From the § Department of Pharmacology and
Department of Chemistry and Biochemistry, University of
California, San Diego, La Jolla, California 92093-0506
Received for publication, December 21, 2000, and in revised form, January 18, 2001
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ABSTRACT |
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Transient state kinetic studies indicate that
substrate phosphorylation in protein kinase A is partially rate-limited
by conformational changes, some of which may be associated with
nucleotide binding (Shaffer, J., and Adams, J. A. (1999)
Biochemistry 38, 12072-12079). To assess whether specific
structural changes are associated with the binding of nucleotides,
hydrogen-deuterium exchange experiments were performed on the enzyme in
the absence and presence of ADP. Four regions of the protein are
protected from exchange in the presence of ADP. Two regions encompass
the catalytic and glycine-rich loops and are integral parts of the
active site. Conversely, protection of probes in the C terminus is
consistent with nucleotide-induced domain closure. One protected probe
encompasses a portion of helix C, a secondary structural element that
does not make any direct contacts with the nucleotide but has been
reported to undergo segmental motion upon the activation of some
protein kinases. The combined data suggest that binding of the
nucleotide has distal structural effects that may include stabilizing
the closed state of the enzyme and altering the position of a critical
helix outside the active site. The latter represents the first evidence
that the nucleotide alone can induce changes in helix C in solution.
Protein kinases are the essential enzymes that direct protein
phosphorylation in the cell. The results of this posttranslational modification on protein structure and function can have extraordinary effects ranging from changes in carbohydrate and neurotransmitter metabolism to organelle trafficking and cell division. Given the general role that protein phosphorylation plays in these and many other
signal transduction pathways, understanding how these enzymes process
substrates has become key to understanding cell function. The insights
derived from biophysical studies will support the intense consideration
that this enzyme family is now being given as chemotherapeutic targets
(1). As essential components for normal cell function, protein kinases
are tightly regulated through a broad host of processes including
phosphorylation (2), second messengers such as cAMP and
Ca2+, fatty acylation, protein-protein and domain-domain
interactions, and localization through scaffolding and adaptor proteins
(3, 4). These processes ensure that the correct protein kinase is
activated or repressed at the appropriate time and at the correct location in the cell. Indeed, mutations in protein kinases that alter
their regulation are frequently linked to disease (5-10).
Protein kinases possess a well conserved core composed of a small ATP
binding domain and a larger substrate binding domain as exemplified in
Fig. 1 for the catalytic subunit
(C-subunit)1 of protein
kinase A (PKA) (11, 12). The active site lies between these two domains
with ATP embedded deep within the pocket and the substrate fixed toward
the periphery. Protein kinases are conformationally dynamic, and
several movements within the core kinase structure have been observed.
For example, PKA has been crystallized in both open and closed (Fig.
1) forms that differ primarily by domain
rotation (13). Small angle x-ray scattering methods suggest these
conformational dynamics may also occur in solution (14). In addition,
phosphorylation of the activation loop segment in Cdk2 and the
kinase domain of insulin receptor kinase lowers B factors and causes an
ordering of this region (15-17). While it is unlikely that the
activation loop serves the universal function of an autoinhibitor (18,
19), loop motion has been linked to other interesting changes in
structure that may have a general role in regulation. For protein
kinase structures that have been solved in both phosphorylated (active) and dephosphorylated (inactive) states, the ordering of the activation loop upon phosphorylation results in a notable shift in helix C (16,
20, 21). This is thought to be a key element in kinase activation,
since this movement places a conserved glutamate in this helix
(Glu91 in PKA) within hydrogen bonding distance of a
conserved lysine (Lys72 in PKA) residue in the active site.
This lysine chelates either the
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
or the
/
phosphates of ATP
and upon mutation results in a low activity mutant (22-24). Based upon
these findings, it is thought that protein kinase regulation through
activation loop phosphorylation is linked to the formation of this
essential glutamate-lysine dyad.
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Fig. 1.
Closed form of the C-subunit of PKA.
