©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
How Potassium Affects the Activity of the Molecular Chaperone Hsc70
I. POTASSIUM IS REQUIRED FOR OPTIMAL ATPase ACTIVITY (*)

(Received for publication, June 25, 1994; and in revised form, November 2, 1994)

Melanie C. O'Brien David B. McKay (§)

From the Beckman Laboratories for Structural Biology, Department of Cell Biology, Stanford University School of Medicine, Stanford, California 94305

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Several functions of the 70-kilodalton heat shock cognate protein (Hsc70), such as peptide binding/release and clathrin uncoating, have been shown to require potassium ions. We have examined the effect of monovalent ions on the ATPase activity of Hsc70. The steady-state ATPase activities of Hsc70 and its amino-terminal 44-kDa ATPase fragment are minimal in the absence of K and reach a maximum at 0.1 M [K]. Activation of the ATPase turnover correlates with the ionic radii of monovalent ions; those that are at least 0.3 Å smaller (Na and Li) or larger (Cs) than K show negligible activation, whereas ions with radii differing only 0.1 Å from that of K (NH(4) and Rb) activate to approximately half the turnover rate observed with K. Single turnover experiments with Hsc70 demonstrate that ATP hydrolysis is 5-fold slower with Na than with K. The equilibrium binding of ADP or ATP to Hsc70 is unperturbed when K is replaced with Na. These results are consistent with a role for monovalent ions as specific cofactors in the enzymatic hydrolysis of ATP.


INTRODUCTION

The 70-kDa heat shock protein family is a highly conserved, ubiquitous group of proteins believed to function as ``chaperones,'' facilitating folding, assembly, or disassembly of other proteins but not forming part of the finished product (for reviews, see (1, 2, 3, 4) ). All members of the family have an ATPase activity, which regulates cycles of binding and release from unfolded proteins, although the exact mechanism is still unclear. Hsc70 (^1)is a constitutively expressed mammalian member of this family; it was originally described as a clathrin-uncoating protein(5) .

The Hsc70 protein can be divided into several functional domains: an amino-terminal 44-kDa ATPase domain, an 18-kDa peptide binding domain, and a carboxyl-terminal 10-kDa domain of unknown function(6, 7) . The crystal structure of the 44-kDa ATPase domain has been determined(8) . The tertiary fold is similar to that of actin, which is also an ATPase. Furthermore, the structures of the ATP binding domains of hexokinase and glycerol kinase, two other phosphotransferases, are similar to those of actin and the Hsc70 ATPase fragment(9) .

There have been several reports that monovalent cations affect activities of Hsc70. Recently, it was shown that both K and ATP are required for the dissociation of denatured proteins from DnaK (the Escherichia coli Hsc70 homolog) and that Na will not substitute for K(10) . Potassium ion is required for in vitro reconstitution of a complex of Hsc70, steroid receptor, and Hsp90(11) . In this case, NH or Rb cations could substitute for K, but Na and Li could not. In the original biochemical characterization of the clathrin uncoating activity of Hsc70, it was noted that K or NH above a threshold concentration of approximately 10-20 mM was required for uncoating activity(12) .

All of these reports describe functions of Hsc70 that involve a combination of polypeptide binding, ATPase activity, and a coupling between them; they do not indicate which specific activity is dependent on monovalent cation. In this context, we have examined the question of whether the ATPase activity of Hsc70, in particular, is dependent on monovalent cations and have concluded that both the rate of ATP hydrolysis and the rates of binding and release of nucleotide are affected by the type and concentration of ion. The characteristics of the stimulation of the Hsc70 ATPase activity by monovalent cations are similar in ion specificity and concentration dependence to those displayed by a number of enzymes, including many phosphotransferases. The direct participation of monovalent ions as cofactors in the ATPase activity of Hsc70, which we postulate from the kinetic and structural results presented here and in the accompanying manuscript(28) , may represent a phenomenon that is an essential feature of a significant number of phosphotransferase reactions.


MATERIALS AND METHODS

Protein Expression and Purification

Bovine recombinant Hsc70 and its 44-kDa ATPase domain (amino acid residues 1-386) were expressed (13) and purified (14) as described previously. All protein preparations were shown to be free of contaminating DnaK by Western blot.

ATPase Assays

Rates of steady-state ATP hydrolysis were determined at 37 °C in 20-µl reaction mixtures containing 100 nM enzyme, 10 mM HEPES, pH 7.0, 4.5 mM magnesium acetate, 5 µM ATP, 0.5 µCi of [alpha-P]ATP, and chloride salts of monovalent cations as indicated. Aliquots were spotted on polyethyleneimine-cellulose TLC plates at 4-8-min intervals, until about 10% total hydrolysis had occurred, which corresponds to five turnovers per enzyme molecule. Product ADP was determined by TLC as described previously(14, 15) , except that quantitation of radioactivity was done with a PhosphorImager (Molecular Dynamics). ATP hydrolysis rates were computed as straight line fits to ADP produced versus time (KaleidaGraph).

