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Cyclophilins accelerate slow protein folding reactions in vitro by catalyzing the cis/trans isomerization of peptidyl-prolyl bonds. Cyclophilins were reported to be involved in a variety of cellular functions, including the promotion of protein folding by use of the substrate mouse dihydrofolate reductase (DHFR). The interaction of cyclophilin with DHFR has only been studied under limited conditions so far, not taking into account that native DHFR exists in equilibrium with a non-native late-folding intermediate. Here we report a systematic analysis of catalysis of DHFR folding by cyclophilins. The specific ligand methotrexate traps DHFR in its native state, permitting a specific analysis of the action of cyclophilin on both denatured DHFR with non-native prolyl bonds and denatured DHFR with all-native prolyl bonds. Cyclophilins from yeast and Neurospora crassa as well as the related prolyl isomerase b from Escherichia coli promote the folding of different forms of DHFR to the enzymatically active form, demonstrating the generality of cyclophilin-catalyzed folding of DHFR. The slow equilibrium between the late-folding intermediate and native DHFR suggests that prolyl isomerization may be required for this final phase of conversion to native DHFR. However, by reversible trapping of the intermediate, we analyze the slow interconversion between native and late-folding conformations in the backward and forward reactions and show a complete independence of cyclophilin. We conclude that cyclophilin catalyzes folding of DHFR, but surprisingly not in the last slow folding step.

The molecular chaperone DnaK, the Hsp70 homolog of Escherichia coli, acts in concert with the co-chaperones DnaJ and GrpE. The aim of this study was to identify the particular phase of the peptide binding-release cycle of the DnaK/DnaJ/GrpE system that is directly responsible for the chaperone effects. By real-time kinetic measurements of changes in the intrinsic fluorescence of DnaK and in the fluorescence of dansyl-labeled peptide ligands, the rates of the following steps in the chaperone cycle were determined: (1) binding of target peptide to fast-binding-and-releasing, low-affinity DnaK·ATP; (2) DnaJ-triggered conversion of peptide·DnaK·ATP (T state) to slowly-acting, high-affinity peptide·DnaK·ADP·Pi (R state); (3) switch from R to T state induced by GrpE-facilitated ADP/ATP exchange; (4) release of peptide. Under conditions approximating those in the cell, the apparent rate constants for the T→R and R→T conversion were 0.04 s−1 and 1.0 s−1, respectively. The clearly rate-limiting T→R conversion renders the R state a minor form of DnaK that cannot account for the chaperone effects. Because DnaK in the absence of the co-chaperones is chaperone-ineffective, the T state has also to be excluded. Apparently, the slow, ATP-driven conformational change T→R is the key step in the DnaK/DnaJ/GrpE chaperone cycle underlying the chaperone effects such as the prevention of protein aggregation, disentangling of polypeptide chains and, in the case of eukaryotic Hsp70 homologs, protein translocation through membranes or uncoating of clathrin-coated vesicles.

GrpE is the nucleotide-exchange factor of the DnaK chaperone system. Escherichia coli cells with the classical temperature-sensitive grpE280 phenotype do not grow under heat-shock conditions and have been found to carry the G122D point mutation in GrpE. To date, the molecular mechanism of this defect has not been investigated in detail. Here, we examined the structural and functional properties of isolated GrpE(G122D) in vitro. Similar to wild-type GrpE, GrpE(G122D) is an elongated dimer in solution. Compared to wild-type GrpE, GrpE(G122D) catalyzed the ADP/ATP exchange in DnaK only marginally and did not compete with wild-type GrpE in interacting with DnaK. In the presence of ADP, GrpE(G122D) in contrast to wild-type GrpE, did not form a complex with DnaK detectable by size-exclusion chromatography with on-line static light-scattering and differential refractometry. Apparently, GrpE(G122D) in the presence of ADP binds to DnaK only with much lower affinity than wild-type GrpE. GrpE(G122D) could not substitute for wild-type GrpE in the refolding of denatured proteins by the DnaK/DnaJ/GrpE chaperone system. In the crystal structure of a (Delta1-33)GrpE(G122D).DnaK-ATPase complex, which as yet is the only available structure of a GrpE variant, Asp122 does not interact directly with neighboring residues of GrpE or DnaK. The far-UV circular dichroism spectra of mutant and wild-type GrpE proved slightly different. Possibly, a discrete change in conformation impairs the formation of the complex with DnaK and renders GrpE(G122D) virtually inactive as a nucleotide exchange factor. In view of the drastically reduced ADP/ATP-exchange activity of GrpE(G122D), the heat sensitivity of grpE280 cells might be explained by the ensuing slowing of the chaperone cycle and the increased sequestering of target proteins by high-affinity, ADP-liganded DnaK, both effects being incompatible with efficient chaperone action required for cell growth.

