©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
How Potassium Affects the Activity of the Molecular Chaperone Hsc70
II. POTASSIUM BINDS SPECIFICALLY IN THE ATPASE ACTIVE SITE (*)

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

Sigurd M. Wilbanks 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

Crystallographic anomalous scattering from potassium at 1.7 Å resolution reveals two monovalent ions that interact with MgADP and P(i) in the nucleotide binding cleft of wild-type recombinant bovine Hsc70 ATPase fragment. K at site 1 interacts with oxygens of the beta-phosphate of ADP, whereas K at site 2 interacts with an oxygen of P(i). Both K ions also interact with specific H(2)O molecules in the first hydration shell of the octahedrally coordinated Mg ion and with specific protein ligands. In crystals that have Na present, K is replaced by a Na ion at site 1 and by a NaH(2)O pair at site 2. The K ions are positioned where they could stabilize binding of a beta,-bidentate MgATP complex with Hsc70, as well as a transition state during ATP hydrolysis, suggesting that monovalent ions act as specific metal cofactors in the ATPase reaction of Hsc70.


INTRODUCTION

Hsc70 (^1)is a molecular chaperone of the 70-kilodalton heat shock protein family (for reviews, see (1, 2, 3) ). For this group of proteins, binding and release of target polypeptides is tightly coupled to binding and hydrolysis of ATP. Several of the biochemical activities of Hsc70 (uncoating of clathrin coated vesicles (4) , assembly of glucocorticoid receptor complexes(5) , release of denatured polypeptides(6) ) manifest a requirement for K to optimize activity. Whether K is directly involved in peptide binding has not been studied in detail. However, as shown in the accompanying manuscript(34) , Hsc70 ATPase activity is stimulated by monovalent ions at concentrations 0.1 M, with K giving maximal activity, NH(4) and Rb showing approximately half as much activity and Na, Li, and Cs showing minimal activation(34) .

The ATPase activity of Hsc70 resides in a separable, amino-terminal fragment (7) (e.g. amino acid residues 1-386(8) ) of the protein. The dependence of the steady-state, peptide-independent ATPase activity of this fragment on monovalent ions parallels that of full-length Hsc70(34) . The x-ray crystallographic structure of this fragment has been solved(9) . The tertiary fold is almost identical to that of actin(10, 11) ; furthermore, the nucleotide binding subdomains of actin and the Hsc70 ATPase fragment are similar to those of hexokinase (12) and glycerol kinase(13) , identifying these molecules as members of a structural superfamily of phosphotransferases that use a similar tertiary fold to bind ATP. Many of the amino acid residues in the nucleotide binding sites of actin and Hsc70 are identical(11) , suggesting that their hydrolytic mechanisms may be similar; features of this mechanism may extend to the phosphotransferase mechanisms of the structurally related kinases. Crystallographic and kinetic studies of both wild-type Hsc70 ATPase fragment and a series of active site point mutants have shown how the hydrolysis products, MgADP + P(i), bind at the active site; they have also shown at least one conformation in which MgATP and nonhydrolyzable ATP analogs bind(8, 14, 15) . These results provided the basis for a suggested mechanism of ATP hydrolysis involving in-line attack by an H(2)O molecule or OH ion on the -phosphate of the nucleotide; the role of Mg to form a beta,-bidentate complex with the nucleotide and stabilize a transition state was essential to the suggested mechanism. No efforts had been made to identify monovalent ions in previous structural studies; here, we report anomalous scattering measurements that identify specifically bound K ions in the active site region of the ATPase fragment and suggest how K may be involved in the hydrolytic mechanism.

A number of enzymes, including many phosphotransferases, require particular monovalent ions for optimal activity (see (16) for review); the structural results presented here, in conjunction with the accompanying kinetic studies(34) , suggest that direct participation of monovalent ions as cofactors in phosphotransferase reactions may be a common phenomenon.


