorescence of Ssa1p and exposed numerous trypsin-sensitive sites in its nucleotide-binding domain. The intrins ic fluorescence of Ssa1p's only tryptophan residue, which is located i n a C-terminal alpha-helical region adjacent to the polypeptide-bindin g cleft, was quenched in the presence of ATP, hut not ADP. NEM modific ation altered nucleotide-dependent changes in the intrinsic fluorescen ce of Ssa1p. Together, these results demonstrate that NEM alters the c onformation of Ssa1p and disrupts, but does not eliminate, interdomain communication. Furthermore, the results provide evidence for a model in which the polypeptide-binding cleft of Hsp70s is covered by an alph a-helical lid that is open in the presence of ATP, but closed in the p resence of ADP.