The gene encoding the dihydropteroate synthase of staphylococcus aureus has been cloned, sequenced and expressed in Escherichia coli. The protein has been purified for biochemical characterization and X-ray crystallographic studies. The enzyme is a dimer in solution, has a steady state kinetic mechanism that suggests random binding of the two substrates and half-site reactivity. The crystal structure of apo-enzyme and a binary complex with the substrate analogue hydroxymethylpterin pyrophosphate were determined at 2.2 A and 2.4 A resolution, respectively. The enzyme belongs to the group of “ TIM-barrel” proteins and crystallizes as a non-crystallographic dimer. Only one molecule of the substrate analogue bound per dimer in the crystal. Sequencing of nine sulfonamide-resistant clinical isolates has shown that as many as 14 residues could be involved in resistance development. The residues are distributed over the surface of the protein, which defies a simple interpretation of their roles in resistance. Nevertheless, the three-dimensional structure of the substrate analogue binary complex could give important insight into the molecular mechanism of this enzyme.

GroEL and DnaK with their cofactors constitute the major chaperone systems promoting protein folding in theEscherichia colicytosol. The ability of GroEL to bind and promote folding of a substrate released from DnaK led to the proposal that the DnaK and GroEL systems act successively along a protein folding pathway. Here we have investigated the role of both systems in preventing aggregation and assisting refolding of firefly luciferase denatured by guanidinium chloride and heat. We find that DnaK and GroEL compete with each other for binding to non-native luciferase. Addition of ATP and co-operating proteins results in release of luciferase from either chaperone in a non-native conformation. Only a small fraction of luciferase molecules released from GroEL can reach the native state. Instead, the released luciferase must bind repeatedly to the DnaK system, and only then is it able to fold to the native state. Thus, during a folding reaction, DnaK and GroEL do not obligatorily act in succession by promoting earlier and later protein folding steps, respectively. Rather, the two chaperone systems and perhaps others can form a lateral network of co-operating proteins. This chaperone network is proposed to be of particular importance for the assisted refolding of proteins that are unfolded by stress treatment such as heat shock and whose size is too large to allow folding inside the substrate binding cavity of the GroEL ring underneath GroES.

The E. coli heat shock response is regulated at the transcriptional level through stress-dependent controls of the heat shock promoter-specific sigma32 subunit of RNA polymerase. A key aspect of this regulation, the sensing of stress and transmission of this information to sigma32, involves the chaperone system formed by the DnaK, DnaJ and GrpE heat shock proteins. This system mediates stress- dependent controls of levels and activity of sigma32 which rely, at least in part, on direct association of DnaK and DnaJ with sigma32. We identified DnaK binding sites within the sigma32 sequence by probing a cellulose-bound peptide library scanning sigma32. Two sites with high affinity for DnaK, containing the motifs RKLFFNLR and LRNWRIVK, were located centrally and peripherally, respectively, to the region C of sigma32, previously implicated genetically in chaperone-dependent control of sigma32 levels. Cloning and sequencing of rpoH homologs from five Gram-negative proteobacteria revealed that region C, including the DnaK binding motif central to it, is highly conserved among sigma32 homologs but missing in the other sigma factors. We propose that binding of DnaK to region C is central to a conserved regulatory mechanism allowing the sensing of stress by the heat shock gene transcription machinery.