Recognition of a lambda pre-replicative complex by the Escherichia coli DnaJ molecular chaperone.

摘要

E. coli DnaJ is a member of the universally conserved Hsp40 family of molecular chaperones. Acting in concert with DnaK and GrpE, DnaJ plays a key role in a diverse set of cellular processes. Though traditionally known for their ability to prevent the aggregation of unfolded or partially denatured polypeptide substrates, it has recently become clear that the DnaJ/DnaK/GrpE chaperone system also functions in a variety of processes that require interactions with select native protein substrates. DnaJ regulates the initial step in these processes by binding client proteins and targeting DnaK to its site of action. Therefore, it is important to understand how DnaJ recognizes such a diverse set of polypeptide substrates.; During my dissertation research, I sought to characterize DnaJ's interaction with a model protein substrate known as the lambda pre-replicative complex (preRC). The preRC is composed of the viral initiator proteins lambda O and lambda P as well as the E. coli replicative helicase, DnaB. I have defined the stoichiometry of DnaJ associated with the preRC and find that approximately three DnaJ dimers remain bound to the complex through a short gel filtration column. DnaJ-preRC interactions are likely mediated through the C-terminal region of lambda O and appear to be very strong. In fact, Biacore analysis revealed that DnaJ bound a model preRC with an apparent KD of 1.6 x 10-9 M. This interaction may be stabilized by oligomerization of distinct DnaJ dimers on the surface of the complex. Indeed, examination of DnaJ's quaternary structure suggests that while the chaperone existed almost exclusively as a dimer at physiological concentrations, it self-associated to form higher-order structures at increased protein concentrations. The dimer-tetramer transition occurred between 1.2 and 12 muM, well below the anticipated concentration of DnaJ associated with the complex. Finally, mutational analysis has identified amino acids within DnaJ's putative substrate binding pocket important to its interaction with the preRC. These include residues Y119, I136 and I138. Of these, Y119 has not previously been implicated in substrate recognition. Together, these studies provide insights into the mechanism by which DnaJ recognizes the preRC and may shed light upon Hsp40 substrate binding, in general.