A, the standard representation of the C-subunit derived from
the ternary complex with ATP and a peptide inhibitor (not shown). The
arrow indicates the direction of domain movement observed in
the open conformation derived from a binary complex with a peptide
inhibitor (13). The open and closed forms are related by a 15°
rotation of the -sheet in the small domain relative to the larger
domain. The activation loop is shown in magenta, and
helix C is shown in blue. Co-crystallized ATP and
phosphorylated Thr197 in the activation loop are shown in
ball and stick representations.
B, rotation of the closed C-subunit structure in
A by 90 °C in the z axis. The
figure was prepared using Protein Data Bank accession
number 1ATP (Research Collaboratory for Structural Bioinformatics,
Rutgers University, New Brunswick, NJ).
Detailed kinetic studies of the paradigm protein kinase, PKA,
reveal that conformational changes not only are part of normal catalysis but also may be slow relative to turnover and provide a means
of regulating enzyme function. Two conformational changes (one before
and one after the phosphoryl transfer step) partially control
kcat in wild-type PKA under physiological
concentrations of magnesium (25, 26). At least one of these steps
appears to be linked to nucleotide binding. Stopped-flow experiments
performed on a fluorescently labeled mutant of PKA demonstrate that
turnover is partially limited by a conformational change occurring
after the phosphoryl transfer step (27). That some of these structural changes are linked to nucleotide binding is further supported by
stopped-flow binding studies using fluorescently labeled mant derivatives of ATP and ADP (28). The binding of mant-ADP is accompanied
by slow conformational changes that are close in value to the turnover
rate. In this paper, we now describe structural methods for attaining a
molecular description of these conformational changes. By applying
hydrogen-deuterium (H-D) exchange techniques coupled with MALDI-TOF
mass spectrometric detection (29), we have shown that four distinct
polypeptide regions in PKA alter their protection from amide proton
exchange upon ADP binding. Two of these regions are located in the
active site, while two are distal to the nucleotide pocket. The latter
regions contain part of the C-terminal tail and helix C, the secondary
structural element containing one essential member of the
glutamate-lysine dyad, namely Glu91. The data suggest that
when nucleotides bind to PKA, helix C changes conformation, perhaps
causing formation or strengthening of the dyad, and the closed form of
the enzyme is stabilized.
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EXPERIMENTAL PROCEDURES |
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Materials--
PD10 columns for buffer exchange were obtained
from Amersham Pharmacia Biotech. Dipotassium-ADP was obtained from ICN
Biomedicals Inc. D2O (99.9% deuterium) was obtained
from ISOTEC Inc. Pepsin immobilized on 6% beaded agarose was obtained
from Pierce. Trifluoroacetic acid and acetonitrile were obtained from
Fisher and were of peptide synthesis grade and optima grade,
respectively. -Cyano-4-hydroxycinnamic acid was obtained from
Aldrich and recrystallized once from ethanol. ATP, Mops, lactate
dehydrogenase, pyruvate kinase, reduced nicotinamide adenine
dinucleotide (NADH), and phosphoenolpyruvate were purchased from Sigma.
The substrate peptide, LRRASLG (Kemptide), was synthesized at the USC
Microchemical Core Facility using Fmoc
(N-(9-fluorenyl)methoxycarbonyl) chemistry and purified by
C-18 reverse phase HPLC.
Protein Preparation and Activity Assays-- The C-subunit of murine PKA was expressed in E. coli and purified as previously described (30). Isozyme I, the first isoform eluting from the cation exchange column, was used for all experiments. The buffer was changed to 100 mM KPi, pH 7.0, 5 mM 2-mercaptoethanol on a PD10 column, and the protein was concentrated to 192 µM. The protein was stored at 4 °C and used in the experiments without lyophilizing. The activity of the C-subunit was measured using a spectrophotometric coupled enzyme assay (31). The C-subunit was preequilibrated with 1 mM ATP and 11 mM MgCl2 in 100 mM Mops (pH 7), and the reaction was initiated with 0.5 mM Kemptide.
