MGATP BINDING TO THE NUCLEOTIDE-BINDING DOMAINS OF THE EUKARYOTIC CYTOPLASMIC CHAPERONIN INDUCES CONFORMATIONAL-CHANGES IN THE PUTATIVE SUBSTRATE-BINDING DOMAINS

The eukaryotic cytosolic chaperonins are large heterooligomeric comple xes with a cylindrical shape, resembling that of the homooligomeric ba cterial counterpart, GroEL. In analogy to GroEL, changes in shape of t he cytosolic chaperonin have been detected in the presence of MgATP us ing electron microscopy but, in contrast to the nucleotide-induced con formational changes in GroEL, no details are available about the speci fic nature of these changes. The present study identifies the structur al regions of the cytosolic chaperonin that undergo conformational cha nges when MgATP binds to the nucleotide binding domains. It is shown t hat limited proteolysis with trypsin in the absence of MgATP cleaves e ach of the eight subunits approximately in half, generating two fragme nts of similar to 30 kDa. Using mass spectrometry (MS) and N-terminal sequence analysis, the cleavage is found to occur in a narrow span of the amino acid sequence, corresponding to the peptide binding regions of GroEL and to the helical protrusion, recently identified in the str ucture of the substrate binding domain of the archeal group II chapero nin. This proteolytic cleavage is prevented by MgATP but not by ATP in the absence of magnesium, ATP analogs (MgATP gamma S and MgAMP-PNP) o r MgADP. These results suggest that, in analogy to GroEL, binding of M gATP to the nucleotide binding domains of the cytosolic chaperonin ind uces long range conformational changes in the polypeptide binding doma ins. It is postulated that despite their different subunit composition and substrate specificity, group I and group II chaperonins may share similar, functionally-important, conformational changes. Additional c onformational changes are Likely to involve a flexible helix-loop-heli x motif, which is characteristic for all group II chaperonins.