(Received for publication, November 3, 1995; and in revised form, November 25, 1995)
From the
Structural basis for ligand-induced protein stabilization was
investigated in the case of an acid phosphatase (red kidney bean purple
acid phosphatase (KBPAP)) from red kidney bean. Phosphate, a
physiological ligand, increases the stability against solvent
denaturation by 3.5 kcal/mol. Generality of phosphate stabilization was
shown by similar effects with other KBPAP ligands viz. adenosine 5`-O-(thiotriphosphate), a nonhydrolyzable
ligand, and arsenate, an inhibitor. The dissociation constant of
phosphate obtained from denaturation curves matches with the
dissociation constant estimated by conventional methods. The
guanidinium chloride-mediated denaturation of KBPAP was monitored by
several structural and functional parameters viz. activity,
tryptophan fluorescence, 8-anilinonaphthalene 1-sulfonic acid binding,
circular dichroism, and size exclusion chromatography, in the presence
and absence of 10 mM phosphate. In the presence of phosphate,
profiles of all the parameters shift to a higher guanidinium chloride
concentration. Noncoincidence of these profiles in the absence of
phosphate indicates multistate unfolding pathway for KBPAP; however, in
the presence of phosphate, KBPAP unfolds with a single intermediate.
Based on the crystal structure, we propose that the Arg
may have an important role to play in stabilization mediated by
phosphate.
Increase in the free energy upon ligand binding to a protein could translate into specific conformational changes that may have stabilizing or destabilizing effects on the protein(1, 2) . Native proteins are only marginally more stable than the denatured proteins (3) . Since unfolded proteins are better substrates for proteolytic degradation, ligand-mediated stabilization was considered to have influence on the protein turnover(3, 4) . Physiological ligands such as substrate analogues, inhibitors, etcetera were shown to enhance enzyme stability(5, 6, 7) . Conformational rearrangements in a protein on ligand binding was documented, and in several instances ligand-protein complexes were crystallized(8, 9) . Ligand-induced enzyme stabilization was quantitated by the protective role ligand binding plays against denaturation, e.g. heat or denaturant, and the reported values of the energy of stabilization were in the range of 1-4 kcal/mole(10, 11) . However, the causal relation between the ligand-induced structural changes and the ensuing stability was not addressed. It would be of interest to know whether the ligand merely shifts the unfolding pathway to a higher value on a temperature/denaturant scale or if it alters the way the protein responds to temperature/denaturant, i.e. alters the unfolding pathway. Such information would be of interest in our attempt to design enzymes with improved stability(6) .
Acid phosphatase from
red kidney beans (KBPAP) ()is a homodimeric glycoprotein
with a subunit molecular mass of 55
kDa(12, 13, 14, 15) . The binuclear
metal center of the protein with a tyrosine to Fe(II) charge transfer
transition was responsible for its characteristic purple
color(15) . Based on amino acid sequence alignment, it was
shown that the protein has
motif and has same
amino acids ligating the metal ions as in uteroferrin, its mammalian
counterpart(16) . The N-terminal 120-amino acid stretch was
absent in uteroferrin and does not have any active site or metal
ion-ligating amino acid residues(16) . The enzyme has limited
subunit interactions between the
5 helix and a loop formed by
amino acids from 253 to 260 beside an intersubunit disulfide bond at
Cys
(17) . KBPAP shows a rather narrow substrate
specificity toward ATP, unlike other plant acid phosphatases, and its
exact physiological function has not been ascertained until recently (18) . Plant acid phosphatases have been observed to be
intimately involved in plant phosphate metabolism(19) . We
observed significant enhancement in the stability of KBPAP toward
solvent and heat denaturation in the presence of phosphate. In this
communication, we report data on the influence of phosphate binding on
the denaturation pathway of KBPAP and demonstrate that phosphate not
only stabilizes KBPAP to denaturation but also alters the unfolding
pathway.