Single turnover rates of ATP hydrolysis were determined at 37 °C in 36-µl reaction mixtures containing 10 mM HEPES, pH 7.0, 4.5 mM magnesium acetate, 4.3 µCi of [alpha-P]ATP (40 nM), 100 mM KCl or NaCl, and an excess of Hsc70 (2 or 10 µM, as noted). Aliquots of the reaction were stopped at specific times by addition of an equal volume of ice-cold 12% trichloroacetic acid, followed rapidly by neutralization with 10 mM HEPES, pH 7.0, 0.4 M KOH. 0.5 µl of each stopped reaction was spotted onto a polyethyleneimine-cellulose TLC plate; TLC was performed and product ADP determined as above.

Filter Binding Assays

Preparations of 70-kDa Hsc70 were determined to be nucleotide-free by perchloric acid extraction (16) and spectrophotometric analysis. Other investigators have found that Hsc70 is not nucleotide-free following purification on an ATP-agarose column (17) ; our procedure includes further steps, such as a chromatofocusing column, that result in absence of nucleotide in our Hsc70 preparations.

The rate of association of nucleotide-free Hsc70 and labeled ATP was assayed at 37 °C in 10 mM HEPES, pH 7.0, 4.5 mM magnesium acetate, and 100 mM KCl or NaCl. The binding reactions were initiated by mixing equal volumes of prewarmed solutions of 2 times enzyme and 2 times buffer + 2 nM [alpha-P]ATP in a reaction volume of 100 µl. At the indicated time points, 10-µl aliquots of the solution were spotted onto BA85 nitrocellulose membrane filters (Schleicher and Schuell) and washed with 1-2-ml reaction buffer. For each zero time point, 10 µl of ATP solution with no protein present was spotted onto a filter and washed as above. Filters were dried and Cerenkov radiation was counted in a Beckman LS 5000TA scintillation counter. The apparent association rate k was computed by fitting a single exponential of the form P(1 - e^kobs^t) to the fraction of background-corrected counts retained on filters versus time, where P is the asymptotic value of the function.

To determine equilibrium binding constants (K(d)) for ADP, [alpha-P]ADP, prepared as described, (^2)was mixed with Hsc70 ranging in concentration 0.125-8.0 µM in the buffers described above. The solution was filtered, and K(d) was determined by a least squares fit of the function,

to the data, where is the fraction of counts retained on the filter and is the asymptotic value at high [Hsc70], which is the filter efficiency.


RESULTS

The ATPase cycle for Hsc70 and its 44 kDa ATPase domain can be described by the following scheme.

where ATP hydrolysis is essentially irreversible (k approx 0) and product release is ordered.^2 We use this scheme to interpret the results presented below.

Steady-state ATPase Activity

We first examined the effect of different monovalent ions on the steady-state ATPase activity by Hsc70. We observed a marked difference in the rates of ATP hydrolysis with NaCl versus KCl present (Fig. 1A). The ATPase activity increased from its basal level with increasing KCl concentration and reached a plateau at 150 mM KCl. In the presence of NaCl, a relatively low basal rate of hydrolysis that showed no significant dependence on the concentration of NaCl was observed (Fig. 1A). The steady-state ATPase rate in the presence of NaCl was 0.0002-0.0004 mol of ATP hydrolyzed (mole of enzyme) s, which is approximately 10-fold lower activity than observed under optimal KCl conditions.


Figure 1: Effect of increasing monovalent cation concentration on ATP hydrolysis by Hsc70. The error bars represent the residual error in the slope of a straight line fit to these time points; they should be regarded as minimum estimates of error. A, full-length Hsc70 with NaCl (bullet), NaCl + 2 mM VHLTPVGK (circle), KCl (times), or KCl + 2 mM VHLTPVGK (). B, 44-kDa ATPase fragment of Hsc70 with NaCl (bullet) or KCl (times).



The ATPase activity of full-length Hsc70 can be stimulated in vitro 2-3-fold by addition of denatured protein (18) or short peptides(19) . When 2 mM of a peptide known to stimulate ATPase activity (VHLTPVGK, Sigma) was added to the reactions, all hydrolysis rates were increased approximately 2-fold (Fig. 1A). A marked preference for KCl over NaCl was again observed.

We next tested the effect of monovalent cations on the steady-state ATPase activity of the 44-kDa ATPase fragment of Hsc70 (Fig. 1B). KCl was preferred to NaCl at all concentrations, and the observed rates in the presence of NaCl did not increase significantly with increasing NaCl. The apparent hydrolysis rate reached a maximum at 250 mM KCl; at salt concentrations above this, distortion of the TLC separations by the salt made quantitation of results unreliable.