Deuterium Exchange Experiments--
All exchange mixtures for
the C-subunit contained the following: 16 µM C-subunit,
40 mM KPi, 50 mM KCl, and 10 mM MgCl2. Final pH* was 6.9, and final
percentage of D2O was 87.5%. Exchange experiments performed in the presence of nucleotide included 1 mM ADP
and 11 mM MgCl2. The C-subunit was
preequilibrated with ADP in H2O before starting the
deuterium exchange by diluting into D2O. The D2O mixtures were prepared as follows (numbers are per
12-µl aliquot). The amounts of 100 mM KPi and
4 M KCl needed were mixed, dried in a Speedvac,
dissolved in D2O, and mixed with D2O solutions of ADP and MgCl2. The final volume was 10.5 µl, and it
contained 36 mM KPi, 57 mM KCl, and
either 10 mM MgCl2 or 1 mM ADP and
11 mM MgCl2. The H2O mixtures (1.5 µl per 12-µl aliquot) contained 128 µM C-subunit, 67 mM KPi, and either 10 mM
MgCl2 or 1 mM ADP and 11 mM
MgCl2. The deuterium exchange was initiated by combining the H2O and D2O solutions. The solutions were
incubated at 20 °C. At various times a 12-µl aliquot was added to
an ice-cold tube containing 36 µl of 0.19% trifluoroacetic acid and
25 µl of pepsin bead slurry (previously washed two times in 1 ml of cold 0.05% trifluoroacetic acid). This brought the pH* of the C-subunit solution down to 2.5 and quenched the deuterium exchange. The
mixture was incubated on ice with occasional mixing for 5 min to
facilitate pepsin proteolysis of the C-subunit. The mixture was then
centrifuged for 20 s at 12,000 × g at 4 °C to remove the
pepsin beads, and the solution was divided in aliquots and frozen in
liquid N2. The samples were stored at 80 °C until
MALDI-TOF MS analysis.
The mixtures for deuterium exchanging C-subunit at pH* 5.9 were prepared similarly to the pH* 6.9 samples. A predetermined amount of phosphoric acid that would bring the solution to pH* 5.9 was added together with KCl and KPi before drying in the Speedvac. The concentration of trifluoroacetic acid used to quench the reaction was adjusted accordingly to reach pH* 2.5.
In- and Back-exchange Controls-- In-exchange of deuterium under quench conditions was measured by adding the protein solution directly to a mix of the D2O solution, quench solution, and pepsin and performing the remaining procedure as normal. This sample corresponds to time point 0. The back-exchange occurring during the procedure was measured essentially as in Ref. 32 by using a previously pepsin-digested protein sample, drying it completely, redissolving it in labeling buffer, and incubating at 20 °C for 1 h to achieve complete exchange of backbone amide protons for deuterium. To assess the amount of label lost during sample workup (back-exchange) the deuterated samples were treated to quench and MALDI-TOF MS analysis as described above. This control measures, for each peptide, the maximal experimental mass that corresponds to a fully exchanged peptide.
MALDI-TOF MS--
MALDI-TOF MS was performed essentially as
described previously (29), under which conditions the H-D exchange is
kept at a minimal rate. Samples were kept on finely crushed dry ice,
and target plates were kept at 4 °C. The matrix solution consisted of 5 mg/ml -cyano-4-hydroxycinnamic acid in 1:1:1 acetonitrile, ethanol, 0.52% trifluoroacetic acid (final pH 2.0). The samples were
thawed quickly, and 5 µl were mixed with a 5-µl aliquot of 4 °C matrix solution. One µl was spotted on the target plate at 4 °C and dried in 1.5 min under moderate vacuum. Mass spectra were
acquired on a PerSeptive Biosystems Voyager DE STR MALDI-TOF. Data were acquired at a 2-GHz sampling rate, 100,000 data channels, with a 20,000-V accelerating voltage, 78% grid voltage, and 0.012% guide wire voltage and using delayed extraction with a 100-ns pulse
delay. 256 scans were averaged in ~3 min.
Data Analysis-- The mass spectra were calibrated in the software GRAMS using the 1194.6485 and 1793.9704 mass peptides. The spectra were then base line-corrected, and centroids of each peak were determined using in-house software (29). The number of deuteriums in-exchanged at time t was calculated as in Ref. 33 using Equation 1,
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(Eq. 1) |
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RESULTS |
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Experimental Design--
H-D exchange reactions are exquisitely
sensitive to changes in structure and dynamics that accompany protein
folding, ligand binding, or formation of protein-protein interactions.