Potassium iodide quenching of
tryptophan fluorescence was done on the protein incubated at various
concentrations of GdmCl for 6 h at 25 °C. A stock of potassium
iodide was prepared in buffer containing the respective concentration
of GdmCl. Small aliquots of this stock were added to the denatured
protein up to a final potassium iodide concentration of 0.25 M. Fluorescence spectra were recorded after each addition. The
intensities at 340 nm were corrected for the increase in volume after
addition of potassium iodide. Stern-Volmer quenching constants (K) were calculated by established
procedures(21) .
ANS fluorescence spectra were recorded after adding twice-crystallized ANS to the protein sample to a final concentration of 112 µM. Excitation wavelength was set at 350 nm with a slit width of 10 nm. Emission spectra were recorded between 450 and 570 nm with a slit width of 5 nm. Base-line corrections were done with buffer without protein in all cases.
Figure 1:
Enzymatic
activity as a function of GdmCl concentration in the presence of
ligands or effectors. A, presence (solid circles) and
absence (open circles) of 10 mM phosphate in the
denaturation mix. B, presence of 1 mM ATPS (triangles) or 10 mM sodium arsenate (squares) in the denaturation mix. C, denaturation in
the presence (solid circles) or absence (open
circles) of 10 mM phosphate (24 h) was followed by
incubation with 50 mM
-mercaptoethanol (3 h). D,
plot of the midpoints of denaturation transition (D
, M) as a function of phosphate
concentration. The D
values were obtained from
experiments similar to A at several phosphate concentrations
(data not shown).
KBPAP is a homodimer with an intersubunit disulfide bond at
Cys, and an intact disulfide is necessary for native
conformation and activity(13) . To study the accessibility of
the intersubunit disulfide after denaturation for 24 h at 25 °C, we
incubated the protein in the denaturing mix for further 3 h after
addition of
-mercaptoethanol to a final concentration of 50
mM. In the absence of phosphate, the D
decreased to 1.6 M GdmCl, while in the presence of
phosphate the D
remained unchanged (Fig. 1C and Table 1). Even in the samples
without GdmCl, a 32% loss in the specific activity was caused by
-mercaptoethanol in the absence of phosphate and only a 7% loss in
the presence of phosphate (data not shown).
Based on theoretical
calculations, it was predicted that the binding constants of ligands
could be calculated from the denaturation curves (23) . We
calculated the binding constant of phosphate to KBPAP by plotting the D values against phosphate concentration (Fig. 1D) and obtained a binding constant of 1.2
mM, which was similar to the inhibition constant (K
= 1.8 mM) obtained by activity
inhibition studies. The equilibrium binding constants were calculated
by the non-parametric method described by Cornish-Bowden(20) .
Protection by phosphate was also observed against thermal denaturation,
wherein the midpoint of transition was shifted by 5 °C (data not
shown).
Figure 2:
Tryptophan fluorescence emission
wavelength maxima (max) plotted as a function of GdmCl
concentration. Open circles indicate absence and solid
circles indicate presence of 10 mM phosphate in the
denaturation mix. The inset shows the plot of
G versus GdmCl concentration. The
G(H
O) was the
intercept on the the y axis.
Figure 3:
Accessibility of KBPAP tryptophan to a
polar quencher, potassium iodide, measured as Stern-Volmer quenching
constants (K) was plotted as a function of GdmCl
concentrations. Open circles indicate absence and solid
circles indicate presence of 10 mM phosphate in the
denaturation mix.
Figure 4: Binding of ANS to denatured KBPAP in various GdmCl concentrations. Fluorescence of protein-bound ANS increases by severalfold compared to the free ANS, and relative fluorescence intensity of ANS at 490 nm was plotted against GdmCl concentration. Relative fluorescence intensity of samples without GdmCl were taken as unity.
Figure 5:
Change in mean residue ellipticity
([]
) at 220 nm as a function of GdmCl. Open circles indicate absence and solid circles indicate presence of 10 mM phosphate in the denaturation
mix. CD spectra were obtained at a concentration of 0.1 mg/ml using a
pathlength of 1 mm.
