(Received for publication, June 25, 1994; and in revised form, November 2, 1994)
From the
Crystallographic anomalous scattering from potassium at 1.7
Å resolution reveals two monovalent ions that interact with MgADP
and P in the nucleotide binding cleft of wild-type
recombinant bovine Hsc70 ATPase fragment. K
at site 1
interacts with oxygens of the
-phosphate of ADP, whereas
K
at site 2 interacts with an oxygen of P
.
Both K
ions also interact with specific H
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
Na
H
O pair at site 2. The K
ions are positioned where they could stabilize binding of a
,
-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.
Hsc70 ()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
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, 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
O molecule or OH
ion on the
-phosphate of the nucleotide; the role of Mg
to
form a
,
-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.
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.
Our earlier model for wild-type
ATPase fragment with MgADP and P 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
, 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
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
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.
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 -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 (f" = 0.43 e
at
= 1.54 Å,(21) ), sulfur (
f" =
0.56), chlorine (
f" = 0.70), or potassium
(
f" = 1.07) atoms. The elements carbon, nitrogen,
oxygen, and sodium have
f" < 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
,
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.
Figure 1:
Stereo view active site. a,
electron density maps: 2F - F
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 P
and the
P
-P
bridging oxygen of ADP. The H
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
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
O molecules of the
Mg
cluster, the internal H
O molecule, and
one oxygen of the
-phosphate of ADP. However, the phosphates of
the ADP are rotated such that neither the second oxygen of the
-phosphate nor the bridging oxygen between P
and P
is
close enough to coordinate the Na
ion. Thus, the
interactions of Na
and K
with the
protein and the H
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
that is in the -x position of the Mg
cluster octahedron; it also
ligates the H
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
O molecule at
the other (Fig. 3b). The Na
ion
coordinates one oxygen each of the ADP P
and P
, the
H
O at the -y site of the Mg
cluster, and the hydroxyl of Thr-204, as well as the
H
O molecule in the second electron density peak. This
H
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 Na
H
O pair
in this case, with the Na
binding closer to the ADP
and P
ligands, whereas the H
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 bound,
the first site is at the interface between the protein and the
-phosphate of the MgADP complex; the second is at the interface
between protein and P
.
Figure 4:
Chloride ion under the insertion loop.
The -helix from residues 122-135,
-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.
The anomalous scattering signals of potassium and chlorine
have been used to distinguish monovalent ions from solvent
HO 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
-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
-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
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 P
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 Na
H
O pair,
with the Na
ion binding oxygens of P
and
P
, the hydroxyl of Thr-204, and the H
O molecule of the
Mg
cluster; although the H
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 ,
-bidentate complex between the
triphosphate and Mg
, followed by in-line nucleophilic
attack by an H
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
,
-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
- and
-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
-phosphate and may bridge between the
- and
-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
- and
-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
,
-bidentate complex with adenyl-5`-yl
,
-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
O for
in-line attack) which are better met by K
than
Na
. The repositioning of the
-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.