MATERIALS AND METHODS

Expression, Purification, and Crystallization

Recombinant protein was prepared as described previously(8, 14) . Briefly: the amino-terminal ATPase domain (amino acid residues 1-386) of bovine Hsc70 was expressed in Escherichia coli from a pT7-7-based vector. Protein was purified by anion exchange chromatography (DE52; Whatman), affinity chromatography (ATP-agarose; Sigma), chromatofocusing (Mono-P; Pharmacia, Uppsala, Sweden), and gel-filtration (Superdex-75, Pharmacia). Protein was essentially homogenous as judged by Laemmli gels and uncontaminated by DnaK as judged by Western blot.

Crystals were grown essentially as described (17) (crystallized from 20% PEG-8000, 1.0 M NaCl, 50 mM CAPS adjusted to pH 9.5 with NaOH, with 1 mM MgATP) and appeared identical to those obtained previously. To adapt crystals to more physiological conditions (pH 7.0 in the presence of potassium) and further to adapt crystals to a cryosolvent that allows flash-freezing, they were shifted to 5% ethylene glycol, 20% PEG-8000-1.0 M KCl, 50 mM HEPES (adjusted to pH 7.0 with KOH), 1 mM MgATP and then to progressively higher ethylene glycol (10, 15, and 20%) in the same mother liquor. Crystals were soaked at least one hour at each intermediate ethylene glycol concentration to avoid cracking.

Data Collection and Reduction

A crystal that had been stored 12 days in 20% ethylene glycol mother liquor at 4 °C was flash-frozen to 100 K in a stream of cold N(2) gas. Data were collected with a Rigaku R-axis image plate system using Cu K radiation monochromatized with graphite. Data collection parameters were: crystal-to-detector distance, 115 mm; detector swing, 13°; oscillation range and exposure time, 1.2° and 30 min when a* axis was greater than 60° from direct beam and 0.7° and 18 min when a* axis was less than 60° from direct beam. Crystal orientation and unit cell parameters were determined with the Molecular Structure Corp. R-axis software; the unit cell axes of the frozen crystals were a = 143.9 Å, b = 63.7 Å, c = 46.2 Å, which are changed significantly from the room temperature values of a = 145.3 Å, b = 65.0 Å, c = 46.9 Å. Integrated intensities were extracted from data frames and scaled with DENZO, giving R = 0.054 for data for 174,376 measurements with I geq 2 encompassing 39,253 unique reflections to a resolution of 1.7 Å (the resolution shell at which observed data are 50% complete).

Model Refinement

Standard crystallographic computations were performed using PROTEIN(18) . XPLOR (19) was used for model refinement by both simulated annealing and positional restrained least squares minimization. Model building was done with the program CHAIN (Rice version ESVAIN 2.2) on an Evans and Sutherland ESV(20) .

Our earlier model for wild-type ATPase fragment with MgADP and P(i) bound (15) was positioned in the asymmetric unit of the frozen crystal by rigid body refinement in XPLOR; this required translations of -0.19, -0.67, +0.08 Å in the x, y, and z directions, respectively, and rotations of +0.08°, +0.20°, and +0.78° around those same axes. One cycle of simulated annealing using data from 6.0 to 2.0 Å reduced the R factor to below 0.26. Multiple rounds of manual, positional and B-factor refinement were used to adjust the protein model, place metal ions that could be confirmed on the basis of anomalous scattering signals and reasonable ligand distances, and place water molecules in well defined peaks of density found at reasonable distances (2.5-3.1 Å) from polar groups. The current model includes amino acid residue 4-381, one ADP, one P(i), one Mg ion, two K ions, a single Cl ion, and 431 water molecules. The position of side chains of the following surface residues could not be determined and are modeled as alanine: Arg-77, Arg-100, Lys-137, Lys-188, Lys-248, Lys-250, and Arg-264. Refinement statistics for the model are: R-factor = 0.205 for 38,150 reflections with I geq 2 between 6.0 and 1.7 Å resolution with root mean square deviation from ideal geometry of 0.007 Å for bonds and 1.33° for angles. Luzzati plots estimate the coordinate error as <0.20 Å for the model.