Figure 6: Elution profiles of KBPAP, on a Superose 12 column, denatured in various concentrations of GdmCl in the absence (A) or presence (B) of 10 mM phosphate in the denaturing mix. The column was preequilibrated with the solvent components identical to the incubation conditions. Peak 1 has elution volume equivalent to the void volume (5.7 ± 0.07 ml), peak 2 indicates where fully denatured protein appears, and peak 3 indicates the position of the native protein.
Figure 7: Areas under the three elution peaks at each GdmCl concentration were plotted as a function of GdmCl concentration (see Fig. 6). In the absence (A) and in the presence (B) of 10 mM phosphate. Peak 1, circles; peak 2, squares; peak 3, triangles.
To understand the structural basis of ligand-induced
stabilization in KBPAP, equilibrium solvent denaturation was studied by
various structural and functional parameters in the presence of its
physiological ligand, phosphate. In the presence of 10 mM phosphate, D, i.e. the denaturant
concentration at 50% denaturation, shifted to higher GdmCl
concentration by more than 1 M, indicating substantial
stabilization of KBPAP toward solvent denaturation (Table 1). A
(
G) of 3.5 kcal/mol was obtained for phosphate protection,
according to
G calculations based on fluorescence data (see inset in Fig. 2). Reported ligand binding energies were
in the order of 1-4
kcal/mol(2, 4, 27, 28) . Observed
stabilization with ATP
S, a nonhydrolyzable substrate analogue, and
arsenate, an inhibitor, indicated that the stabilization were general
and not specific for phosphate alone. In all these assays, enzyme was
preincubated in the presence or absence of phosphate/ligand and GdmCl
and assayed in the absence of phosphate/ligand and GdmCl. Phosphate was
used at about 10 times its inhibitory constant in the stability
experiments. We also observed that the inhibitory constant of phosphate
increases with GdmCl (data not shown). Similar information was not
available with ATP
S as ligand, and the enhanced specific
activities in 0-3 M GdmCl region may be due to decreased
binding of ATP
S. Phosphate inhibition was competitive in nature
when studied with ATP and p-nitrophenyl phosphate as
substrates, indicating that primary effect of phosphate was by binding
to the active site. Ligand binding energy could, for reasons of energy
conservation, have structural consequences that might stabilize or
destabilize a protein (2, 4) . Such substrate effects
were implicated in the protein turnover in vivo(3) .
In this case, dissociation and denaturation of KBPAP mediated by GdmCl
was delayed by phosphate ligation. The crystal structure of KBPAP has
been reported at 2.9-Å resolution and was shown to be a
homodimeric glycoprotein with an intersubunit disulfide bond (13, 17) . KBPAP has a limited subunit interface at
5 helix and a loop (amino acids 253-260) participating in
the intersubunit interaction beside the intersubunit
disulfide(17) . Reduction of the disulfide bond results in
inactivation and aggregation of the protein. (
)Here, we
observe that in the presence of phosphate, accessibility of the
intersubunit disulfide bond was reduced, indicating that the active
site bound ligand can stabilize subunit interactions.
By overlapping various parameters on a common GdmCl scale, one could identify and approximately characterize the intermediates in the unfolding pathway(29) . Unfolding pathway of KBPAP was multistate in nature, with all the parameter profiles showing clear dependence on the presence of phosphate. At low concentrations of GdmCl, surface hydrophobicity of the KBPAP increased significantly resulting in a 12-fold increase in ANS fluorescence. Substantial suppression of ANS binding in the presence of phosphate suggests prevention of underlying structural changes by phosphate. In this concentration range of GdmCl, i.e. up to 1.5 M, except ANS binding, other parameters including activity, the most susceptible parameter to denaturation, were invariant. KBPAP has an N-terminal 120-amino acid domain, which does not have any active site amino acids and was shown to be absent in the mammalian counter part of KBPAP, uteroferrin(16) . The structural changes occurring in 0-1.5 M range of GdmCl may be limited to the N-terminal domain.