LiCl, NH(4)Cl, RbCl, and CsCl were also tested for their ability to stimulate the steady-state ATPase activity of Hsc70. Data for Hsc70 ATPase activities in the presence of 150 mM monovalent cation are shown in Fig. 2; the activities showed the same order of cation preference at concentrations of 75 mM and 225 mM as well (data not shown). K stands out as the monovalent cation that is most effective in stimulating the ATPase activity of Hsc70. NH and Rb, with ionic radii differing less than 0.2 Å from that of K, are partially effective, whereas cations whose ionic radii differ from that of K by > 0.3 Å (either larger or smaller) are ineffective in stimulating the ATPase activity.


Figure 2: Rate of ATP hydrolysis by Hsc70 in the presence of various monovalent cations (chloride salts at 150 mM). Ionic radii are from (27) .



Association and Dissociation Rates for ATP

We used a filter binding assay to investigate binding of ATP to nucleotide-free Hsc70 in 100 mM KCl or NaCl. In the absence of protein, negligible background counts remain on filters spotted with aliquots of [alpha-P]ATP and washed as described under ``Materials and Methods.'' Data for the fraction of counts retained as a function of time at several protein concentrations (Fig. 3, A and C) were fit with a single exponential to give an observed rate of Hsc70-ATP association (k), as described under ``Materials and Methods.'' Under conditions where [Hsc70] [ATP], so that the concentration of free enzyme is essentially constant, k is expected to have a first-order dependence on [Hsc70]. If hydrolysis is minimal during the time course of the binding experiment, only binding and release of nucleotide will contribute to k; numbers reported here for the hydrolysis rate, in conjunction with a value^2 of 0.25 µM for the single turnover K(m), yield an estimate of leq10% ATP hydrolysis before ATP binding reaches its plateau under our conditions. Under these conditions, k has a linear dependence on enzyme concentration, with slope equal to the association rate (k(1)) and intercept equal to the dissociation rate (k)(20) . Table 1shows values for k(1) and k as well as the calculated equilibrium dissociation constant, K(d) = k/k(1), in the presence of KCl or NaCl. Both the association and dissociation rate for ATP are an order of magnitude slower in 100 mM NaCl than in 100 mM KCl. Our K(d) values are in agreement with a value determined for nucleotide-free protein by other investigators(16) .


Figure 3: Binding kinetics of ATP and Hsc70 in the presence of 100 mM KCl (A, B) or 100 mM NaCl (C, D). Experiments were performed as described under ``Materials and Methods.'' Enzyme concentrations were 20 nM (bullet), 40 nM (circle), 60 nM (times), 80 nM () (A); 50 nM (bullet), 100 nM (circle), 200 nM (times), 400 nM () (C). k values, determined from exponential fits as described in the text, were plotted against enzyme concentration and fit to a linear equation to determine kinetic constants (B, D).





Equilibrium Binding of ADP

We measured the equilibrium binding of ADP to Hsc70 in the presence of both KCl and NaCl, as described under ``Materials and Methods'' (Fig. 4). The binding curves in the presence of the two different ions are identical within experimental error. Values of K(d) for ADP computed from these data are given in Table 1.


Figure 4: Equilibrium binding of [alpha-P]ADP to Hsc70 in the presence of 100 mM KCl (circle) or 100 mM NaCl (bullet). Curves show the result of a least squares fit of a hyperbolic function to the data.



Rate of ATP Hydrolysis

We investigated the rate of ATP hydrolysis (k(2)) by Hsc70 in the presence of 100 mM KCl or NaCl under single turnover conditions. We used concentrations of Hsc70 (geq2 µM) that substantially exceeded the K(d) values of the Hsc70-ATP complexes in both KCl and NaCl, as well as the anticipated [Hsc70] that gives half-maximal rate of pre-steady-state hydrolysis (approx(k(2) + k)/k(1); approx 0.08 µM in KCl and 0.24 µM in NaCl, computed from values determined here). Under our experimental conditions, the nucleotide will be saturated with protein, and the hydrolysis rate will be maximal. Since ATP hydrolysis by Hsc70 is essentially irreversible, the time dependence of ADP formation will be adequately described by a single exponential equation^2 where the exponential term is -k(2)t.

Data showing the time course of ADP formation under single turnover conditions are shown in Fig. 5. The rate of hydrolysis is substantially slower in the presence of 100 mM NaCl than in the presence of 100 mM KCl. The observed rate of ATP hydrolysis will reach a plateau as the enzyme concentration exceeds the K(m). The K(m) for ATP in the presence of KCl is known to be about 0.5 µM(13, 14) , but the K(m) in the presence of NaCl has not been determined. We therefore tested enzyme concentrations of 2 µM and 10 µM in the presence of NaCl; the data points fall on essentially the same curve, and therefore the observed hydrolysis rate has reached a plateau. Values of k(2) determined from fits to the single exponential equation are 0.013 ± 0.002 s in 100 mM KCl, and 0.0023 ± 0.0004 s in 100 mM NaCl (average of 2 and 10 µM determinations).