The unambiguous identification of two K sites in the ATPase fragment structure, based on anomalous difference Fourier peaks, allowed identification of Na sites in the earlier model of the ATPase fragment derived from data collected using crystals in a mother liquor containing 1 M NaCl. Two Na ions placed in electron density peaks near the K ion sites have interaction distances to nearby oxygens that are too short for H(2)O molecules and closer to what is expected for a Na ion (2.4-2.8 Å). One cycle of positional restrained least squares refinement of the model gave R-factor = 0.210 for 21,926 reflections between 6.0 and 1.9 Å resolution (resolution shell of 50% completeness = 2.1 Å), with root mean square deviation from ideal geometry of 0.007 Å for bonds and 1.37° for angles.

Coordinates for both models have been deposited in the Brookhaven Protein Data Bank.


RESULTS

In conjunction with our studies of the effect of monovalent ions on the ATPase activity of Hsc70 and its 44-kDa ATPase fragment, we have examined the crystallographic structure of wild-type Hsc70 ATPase fragment in the presence of 1.0 M KCl, at pH 7.0 (referred to below as the ``KCl structure''), and compared it with the structure previously determined in the presence of 1.0 M NaCl at pH 9.5 (15) (referred to as the ``NaCl structure''). As stated explicitly in the previous publication(15) , no attempt had been made to distinguish between solvent molecules and monovalent ions in the NaCl structure. Flash-freezing of crystals has allowed us to collect data to significantly higher resolution, 1.7 Å, as compared with 2.1 Å for the NaCl structure determined at room temperature. This has resulted in a substantially more precise molecular model. There is an apparent shift of 0.70 Å in the position of the molecule in the crystal due primarily to the change of the unit cell upon freezing. The root mean square difference in position between the backbone atoms of the room temperature NaCl model and the KCl model derived from crystals chilled to 100 K is 0.41 Å. The major differences between the two structures are 1) reorientation of surface exposed loops which have relatively high temperature factors in the NaCl structure and 2) rotation of the beta-phosphate of ADP (discussed in detail below). Beyond this, the overall tertiary conformation of the molecule shows little change between the KCl structure and the NaCl structure.

Data collection of Bijvoet pairs of reflections allowed us to compute an anomalous difference Fourier which show peaks that may be ascribed to phosphorus (Deltaf" = 0.43 e at = 1.54 Å,(21) ), sulfur (Deltaf" = 0.56), chlorine (Deltaf" = 0.70), or potassium (Deltaf" = 1.07) atoms. The elements carbon, nitrogen, oxygen, and sodium have Deltaf" < 0.12 and are not expected to show significant anomalous scattering. Significant peaks in the anomalous difference Fourier, taken as those positive peaks that are larger than 5 ( = standard deviation of the map from its mean), the absolute value of the largest negative peak, are shown in Table 1.



The strongest three peaks in the anomalous difference Fourier have been assigned to K or Cl, based on both ligand distance criteria, peak height, and whether the nearby ligands are predominantly electronegative or electropositive. By these criteria, two peaks of height 22.8 and 22.3 have been assigned to K ions. One peak of height 13.9 has been assigned to a Cl ion bound in a loop region on the surface of the protein. The peaks of height 11.8-5.9 include those that can be assigned to the three phosphorus atoms of ADP and P(i), both sulfur atoms of the 2 cysteine residues, and all but one of the sulfur atoms of the methionines. The side chain of Met-93, whose sulfur does not show a significant peak by this criterion, is on the surface of the protein and is somewhat disordered. A peak of height 5.6 has been attributed to the sulfur of an ordered molecule of HEPES; the remainder of the HEPES molecule is apparent in difference Fourier maps. There are seven other peaks ranging 8.4-5.2 on the surface of the protein; these appear to be weakly bound ions with low occupancy or high temperature factors. They have been modeled as solvent at this time.

K in the Active Site

Wild-type ATPase fragment has ADP, P(i), and an octahedrally coordinated Mg ion in the active site. The Mg ligands include one oxygen of the beta-phosphate of ADP, one oxygen of P(i), and four H(2)O molecules; we refer to the Mg ion and its six oxygen ligands as the ``Mg cluster.'' The anomalous difference Fourier (shown in Fig. 1a) clearly shows peaks for two K ions in the active site, along with three smaller peaks centered on the phosphorus atoms of ADP and P(i). The two K ions on opposite sides of the Mg cluster are prominent as the highest peaks in map.