H-D exchange in small proteins can be monitored at the residue-specific
level by combining H-D exchange with NMR detection. In the case of PKA, a 40-kDa protein, we utilize a medium resolution method that gives region-specific information by combining H-D exchange, pepsin fragmentation, and MALDI-TOF MS detection. A schematic for this technique is displayed in Fig. 2. The
C-subunit, preequilibrated with or without the nucleotide, is incubated
in D2O up to 3 h. After designated time periods, the
exchange is quenched at pH* 2.5, and the protein is digested with
pepsin. During the exchange reaction, the nucleotide concentration is
~100-fold higher than the Ki for ADP (25),
assuring more than 99% binding at all times. In addition, the observed
association rate for ADP is estimated to be 2000 s1 (34), whereas the average intrinsic H-D
exchange rate is slower (3.4 and 0.34 s
1 at
20 °C and pH* 6.9 and 5.9, respectively (35)), ensuring that H-D
exchange does not outcompete the ligand binding reaction.
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The peptides appearing in the mass spectrum after pepsin fragmentation
(Fig. 3) have been assigned to the amino
acid sequence of the C-subunit. They cover ~65% of the primary
structure (29). Due to the nature of our exchange protocol, we
monitored a subset of these peptides (Table
I). The incorporation of deuterium
necessarily broadens the peaks (Fig. 3, lower
panel) with resulting lower signal-to-noise ratio. Three
sets of peaks separated only by 3-6 mass units in the H2O
samples became, upon deuteration, predominantly overlapping and not
suitable for analysis (data not shown).
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Data Analysis and In- and Back-exchange Controls--
The average
mass of each peptide was determined by integrating over the full
envelope of peaks (29), and the mass was converted into number of
in-exchanged deuteriums using Equation 1. The in- and back-exchange
controls set the zero and infinite time points for D(t). The
in-exchange controls ranged from 13-20% (16% average), whereas the
back-exchange controls ranged from 73 to 91% (83% average) for the
specific peptides. Since each peptide fragment can contain a number of
ligand-sensitive exchangeable protons, with different intrinsic
exchange rates, a visual inspection of the curves was used to evaluate
the data. The C-subunit is not stable for more than a day under our
experimental conditions, which excludes measurement of the slowest
exchanging protons that are only exposed with the global unfolding of
the protein. For deuterium in-exchange experiments (Fig. 2), an
apparent equilibrium is generally observed after 100-200 min (Fig.
4), as has also been reported for other
systems (32, 33, 36, 37). Thus, the focus of this study is the specific
identification of structural elements that display differences in
exchange when the C-subunit has ADP bound.
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Time-dependent Deuterium Incorporation at pH* 6.9-- The extent of deuteration of each peptide probe was followed as a function of time at pH* 6.9. Fig. 4 is a presentation of several probes typical of the results obtained. Over the time course of the exchange experiments, the masses of the probes tend to increase in a biphasic manner. The presence of ADP either has no effect or protects amide protons from H-D exchange compared with the apoenzyme, as is evident in the decreased level of label incorporation. The amide protons within probes covering residues 92-100 and 164-174 are clearly protected from H-D exchange over a 3-h period in the presence of ADP (Fig. 4, A and B, and Table I). In comparison, many probes are largely unaffected by nucleotide within experimental error (Fig. 4, C and D, and Table I). At pH* 6.9, a total of three probes covering two regions in the C-subunit displays altered solvent accessibility in the presence of nucleotide. The results for all probes are summarized in Table I. Given the experimental error in the technique, we designate a probe as protected if the mass is 1 or more units lower compared with the free C-subunit after 3 h of exchange.
Time-dependent Deuterium Incorporation at pH* 5.9-- The previously described experiments were performed at pH* 6.9, where the average half-life for exchange of an amide proton is ~200 ms (35). We repeated the experiments with the pH* of the H-D exchange reaction lowered by 1 unit, which decreases the intrinsic H-D exchange rate by 10-fold. Thus, more subtle differences in amide exchange protection can be detected over the same time period. Lowering the pH value further is not possible due to instability of the C-subunit at pH* values below 5.5.