On further denaturation in the absence of phosphate, i.e. in 1.5-3 M GdmCl, we observed a 90%
decrease in enzyme activity, the mole ratio of the native protein, ANS
binding, and only a marginal change in fluorescence and ellipticity. In
this region, KBPAP may be an inactive, swollen (probably aggregated to
a limited extent) intermediate with reduced surface hydrophobicity and
little change in the secondary structure but substantial loss in
tertiary and quarternary structure. Phosphate at 10 mM concentration prevents all the changes induced in the range of
1.5-3 M GdmCl and apparently shifts the equilibrium of N
&rlhar2; I(1) more to the left. Thus, on phosphate ligation, KBPAP in 3 M GdmCl has all the properties as a native enzyme, whereas
nonligated enzyme loses most of its tertiary and quarternary structure.
In the range of 3-5 M GdmCl, both in the presence and
absence of phosphate, significant loss in the secondary structure and
``inside out'' opening of the protein indicated by Trp
fluorescence and quenchability by a polar quencher occurs. These
structural rearrangements resulted in an intermediate with a 5-fold
enhancement in ANS binding and a significant portion of the protein
appearing as an aggregate. Both of these events were substantially
reduced by the presence of phosphate (see Fig. 4, Fig. 6,
and Fig. 7). In presence of phosphate, even in 6 M GdmCl, KBPAP has residual structure, indicated by higher negative
[]
value compared to the value in the
absence of phosphate.
In the absence of phosphate, the
nonoverlapping profiles of ANS binding, activity, SEC, fluorescence,
and CD demonstrate that unfolding pathway of KBPAP was multistate with
at least three intermediates populating the pathway. In the presence of
phosphate, the D of all of these profiles was in
the narrow range of 4-4.5 M. However, SEC analysis of
KBPAP denaturation was rewarding. In the range of 2-4 M GdmCl, even in the presence of phosphate, a small fraction
(<20%) of the protein was in aggregated form. SEC demonstrates that
the phosphate ligation to KBPAP alters the unfolding pathway with three
intermediates into a pathway with a single intermediate. Additionally,
in the absence of GdmCl, phosphate-induced structural changes reduce
the accessibility of the intersubunit disulfide bond to the reducing
agents.
To stabilize the pentacoordinate phosphorous transition
state in KBPAP, His and His
from one
subunit and Arg
from other subunit
participate(17) . It appears that the stabilizing effect of
phosphate on binding to active site of KBPAP was steric. By restricting
the movement of the loop containing Arg
, opening up of
the subunit interface could be prevented. In the phosphate-stabilized
protein, the restriction on movement of various segments restricts the
number of intermediates that could form, and consequently it unfolds
with a single intermediate. Similar ligand stabilizations were observed
with the catalytic trimer of Escherichia coli aspartate
transcarbamylase in the presence of carbamyl phosphate (2) and
in E. coli dehydroquinase with covalently bound
substrate(10, 28) . In the case of
20-
-hydroxysteroid dehydrogenase, NADH was shown to alter the
unfolding pathway(30) . The effects reported in this paper were
observed at a phosphate concentration in the range of its inhibition
constant (i.e. 1.8 mM). The dissociation constant of
phosphate calculated based on the denaturation curves was similar to
the K
value obtained based on activity inhibition.
Further, these effects were duplicated with other active site binding
ligands, indicating that phosphate-induced stabilization was strictly
ligand-induced effects and not specific to phosphate. Also, the
protective effect of phosphate toward KBPAP denaturation was observed
even during thermal denaturation. Stability of a protein was known to
be intimately related to its in vivo life times, since
unfolded proteins are considered to be substrates for cytosolic
proteases(3) . Near doubling in stabilization energies indicate
phosphate may have a physiological role of stabilizing KBPAP in
vivo.