Figure 1: Stereo view active site. a, electron density maps: 2F(o) - F(c) Fourier map contoured at 1.2 is shown in blue; anomalous difference Fourier map contoured at 6 is shown in red. The molecular model is shown in stick diagram using the following colors: carbon in green, nitrogen in blue, oxygen and phosphorus in red, magnesium and potassium in yellow. Solvent (both ions and water) are shown as crosses; the magnesium ion and its ionic bonds are shown by the large, yellow octahedral cross at the center. b, ball and stick model, including selected protein residues (n.b. the side chain of Tyr-15 is omitted); probable hydrogen bonds and salt bridges are shown with lines. Carbon is shown in white, nitrogen in blue, non-water oxygen in red, water oxygen in green, phosphorus in yellow, magnesium in orange, and potassium in turquoise. Residues are labeled near their C.



The first site (site 1; Fig. 1b and Fig. 2) corresponds to a peak previously modeled as a solvent molecule in the NaCl structure and labeled ``w1'' in a previous publication(15) . The K ion at site 1 has eight oxygen ligands at distances ranging 2.57-3.22 Å (Table 2); allowing for the level of error in the coordinates (0.20 Å), these values are in general agreement with the range found for K-O distances from high resolution structures of small molecule complexes, 2.6-3.0 Å(22) . Three K ligands are provided by two oxygens of Pbeta and the Palpha-Pbeta bridging oxygen of ADP. The H(2)O molecules at the +x and +y positions of the octahedral Mg cluster provide ligands, as does one carboxyl oxygen of Asp-10. The two additional ligands of the K ion are the carbonyl oxygen of Tyr-15 and a full-occupancy internal H(2)O molecule.


Figure 2: Stereo model of monovalent ion site 1 occupied with K. The view is similar to that used in Fig. 1. Residues (on the C) and certain atoms are labeled. Phosphorus is shown in black, oxygen in gray, and carbon and nitrogen in white.





By comparison, only six of the eight oxygens that coordinate the K ion ligate the Na ion at this site in the NaCl structure. The six Na ion ligands include a carboxyl of Asp-10, the carbonyl oxygen of Tyr-15, the two H(2)O molecules of the Mg cluster, the internal H(2)O molecule, and one oxygen of the beta-phosphate of ADP. However, the phosphates of the ADP are rotated such that neither the second oxygen of the beta-phosphate nor the bridging oxygen between Palpha and Pbeta is close enough to coordinate the Na ion. Thus, the interactions of Na and K with the protein and the H(2)O molecules of the Mg cluster are very similar, whereas their interactions with the ADP phosphates are significantly different. The Na-O distances range 2.32-3.09 Å, consistent with ligand distances of 2.4-2.8 Å expected from small molecule studies(22) , with the exception of the Na-O(+y) distance of 3.09 Å.

The K ion in the second site (site 2; Fig. 1b and 3a), appears to be in a ``looser'' environment; there are seven oxygens at distances ranging 2.67-3.43 Å from it. The K at site 2 coordinates the oxygen of P(i) that is in the -x position of the Mg cluster octahedron; it also ligates the H(2)O molecule in the -y position. Additionally, it coordinates the hydroxyl and carbonyl oxygens of Thr-204 and (at a greater distance) both carboxyl oxygens of Asp-199 and one carboxyl oxygen of Asp-206.

In the NaCl structure, there are two peaks of density in the vicinity of site 2. The most credible model for this density, based on distances to nearest neighbor atoms, is a Na ion at one peak and an H(2)O molecule at the other (Fig. 3b). The Na ion coordinates one oxygen each of the ADP Pbeta and P(i), the H(2)O at the -y site of the Mg cluster, and the hydroxyl of Thr-204, as well as the H(2)O molecule in the second electron density peak. This H(2)O molecule interacts with one carboxyl oxygen each of Asp-199 and Asp-206, the hydroxyl and carbonyl oxygens of Thr-204, and reciprocally, with the Na ion. Hence, the K site is filled by a NaH(2)O pair in this case, with the Na binding closer to the ADP and P(i) ligands, whereas the H(2)O molecule binds more closely to the Asp-199 and Asp-206 ligands.