Fig. 5 displays several exchange studies
at pH* 5.9. As shown in Fig. 5E and summarized in Table I,
exchange protection by ADP is observed at pH* 5.9 for all probes that
are also protected by the nucleotide at pH* 6.9 (i.e. probes
encompassing residues 92-100, 163-172, and 164-174 are protected
from exchange by ADP at pH* 5.9 and 6.9). Most, but not all, other
regions displayed no difference in H-D exchange in the presence or
absence of nucleotide at pH* 5.9 (Fig. 5F, Table I).
However, at the lower pH, additional nucleotide-protected regions are
apparent. As shown in Fig. 5, A-D, two new regions of the
protein, covered by a total of four probes, are protected from exchange
by ADP. These probes include residues 41-54, 44-54, 303-326, and
303-327 (Table I). These additional regions displaying exchange
protection with nucleotide at pH* 5.9 are not likely to be due to
changes in structure at low pH for several reasons. The steady-state
kinetic parameters, Km for ATP and
kcat, are identical over a wide pH range, which
includes the two pH values of this study (38). The rate of phosphoryl
transfer, measured in pre-steady-state kinetic studies, is constant
from pH 6 to 9 (39). Finally, PKA does not undergo a
time-dependent inactivation within the time frame of our
experiments. The specific activity of the C-subunit was followed at pH*
5.9 and 6.9 over 3 h, and it was found that the C-subunit did not lose more than 5% of its original activity (data not shown).
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DISCUSSION |
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Probing hydrogen bonding and structural dynamics in proteins with hydrogen exchange was initiated by Linderstrøm-Lang nearly 50 years ago (40). In recent years, the approach of H-D exchange coupled with NMR analysis has revealed detailed site-specific information on bond formation and changes in bond energetics as a function of folding (41-51), conformational change (52), or ligand binding (53-60). However, these methods are confined to a small (yet growing) subset of proteins that are amenable to NMR or crystallization and neutron diffraction analysis (61, 62). While site-specific information is highly desirable, for the majority of proteins, this approach is not feasible at the present time. Insights into larger protein systems are facilitated by region specific analysis first introduced by Rosa and Richards in which H-D (H-T) exchange is followed by a quench/protease fragmentation methodology and subsequent analysis by HPLC (63, 64) or more recently mass spectrometric techniques (29, 33). The functional labeling techniques of Englander et al. (65, 66) allowed assessment of allosterically active segments of human hemoglobin and elucidated key regulatory structural changes that are coupled to energy transduction. In a similar vein, structural characterization of the conformations of apomyoglobin and a key partially folded form thereof were facilitated by H-D exchange studies of the respective states at equilibrium (41).
In the present study, a variation of the techniques of
Englander et al. (65, 66) and Hughson et al. (41)
were employed to assess structural variations as a function of
nucleotide binding on the solution conformation dynamics of PKA. In
this case, we have applied H-D exchange methodology in combination with
MALDI-TOF MS detection to understand the nature of conformational
changes in solution in the apoC subunit (where no structural data are available to date) of PKA. The goal is to determine whether nucleotide binding alters the conformational dynamics of specific regions of PKA.
We selected ADP to reflect the viable enzyme complex that occurs
subsequent to the phosphoryl transfer step. The transition from
ADP-bound C-subunit to free C-subunit represents a rate-limiting step
in catalysis at 10 mM free Mg2+ and a partially
rate-limiting step at physiological Mg2+ (25). The focus of
our studies is to observe regions displaying different behavior in the
presence of ADP, and the time scale of hours has previously proven
adequate for the detection of exchange differences in other proteins
(32, 36, 37). By following the mass of the probes as a function of
incubation time in D2O, we are able to identify regions of
the polypeptide chain that are either sensitive or insensitive to ADP
(Table I, Figs. 4 and 5). The results obtained were mapped onto the
crystal structure of the C-subunit (Fig.
