The Lid Domain of Caenorhabditis elegans Hsc70 Influences ATP Turnover, Cofactor Binding and Protein Folding Activity

Hsc70 is a conserved ATP-dependent molecular chaperone, which utilizes the energy of ATP hydrolysis to alter the folding state of its client proteins. In contrast to the Hsc70 systems of bacteria, yeast and humans, the Hsc70 system of C. elegans (CeHsc70) has not been studied to date

The second step of ATP binding to DnaK induces peptide release

The interaction of the nucleotide-free molecular chaperone DnaK (Hsp70) from Escherichia coli with nucleotides was studied under equilibrium and transient kinetic conditions. These studies used the intrinsic fluorescence signal of the single tryptophan residue (Trp102) of DnaK, or of novel fluorescent nucleotide analogs of ADP and ATP, N8-(4-N'-methylanthraniloylaminobutyl)-8-aminoadenosine 5'-di- or triphosphate (MABA-ADP and MABA-ATP) as spectroscopic probes. Titration of MABA-ADP with DnaK resulted in a 2.3-fold increase of the fluorescence signal, from which a binding stoichiometry of 1:1, and a dissociation constant (Kd) of 0.09 microM were derived. The intrinsic rate constant of hydrolysis of ATP or MABA-ATP in single turnover experiments was found to be 1.5 x 10(-3) s-1 and 1.6 x 10(-3) s-1, identical with the catalytic rate constant of 1.5(+/- 0.17) x 10(-3) s-1 obtained under steady-state conditions. The dissociation rate constant of ADP was measured to be 35(+/- 7) x 10(-3) s-1 in the absence or 15(+/- 5) x 10(-3) in the presence of 2 mM inorganic phosphate (Pi) and is therefore 10 to 20 times faster than the rate of hydrolysis. These results demonstrated that processes governing ATP hydrolysis are rate-limiting in the DnaK ATPase reaction cycle. The three observed different fluorescent states of the single tryptophan residue were investigated. The binding of ATP gave a decrease of 15% in fluorescence intensity compared with the nucleotide-free state. Subsequent ATP hydrolysis, or the simultaneous addition of ADP and Pi, increased the fluorescence 7% above the fluorescence intensity of the nucleotide-free protein. Changes in the tryptophan fluorescence could not be detected when ADP, Pi or the non-hydrolyzable nucleotide analogs AMPPNP (Kd = 1.62(+/- 0.1) microM) or ATP gamma S (Kd = 0.044(+/- 0.003) microM) were added. These data suggested that DnaK exists in at least three different conformational states, depending on nucleotide site occupancy. The fluorescence increase of DnaK upon ATP binding was resolved into two steps; a rapid first step (Kd 1 = 7.3 microM) is followed by a second slow step (k+2 = 1.5 s-1 and k-2 < or = 1.5 x 10(-3) s-1) that causes the decrease in the tryptophan fluorescence signal. The addition of ATP also resulted in the release of DnaK-bound peptide substrate with koff = 3.8 s-1, comparable with the rate of the second step of nucleotide binding. AMPPNP or ATP gamma S were not able to change the fluorescence signal nor to release the peptide. We therefore conclude that the second step of ATP binding, and not the 1000-fold slower ATP hydrolysis is coupled to peptide release

GrpE accelerates nucleotide exchange of the molecular chaperone DnaK with an associative displacement mechanism

The ATP hydrolysis and protein−binding and release cycle of the molecular chaperone DnaK is regulated by the accessory proteins GrpE and DnaJ. Here we describe a study of the formation of complexes between the molecular chaperone DnaK, its nucleotide exchange factor GrpE, and the fluorescent ADP analog N8−[4−[(N&#39;−methylanthraniloyl)amino]butyl]−8−aminoadenosine 5&#39;−diphosphate (MABA−ADP) by equilibrium and stopped flow kinetic experiments. The catalytic cycle of the GrpE−stimulated nucleotide exchange involves a ternary DnaK x GrpE x ADP complex as well as the binary DnaK x GrpE and DnaK x ADP complexes. The equilibrium data of the interaction of GrpE with DnaK x ADP and the nucleotide−free DnaK can be described by a simple equilibrium system where GrpE reduces the affinity of ADP for DnaK 200−fold. However, transient kinetic studies revealed that the functional cycle of GrpE in addition includes at least two distinct ternary DnaK x GrpE x ADP complexes. Our data indicate that the initial weak binding of GrpE to DnaK x ADP is followed by an isomerization of the ternary complex which leads to weakening of nucleotide binding and finally to its rapid dissociation. The maximal stimulation for nucleotide exchange brought about by GrpE was found to be 5000−fold. We propose that this kinetically observed isomerization represents a structural change (opening) of the nucleotide binding pocket of DnaK that allows for fast nucleotide exchang

Structural Dynamics of the DnaK–Peptide Complex

The molecular chaperone DnaK recognizes and binds substrate proteins via a stretch of seven amino acid residues that is usually only exposed in unfolded proteins. The binding kinetics are regulated by the nucleotide state of DnaK, which alternates between DnaK.ATP (fast exchange) and DnaK.ADP (slow exchange). These two forms cycle with a rate mainly determined by the ATPase activity of DnaK and nucleotide exchange. The different substrate binding properties of DnaK are mainly attributed to changes of the position and mobility of a helical region in the C-terminal peptide-binding domain, the so-called LID. It closes the peptide-binding pocket and thus makes peptide binding less dynamic in the ADP-bound state, but does not (strongly) interact with peptides directly. Here, we address the question if nucleotide-dependent structural changes may be observed in the peptide-binding region that could also be connected to peptide binding kinetics and more importantly could induce structural changes in peptide stretches using the energy available from ATP hydrolysis. Model peptides containing two cysteine residues at varying positions were derived from the structurally well-documented peptide NRLLLTG and labelled with electron spin sensitive probes. Measurements of distances and mobilities of these spin labels by electron paramagnetic resonance spectroscopy (EPR) of free peptides or peptides bound to the ATP and ADP-state of DnaK, respectively, showed no significant changes of mobility nor distance of the two labels. This indicates that no structural changes that could be sensed by the probes at the position of central leucine residues located in the center of the binding region occur due to different nucleotide states. We conclude from these studies that the ATPase activity of DnaK is not connected to structural changes of the peptide-binding pocket but rather only has an effect on the LID domain or other further remote residues

Tuning of chaperone activity of Hsp70 proteins by modulation of nucleotide exchange

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

Workflow interoperability in a grid portal for molecular simulations

Molecular simulations play in important role in diverse research areas like chemistry, biology and physics. The emerging MoSGrid (Molecular Simulation Grid) portal intends to offer various molecular simulation tools in WS-PGRADE, a workflow-enabled Grid portal. Hence, the portal will support also workflows including these tools. UNICORE is a Grid middleware with the additional feature of an included workflow engine. In this work we present a tool to invoke tasks of WS-PGRADE workflows in UNICORE and vice versa, to integrate existing UNICORE workflows in WS-PGRADE work-flows. Researchers are enabled to create and use both kind of Grid workflows without becoming acquainted to the Grid infrastructure or the workflow language