Figure 3: Stereo model of monovalent ion site 2 with K bound (a) and with Na bound (b). These views are both rotated 120° around the vertical axis, relative to Fig. 1and Fig. 2. The shading scheme is the same as Fig. 2.



In summary, there are two monovalent ion sites in the active site of the Hsc70 ATPase fragment. In the wild-type protein with MgADP and P(i) bound, the first site is at the interface between the protein and the beta-phosphate of the MgADP complex; the second is at the interface between protein and P(i).

The Cl- Binding Site

The third highest peak in the anomalous difference Fourier maps lies in the solvent exposed pocket formed by an alpha-helix (residues 122-134) and the residues 31-33, the ``insertion loop'' (Fig. 4). The closest interaction for an atom in this position are with the N of Lys-126 (3.25 Å), the amide of Asp-32 (3.35 Å), and the N of Gln-33 (3.44 Å). A fourth nitrogen, N of Asn-31 (4.23Å), is further away. The magnitude of the anomalous signal, and the overall positive ligand environment, suggest this position is occupied by a Cl ion. Furthermore, the ligand distances are approximately equal to the sum of the Cl ionic radius (1.81 Å) and the effective bonding distance of nitrogen or oxygen (1.4 Å) and are longer than would be expected for K, whose ionic radius is 1.33 Å. This pocket is partially closed by the carboxylate of Asp-32, the oxygens of which are each 3.9 Å distant from the chloride ion. No equivalent electron density was observed in the room temperature NaCl structure at pH 9.5, possibly because Lys-126 may be unprotonated, and unable to form a salt bridge, at the higher pH.


Figure 4: Chloride ion under the insertion loop. The alpha-helix from residues 122-135, beta-sheet strands from 14-24, 28-30 and 36-39, and loops from 25-27 and 34-35 are shown schematically. Residues 31-33 are shown as balls and sticks with carbon and oxygen in white and nitrogen in black. The chloride ion's closest contacts are indicated by dashed lines.




DISCUSSION

The anomalous scattering signals of potassium and chlorine have been used to distinguish monovalent ions from solvent H(2)O molecules in crystals of Hsc70 ATPase fragment. A chloride ion is specifically bound under the insertion loop. The insertion loop resides in an 8-residue insertion in the third beta-strand of the Hsc70 ATPase fragment. This insertion is present in the sequences of other 70-kDa heat shock proteins but absent in actin and hexokinase. The residues within this loop which appear to interact with the chloride ion (NDQ) are completely conserved among the cytosolic eukaryotic hsp70s (and occurs as either NDQ or NEQ in the family members found in the endoplasmic reticulum) but occurs as NXQ (X = S, T, A, or G) in bacterial, mitochondrial, and most chloroplastic dnaKs. This loop is of interest because it is, in dnaK, the site of interaction with the nucleotide exchange factor grpE(23) . Whether the ion binding property of this loop has physiological significance, or is merely a fortuitous circumstance of the crystallization conditions, remains an open question.

The primary purpose of our experiment was identification of ions within the active site. Two specific binding sites for monovalent cations were found in the nucleotide binding site of the protein. K at site 1 interacts with the beta-phosphate oxygens of the nucleotide, as well as with protein ligands and water molecules in the Mg cluster; K at site 2 interacts with a P(i) oxygen, in addition to ligands from the protein and the Mg cluster.

Na has a substantially smaller ionic radius (0.95 Å) than K (1.33 Å) and an accordingly lower preferred coordination number (typically 6 for Na as compared with 8 for K, as revealed by structures of small molecule complexes(22) ). In the presence of 1 M NaCl, Na ions are found in both of the monovalent ion binding sites; however, Na is an imperfect mimic of K. Na at site 1 binds the same ligands from the protein and Mg cluster as K. However, the Pbeta of ADP is rotated such that only one oxygen coordinates Na. Na binding at site 2 differs substantially from K binding; the K ion is replaced by a NaH(2)O pair, with the Na ion binding oxygens of P(i) and Pbeta, the hydroxyl of Thr-204, and the H(2)O molecule of the Mg cluster; although the H(2)O molecule interacts primarily with the protein ligands that would otherwise bind K. At both sites, discrimination between Na and K appears to be based on differences in ionic radius.