6). Many regions showed similar H-D
exchange behavior independent of the presence or absence of nucleotide. Four regions, however, displayed exchange protection in the presence of
ADP. Regions 41-54 and 163-174 covering the glycine-rich and catalytic loops, respectively, and regions 92-100 and 303-327, covering the helix C and the C-terminal tail, respectively, were protected from exchange by ADP. Thus, both regions in the active site
and distal to the active site were affected by the presence of ADP.
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The Active Site--
Two of the regions where exchange protection
is observed lie in the active site of the C-subunit. The catalytic loop
(residues 165-171, RDLKPEN) has, in the crystal structure (67),
several interactions with the bound ATP molecule. The side chain of
Lys168 interacts with the -phosphate of ATP, the
carbonyl oxygen of Glu170 hydrogen bonds to the ribose
ring, and the side chain of Asn171 chelates the second
Mg2+, which in turn interacts with the
- and
-phosphates of ATP. The data suggest that approximately two to three
deuteriums are protected in the nucleotide-bound form compared with the
apoenzyme after 3 h of incubation (Figs. 4B and
5E). In the absence of site-specific information, we will
use a more qualitative analysis and characterize the observed
protection in terms of regional effects. Using this criterion, the
observed lower solvent exchange with ADP in the probes containing the
catalytic loop (regions 163-172 and 164-174; Figs. 5E and
4B, respectively) may result from two possible
effects: (a) reduced access of solvent to the region as a
result of nucleotide binding or (b) stabilization or
re-positioning of the catalytic loop.
The glycine-rich loop (residues 49-57, LGTGSFGRV) also
forms several interactions with the bound ATP. In contrast to the
catalytic loop, many of these contacts are backbone amide interactions. The amides of Phe54 and Gly55 interact with the
- and
-phosphates of ATP, whereas the amide of Ser53
interacts with the
-phosphate of ATP. Furthermore, the side chains
of Leu49 and Val57 are involved in hydrophobic
contacts with the adenine and ribose ring (67, 68). The observed
exchange protection in peptides 41-54 and 44-54 (Fig. 5, A
and B) could have similar origins to that proposed for the
catalytic loop, i.e. reduced access of solvent to the region
as a result of nucleotide binding or stabilization or repositioning of
the loop. The glycine-rich loop is, in the crystal structures, the most
mobile region of the protein, but the binding of nucleotides brings the
loop into a more stable conformation and lowers the B factors (12,
67-70).
A third region consisting of residues 66-83 covers an important
interaction in the active site. The side chain of Lys72,
which is strictly conserved in all protein kinases, interacts with the
- and
-phosphates of ATP. Further, Ala70 is involved
in hydrophobic interactions with the adenosine ring (68).
Interestingly, we do not observe any exchange protection of backbone
amide protons in the 66-83 region with ADP bound. These data are
reminiscent of the behavior of calmodulin in complex with the MLCK
peptide. Backbone and side chain dynamics studies by Wand and
co-workers (71) indicate that only side chains undergo changes in
motion upon peptide binding, with no changes in the backbone dynamics.
These data are consistent with the idea that the entropy cost of
binding is minimized by maintaining motional flexibility in the
backbone, a phenomenon that may also be occurring in PKA.
Helix C--
Exchange protection in the presence of ADP is
detected in the probe covering part of helix C (positions 92-100)
(Fig. 4A). Within 3 h, at least two deuteriums are
protected in the nucleotide bound form. Interestingly, helix C has no
direct interactions with bound nucleotide, and the backbone amides are
more than 12 Å distant from ATP (Fig. 1). Crystal structures of Cdk2,
extracellular signal-regulated kinase 2, and insulin receptor kinase
show extensive movements in helix C and in the activation loop upon
activation of these kinases (16, 17, 20, 21). Cdk2 is activated by binding of cyclin A and activation loop phosphorylation, whereas insulin receptor kinase and extracellular signal-regulated kinase 2 are
activated by phosphorylation in the activation loop. In the former
case, cyclin A is sufficient to induce these structural changes. In all
three examples, the movement of helix C leads to the formation of a
conserved electrostatic dyad between a glutamate in the helix and a
lysine that interacts with the phosphates of ATP (i.e.
glutamate-lysine dyad). The data presented herein demonstrate that the
binding of a nucleotide, ADP, to protein kinase A in itself is
sufficient to cause an altered environment for helix C. This effect may
result in the strengthening of the conserved dyad between
Lys72 and Glu91 (Fig.
