Divergent Effects of ATP on the Binding of the DnaK and DnaJ Chaperones to Each Other, or to Their Various Native and Denatured Protein Substrates

Abstract

Using the native proteins lambda P, lambda O, delta 32, and RepA, as well as permanently unfolded alpha-carboxymethylated lactalbumin, we show that DnaK and DnaJ molecular chaperones possess differential affinity toward these protein substrates. In this paper we present evidence that the DnaK protein binds not only to short hydrophobic peptides, which are in an extended conformation, but also efficiently recognizes large native proteins (RepA, lambda P). The best substrate for either the DnaK or DnaJ chaperone is the native P1 coded replication RepA protein. The native delta 32 transcription factor binds more efficiently to DnaJ than to DnaK, whereas unfolded alpha-carboxymethylated lactalbumin or native lambda P binds more efficiently to DnaK than to the DnaJ molecular chaperone. The presence of nucleotides does not change the DnaJ affinity to any of the tested protein substrates. In the case of DnaK, the presence of ATP inhibits, while a nonhydrolyzable ATP analogues markedly stimulates the binding of DnaK to all of these various protein substrates. ADP has no effect on these reactions. In contrast to substrate protein binding, DnaK binds to the DnaJ chaperone protein in a radically different manner, namely ATP stimulates whereas a nonhydrolyzable ATP analogue inhibits the DnaK-DnaJ complex formation. Moreover, the DnaKc94 mutant protein lacking 94 amino acids from its C-terminal domain, which still possesses at ATPase activity and forms a transient complex with protein substrates, does not interact with DnaJ protein. We conclude that the DnaK-ADP form, derived from ATP hydrolysis, possesses low affinity to the protein substrates but can efficiently interact with DnaJ molecular chaperone.

A disulfide-bonded DnaK dimer is maintained in an ATP-bound state

“…A DnaJ construct fused to BCCP was immobilized on a sensor chip. Consistent with previous observations (Wawrzynow and Zylicz 1995;Suh et al 1998Suh et al , 1999Laufen et al 1999), a robust interaction was observed when WT DnaK was flowing through the sensor chip in the presence of ATP (Fig. 6a).…”

“…ATP hydrolysis may be a natural way to break the dimer during the chaperone cycle. This could be why ATP hydrolysis was observed to be required for productive DnaK-DnaJ interaction (Wawrzynow and Zylicz 1995;Suh et al 1998Suh et al , 1999Laufen et al 1999;Mayer et al 1999). The DnaK-DnaJ interaction is still largely a mystery even after years of extensive efforts.…”

“…On the other hand, the dimer showed a reduced ATPase rate and binding affinity for peptide substrates. Both activities seem to be crucial for the efficient DnaK-DnaJ interaction (Wawrzynow and Zylicz 1995;Gassler et al 1998;Suh et al 1998Suh et al , 1999Davis et al 1999). Moreover, previous mutational studies have shown that ATP binding is not sufficient for a stable DnaK-DnaJ interaction (Suh et al 1999).…”

JPDI, a Novel Endoplasmic Reticulum-resident Protein Containing Both a BiP-interacting J-domain and Thioredoxin-like Motifs

“…4). A requirement for hydrolyzable ATP for the J-protein-Hsp70 chaperone interaction is well documented (32,36). The ATPase activity of BiP next was significantly enhanced by the JPDI J-domain (Fig.…”

“…The following observations indicate that our preparation of JPDI J-domain interacts with BiP as its functional partner rather than as an unfolded protein substrate, which also binds to Hsp70 chaperones to stimulate their ATPase activity (11). First, unlike the case for J-protein partners, the binding of unfolded protein substrates to Hsp70 chaperones is inhibited by ATP (36). Second, amino acid replacement of the highly conserved HPD motif with QPD in the J-domain, which commonly causes dysfunction of various J-proteins (35,37), significantly decreased the ability of the JPDI J-domain to bind to BiP and to stimulate its ATPase activity (Figs.…”