A model for the pathway of ATP hydrolysis which we presented previously (15) suggests that hydrolysis proceeds with formation of a beta,-bidentate complex between the triphosphate and Mg, followed by in-line nucleophilic attack by an H(2)O molecule or OH ion on the -phosphate to form a pentavalent transition state. This scheme can be reconciled with a role for K ions in hydrolysis. A model for the postulated bidentate MgATP complex, showing probable interactions of the two K ions with the phosphate oxygens, is shown in Fig. 5.


Figure 5: Model for possible interaction of K ions with MgATP in beta,-bidentate complex. Interactions of K with the nucleotide that are shown are hypothetical. Orientation and color scheme are that of Fig. 2.



The K ion at site 2 may facilitate an early step of hydrolysis by stabilization of partial negative charges on the oxygens of the -phosphate (through electrostatic interaction), thereby ``deshielding'' the target phosphorus atom and making it more susceptible to nucleophilic attack. Model building suggests that the K ion at site 2 would be likely to coordinate two of the oxygens attached to P for both ATP (as shown in Fig. 5) and for the transition state. Subsequently, the active site cations may contribute to the electrostatic stabilization of the pentavalent transition state, particularly through interaction of both K at site 1 and Mg with the alpha- and beta-phosphates. This is in addition to any contribution made by the mainchain amides: within the loops which most closely approach the phosphate moiety (residues 13-15, 202-204, and 339 and 340) all amide hydrogens point toward the phosphates. K at site 1 would bind an oxygen of the beta-phosphate and may bridge between the alpha- and beta-phosphates to form a bidentate complex similar to that observed in small molecule crystals of K-ADP complexes(24, 25) . It is worthy of mention that other phosphotransferases possess lysine residues with -amino groups situated to act in place of the potassium ions found in Hsc70. The -amino group of Lys-18 in actin forms salt bridges to oxygens of the alpha- and beta-phosphates of ADP(10) ; it could fulfill the identical role proposed for K at site 1 in Hsc70. Hsc70 has cysteine at the residue equivalent to Lys-18 of actin; it does not have a positively charged residue at this site that could perform this function. Similarly, Lys-16 within the conserved phosphate binding loop (P-loop) of H-ras p21 forms a beta,-bidentate complex with adenyl-5`-yl beta,-imidodiphosphate(26) .

The ability of a particular ion to facilitate hydrolysis by either deshielding or electrostatic stabilization is expected to increase as ionic radius decreases, providing a rationale for this enzyme's greater activity in the presence of potassium relative to larger monovalent cations. In addition, the active site may contain geometric constraints (e.g. location of -phosphate and H(2)O for in-line attack) which are better met by K than Na. The repositioning of the beta-phosphate in the sodium structure relative to the potassium structure indicates the importance of cations in establishing phosphate position. The suggestion from the structural results presented here, that facilitation of ATP hydrolysis will depend on the ionic radius of the monovalent cations at the active site of the protein, correlates with the observation that the ATP hydrolysis rate and the steady-state ATPase activity of Hsc70 are activated by monovalent ions and show a preference for K, with ions of either larger or smaller ionic radius being less effective in activation(34) .

The monovalent cation requirement of certain enzymes, including many phosphotransferases has long been recognized (cf. (27) ) and attributed either to a structural requirement for potassium at sites (perhaps on the protein surface) distant from the active site (28) , or to a role for potassium in contact with the substrate and catalytic residues(16) . Toney et al.(29) reported the structural basis for potassium activation of dialkylglycine decarboxylase, basing their identification of K and Na ions on bond distances and the refined temperature factor of the atom. In this case, a specific cation binding site is adjacent to but separate from the active site; it is an example of activation ``at a distance'' by a monovalent cation. K binding at this site activates the enzyme through a local conformational shift; Na deactivates it. Discrimination between the two cations is on the basis of ionic radius; the ligand distances are 2.7 ± 0.1 for K-O and 2.3 ± 0.2 for Na-O, and binding of the smaller Na results in the displacement of one of the six oxygen ligands of K.

