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The Hsp70 chaperone activity in protein folding is regulated by ATP-controlled cycles of substrate binding and release. Nucleotide exchange plays a key role in these cycles by triggering substrate release. Structural searches of Hsp70 homologs revealed three structural elements within the ATPase domain: two salt bridges and an exposed loop. Mutational analysis showed that these elements control the dissociation of nucleotides, the interaction with exchange factors and chaperone activity. Sequence variations in the three elements classify the Hsp70 family members into three subfamilies, DnaK proteins, HscA proteins and Hsc70 proteins. These subfamilies show strong differences in nucleotide dissociation and interaction with the exchange factors GrpE and Bag-1.

Background Quercetinases are metal-dependent dioxygenases of the cupin superfamily. While fungal quercetinases are copper proteins, recombinant Streptomyces quercetinase (QueD) was previously described to be capable of incorporating Ni2+ and some other divalent metal ions. This raises the questions of which factors determine metal selection, and which metal ion is physiologically relevant. Results Metal occupancies of heterologously produced QueD proteins followed the order Ni > Co > Fe > Mn. Iron, in contrast to the other metals, does not support catalytic activity. QueD isolated from the wild-type Streptomyces sp. strain FLA contained mainly nickel and zinc. In vitro synthesis of QueD in a cell-free transcription-translation system yielded catalytically active protein when Ni2+ was present, and comparison of the circular dichroism spectra of in vitro produced proteins suggested that Ni2+ ions support correct folding. Replacement of individual amino acids of the 3His/1Glu metal binding motif by alanine drastically reduced or abolished quercetinase activity and affected its structural integrity. Only substitution of the glutamate ligand (E76) by histidine resulted in Ni- and Co-QueD variants that retained the native fold and showed residual catalytic activity. Conclusions Heterologous formation of catalytically active, native QueD holoenzyme requires Ni2+, Co2+ or Mn2+, i.e., metal ions that prefer an octahedral coordination geometry, and an intact 3His/1Glu motif or a 4His environment of the metal. The observed metal occupancies suggest that metal incorporation into QueD is governed by the relative stability of the resulting metal complexes, rather than by metal abundance. Ni2+ most likely is the physiologically relevant cofactor of QueD of Streptomyces sp. FLA. Electronic supplementary material The online version of this article (doi:10.1186/s12858-015-0039-4) contains supplementary material, which is available to authorized users.