Combining crystallography and EPR: crystal and solution structures of the multidomain cochaperone DnaJ

Abstract

Hsp70 chaperones assist in a large variety of protein-folding processes in the cell. Crucial for these activities is the regulation of Hsp70 by Hsp40 cochaperones. DnaJ, the bacterial homologue of Hsp40, stimulates ATP hydrolysis by DnaK (Hsp70) and thus mediates capture of substrate protein, but is also known to possess chaperone activity of its own. The first structure of a complete functional dimeric DnaJ was determined and the mobility of its individual domains in solution was investigated. Crystal structures of the complete molecular cochaperone DnaJ from Thermus thermophilus comprising the J, GF and C-terminal domains and of the J and GF domains alone showed an ordered GF domain interacting with the J domain. Structure-based EPR spin-labelling studies as well as cross-linking results showed the existence of multiple states of DnaJ in solution with different arrangements of the various domains, which has implications for the function of DnaJ.

Structural insights into mis-regulation of protein kinase A in human tumors

“…1, Table 1, and Table S1): the full-length wild-type C subunit (PKAc), the predominant point mutant (PKAc L205R), the predominant chimeric fusion protein (DnaJ-PKAc), and PKAc with exon 1 residues deleted (PKAc Δexon1). All structures were determined as complexes with ATP and the protein kinase inhibitor (PKI) peptide (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). In each, ATP is bound with two metal ions within the cleft formed between the smaller N-terminal and larger C-terminal lobes of the C subunit, associated as previously described (14).…”

Crowding Modulates the Conformation, Affinity, and Activity of the Components of the Bacterial Disaggregase Machinery

“…The conformational plasticity of this domain is evidenced by its ability to adopt different conformations in distinct DnaJ homologs. The recent X-ray structure of DnaJ from Thermus thermophilus reveals an ordered G/F domain [43], which seems to adopt a rather disordered conformation in its E. coli counterpart [44]. This particular region has been implicated in the interaction with folded protein substrates, although it is dispensable to bind unfolded polypeptides [28], and with the substrate binding cleft of DnaK [21,45].…”

Dynamical Structures of Hsp70 and Hsp70-Hsp40 Complexes

“…Many Hsp40s, including DnaJ, are known to exist as dimers (Figure 1C) (Barends et al, 2013; Kampinga and Craig, 2010; Shi et al, 2005). Numerous crystal and solution structures of Hsp40 J-domains and other fragments have been published (Huang et al, 1999a; Pellecchia et al, 1996; Stark et al, 2014), including a model of full-length E. coli DnaJ (Barends et al, 2013).…”

“…Numerous crystal and solution structures of Hsp40 J-domains and other fragments have been published (Huang et al, 1999a; Pellecchia et al, 1996; Stark et al, 2014), including a model of full-length E. coli DnaJ (Barends et al, 2013). Yet, despite the existence of these Hsp40 structures, structural information regarding Hsp40-Hsp70 complexes lagged for many years.…”