Recently, the structure of rabbit muscle pyruvate kinase bound to pyruvate, Mn, and K was reported(30) . The single K ion does not interact directly with pyruvate, one of the products of the enzymatic phosphorylation of ADP by phosphoenolpyruvate. It remains to be determined whether the K ion interacts directly with the nucleotide or at a distance from both substrates.

In the case of Hsc70, it appears that monovalent ions exert their effect through direct interaction with phosphates of the nucleotide substrate, just as Mg does. This raises the possibility that direct interaction of monovalent ions with substrates may be an essential feature of a significant number of enzymatic phosphotransferase reactions. Data from small molecule crystal structures and solution studies may be cited to corroborate this suggestion. Structures of polyphosphate in complexes with cations show that the geometry of a complex is sensitive to the ionic radius of the cation(24, 25, 31, 32) . Also, non-enzymatic transphosphorylation of orthophosphate by ATP requires both a divalent and monovalent cation, with K being the most effective monovalent cation(33) . Those studies together with the present work provide a structural basis for the hypothesis that the monovalent ion dependence of many phosphotransferase enzymes is a manifestation of precise geometric constraints on the interaction of both divalent and monovalent ions with nucleotide phosphates at the enzyme-substrate interface.


FOOTNOTES

*
This work was supported by Grant GM-39928 from the National Institutes of Health (to D. B. M.) 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 abbreviations used are: Hsc70, 70-kDa heat shock cognate protein; Hsp70, 70-kDa heat shock protein; CAPS, 3-cyclohexylamino-1-propanesulfonic acid; PEG-8000, polyethylene glycol of approximate molecular weight 8000.


ACKNOWLEDGEMENTS

We thank Kevin Flaherty for assistance in all phases of the crystallographic work and Melanie O'Brien for discussions.