Figure 5: Single turnover kinetics of Hsc70 in the presence of 100 mM KCl or NaCl. The amount of ATP hydrolysis at the indicated times was determined as described under ``Materials and Methods,'' with reactions containing 40 nM ATP and an excess of enzyme as noted.




DISCUSSION

We have demonstrated that the steady-state ATPase activity of Hsc70 and its 44-kDa ATPase fragment are stimulated by monovalent cation. The maximum ATPase turnover rate varies with the size, or ionic radius, of the cation, with K giving the highest activity and ions that are either smaller or larger giving lower activity. Since Hsc70 and the 44-kDa ATPase domain show similar dependence on monovalent ions, the effect on ATPase activity can be decoupled from peptide binding and the peptide binding domain.

Comparison of pre-steady-state measurements of ATP binding and hydrolysis shows that both the hydrolysis rate and the binding and release rates are much slower in the presence of Naversus K. Also, the steady-state ATPase rate is 10-fold slower in the presence of Na compared with K. However, the equilibrium dissociation constants for the Hsc70-ADP complex measured in the presence of Naversus K are equal within experimental error, as are the dissociation constants computed from the ATP association rates for the Hsc70-ATP complex. Thus, substitution of Na for K affects the kinetics of several steps of the ATPase cycle, but does not substantially alter the affinity for nucleotides.

The concentration of K required for optimal Hsc70 ATPase activity, 0.1 M, is high compared with, for example, the concentration of Mg that gives optimal activity, 10 µM(13) . It is worth noting in this context that the binding constants for K are anticipated to be substantially weaker than those for Mg, since the electrostatic interactions of monovalent ions with their ligands are much weaker than those of divalent ions. Also, the cytoplasmic concentration of K is 0.1-0.2 M, so that the protein would be maximally active under physiological conditions. The weaker binding affinity of monovalent ions is expected on energetic grounds and does not imply that their interactions with enzymes are less specific than those of divalent ions; on the contrary, in cases where it has been characterized structurally, their binding is very specific(21, 22) , also see accompanying manuscript(28) .

Several dozen enzymes are known to require monovalent cations for their activity(23) . Many of them are phosphotransferases, including a few ATPases. Several patterns of response to monovalent cations are seen among these enzymes(24) . One common pattern is that K (optimal concentration range typically 0.01-0.20 M) gives the greatest activation, NH and Rb give lesser activation, and Na and Li fail to activate. The behavior of Hsc70 is consistent with this pattern. Recently, another example in which K is required to optimize enzymatic ATP hydrolysis has been reported. The E. coli GroEL protein, a molecular chaperone of the Hsp60 family, is fully active in both ribulosebisphosphate carboxylase folding and uncoupled ATP hydrolysis in the presence of K, Rb, and NH; however, it is essentially inactive in the absence of monovalent cation or in the presence of Na, Li, or Cs(25, 26) .

One general question that arises is whether monovalent ions act as allosteric effectors, influencing the overall structure of an enzyme, or whether they interact directly with substrates. Dialkylglycine decarboxylase provides a precedent for the former mode of interaction; K binding at a specific site remote from substrates activates the enzyme, whereas Na binding at the same site inhibits it(22) . The crystallographic observation that two K ions bind in the active site region of the ATPase domain of Hsc70, at sites where they could act as metal cofactors in the ATP hydrolysis reaction(28) , provides an example of the latter mode, activation through direct interaction with substrate. The activities of a substantial number of phosphotransferase enzymes show a pattern of dependence on monovalent ion that parallels what we observe with Hsc70. This suggests that direct participation of K as a metal cofactor, by interaction with the nucleotide substrate, may be a relatively common mechanism by which monovalent ions influence phosphotransferase reactions.


FOOTNOTES

*
This work was supported by Grant GM-39928 (to D. B. M.) and Postdoctoral Fellowship GM-15141 (to M. C. O.) from the National Institutes of Health and by the resources of the Beckman Laboratories for Structural Biology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Cell Biology, Sherman Fairchild Bldg., Stanford University School of Medicine, Stanford, CA 94305-5400.

(^1)
The abbreviation used is: Hsc70, 70-kilodalton heat shock cognate protein.

(^2)
Ha J.-H., and McKay, D. B.(1994) Biochemistry, in press.


ACKNOWLEDGEMENTS

We thank J.-H. Ha, H. Pley, and S. Wilbanks for helpful advice and discussions.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.