REFERENCES

  1. Gething, M. J., and Sambrook, J. (1992) Nature 355, 33-45 [CrossRef][Medline] [Order article via Infotrieve]
  2. McKay, D. B., Wilbanks, S. M., Flaherty, K. M., Ha, J.-H., O'Brien, M. C., and Shirvanee, L. L. (1994) in The Biology of Heat Shock Proteins and Molecular Chaperones (Morimoto, R. I., Tissieres, A., and Georgopoulos, C., eds) pp. 153-177, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  3. Hightower, L. E., Sadis, S. E., and Takenaka, I. M. (1994) in The Biology of Heat Shock Proteins and Molecular Chaperones (Morimoto, R. I., Tissieres, A., and Georgopoulos, C., eds) pp. 179-208, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  4. Schlossman, D. M., Schmid, S. L., Braell, W. A., and Rothman, J. E. (1984) J. Cell Biol. 99, 723-733 [Abstract]
  5. Hutchison, K. A., Czar, M. J., Scherrer, L. C., and Pratt, W. B. (1992) J. Biol. Chem. 267, 14047-14053 [Abstract/Free Full Text]
  6. Palleros, D. R., Reid, K. L., Shi, L., Welch, W. J., and Fink, A. L. (1993) Nature 365, 664-666 [CrossRef][Medline] [Order article via Infotrieve]
  7. Chappell, T. G., Konforti, B. B., Schmid, S. L., and Rothman, J. E. (1987) J. Biol. Chem. 262, 746-751 [Abstract/Free Full Text]
  8. Wilbanks, S. M., DeLuca-Flaherty, C., and McKay, D. B. (1994) J. Biol. Chem. 269, 12893-12898 [Abstract/Free Full Text]
  9. Flaherty, K. M., DeLuca-Flaherty, C., and McKay, D. B. (1990) Nature 346, 623-628 [CrossRef][Medline] [Order article via Infotrieve]
  10. Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F., and Holmes, K. C. (1990) Nature 347, 37-44 [CrossRef][Medline] [Order article via Infotrieve]
  11. Flaherty, K. M., McKay, D. B., Kabsch, W., and Holmes, K. C. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5041-5045 [Abstract]
  12. Fletterick, R. J., Bates, D. J., and Steitz, T. A. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 38-42 [Abstract]
  13. Hurley, J. H., Faber, H. R., Worthylake, D., Meadow, N. D., Roseman, S., Pettigrew, D. W., and Remington, S. J. (1993) Science 259, 673-677 [Medline] [Order article via Infotrieve]
  14. O'Brien, M. C., and McKay, D. B. (1993) J. Biol. Chem. 268, 24323-24329 [Abstract/Free Full Text]
  15. Flaherty, K. M., Wilbanks, S. M., DeLuca-Flaherty, C., and McKay, D. B. (1994) J. Biol. Chem. 269, 12899-12907 [Abstract/Free Full Text]
  16. Suelter, C. H. (1970) Science 168, 789-795 [Medline] [Order article via Infotrieve]
  17. DeLuca-Flaherty, C., Flaherty, K. M., McIntosh, L. J., Bahrami, B., and McKay, D. B. (1988) J. Mol. Biol. 200, 749-750 [Medline] [Order article via Infotrieve]
  18. Steigemann, W. (1974) The Development of a Computational Method and Computer Programs for the Structural Analysis of Proteins, with Application to the Examples of Trypsin-Trypsin Inhibitor Complexes, the Uncomplexed Inhibitors, and L-asparaginase. Ph.D. thesis, Techische Universit ä t M ü nchen
  19. Brunger, A. T., Kuriyan, J., and Karplus, M. (1987) Science 235, 458-460
  20. Jones, T. A., and Thirup, S. (1986) EMBO J. 5, 819-822 [Abstract]
  21. Ibers, J. A., and Hamilton, W. C. (1974) International Tables for X-ray Crystallography , Vol. IV, p. 149, Birmingham, United Kingdom
  22. Glusker, J. P. (1991) Adv. Protein Chem. 42, 1-76 [Medline] [Order article via Infotrieve]
  23. Buchberger, A., Schröder, H., Büttner, M., Valencia, A., and Bukau, B. (1994) Struct. Biol. 1, 95-101
  24. Adamiak, D. A., and Saenger, W. (1980) Acta Crystallogr. B36, 2585-2589 [CrossRef]
  25. Swaminathan, P., and Sundaralingam, M. (1980) Acta Crystallogr. B36, 2576-2584 [CrossRef]
  26. Pai, E. F., Krengel, U., Petsko, G. A., Goody, R. S., Kabsch, W., and Wittinghofer, A. (1990) EMBO J. 9, 2351-2359 [Abstract]
  27. Rose, I. A. (1960) J. Biol. Chem. 235, 1170-1177 [Medline] [Order article via Infotrieve]
  28. Evans, H. J., and Sorger, G. J. (1966) Annu. Rev. Plant Physiol. 17, 47-76
  29. Toney, M. D., Hohenester, E., Cowan, S. W., and Jansonius, J. N. (1993) Science 261, 756-759 [Medline] [Order article via Infotrieve]
  30. Larsen, T. M., Laughlin, L. T., Holden, H. M., Rayment, I., and Reed, G. H. (1994) Biochemistry 33, 6301-6309 [Medline] [Order article via Infotrieve]
  31. Merritt, E. A., and Sundaralingam, M. (1980) Acta Crystallogr. B36, 2576-2584 [CrossRef]
  32. Kennard, O., Isaacs, N. W., Mothrwell, W. D., Coppola, J. C., Wampler, D. L., Larson, A. C., and Watson, D. G. (1972) Proc. R. Soc. Lond. Ser. A 325, 401-438
  33. Lowenstein, J. M. (1960) Biochem. J. 75, 269-274 [Medline] [Order article via Infotrieve]
  34. O'Brien, M. C., and McKay, D. B. (1995) J. Biol. Chem. 270, 2247-2250 [Abstract/Free Full Text]

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