7). Interestingly, the constitutively
activated mitogen-activated protein kinase kinase mutant displayed
increased protection in helix C compared with the repressed form of the
kinase (32). For this enzyme, changes in protection result
from a glutamate mutation in the activation loop that activates this
kinase by mimicking a phosphorylated threonine. The H-D exchange
studies on both PKA and mitogen-activated protein kinase kinase attest to the apparent flexibility of this helix depending upon either the
enzyme's phosphorylation state, as in the case of the latter, or the
ligand-bound state, as in the case of the former.
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C-terminal Tail--
It has been argued that PKA equilibrates
between open and closed forms in solution, but only the open form
permits access of ATP (13). Attempts to monitor changes in the
structure of the C terminus of PKA upon ligand binding in solution by
chemical cleavage or by labeling with fluorescein have failed until now (72, 73). Our studies indicate that ADP protects two probes in the C
terminus from H-D exchange (Fig. 5, C and D,
Table I). Phe327 moves considerably from its position in
the open form and is positioned within 3.5 Å of the adenine ring of
ATP in the closed ternary complex and forms a small element of the
hydrophobic binding pocket (Fig. 8). Only
one of the two probes (residues 303-326 and residues 303-327)
contains Phe327; therefore, the exchange protection also
reflects changes prior to Phe327. When PKA adopts an open
form in the crystalline state, portions of the C terminus, which are
within the two probes, become disordered (Fig. 8). Therefore, the H-D
exchange experiments are likely to monitor opening and closing of the
active site cleft via the C-terminal probes.
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The exchange protection observed in the C-terminal probes could also suggest an active role for this polypeptide segment in nucleotide binding and possibly catalysis. Consistent with this interpretation, cleavage of the C terminus between residues 332 and 333 by the kinase-splitting membranal proteinase leads to an entirely inactive C-subunit (74). The catalytic significance of individual residues in the C terminus is supported by detailed alanine substitution studies of residues 300-350 (75). Several sites are affected, and often the apparent affinities of ATP are considerably lower than that for wild type. In particular, the Lys317 and Lys319 mutants bind ATP with more than 10-fold reduced ATP affinity, while the Glu323 and Phe327 mutants are completely inactive (75). The C termini of other protein kinases are also likely to be important for catalysis but for different reasons. The C terminus of the SR protein kinase, Sky1p, occupies a different surface on the core than that in PKA (77). In Sky1p, the C terminus appears to stabilize the activation loop and play little ostensible role in domain rotation.
Conclusions--
Transient state kinetic studies indicate that the
catalytic cycle in protein kinase A is partially rate-limited by
conformational changes, some of which may be associated with nucleotide
binding and release (25). The H-D exchange results suggest that two key
motions in the C-subunit of PKA may be essential for nucleotide binding. Rotation of the two domains, potentially monitored by the
C-tail probes, may be required for access to the nucleotide binding
pocket. Once this motion occurs, the nucleotide can bind and the closed
state may dominate. Further conformational changes may then occur,
which include movements of helix C and strengthening of the
Glu91-Lys72 electrostatic dyad. The combined
data suggest that the nucleotide has distal structural effects,
including the first evidence that nucleotide alone can induce changes
in the C terminus, stabilizing the closed state of the
enzyme, and alter the position of a critical helix outside the active site.
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ACKNOWLEDGEMENTS |
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We thank Dr. Elizabeth Komives for scientific discussions and assistance with the MALDI-TOF MS instrument and Dr. Jeff Mandell for kindly providing software to measure centroids.
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FOOTNOTES |
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* This work was supported by National Institutes of Health (NIH) Grant GM 54846 (to J. A. A.), NIH Grant GM54038 (to P. A. J.), and a fellowship from the Carlsberg Foundation (to M. D. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 858-822-3360; Fax: 858-822-3361; E-mail: joeadams@ucsd.edu.
Published, JBC Papers in Press, January 19, 2001, DOI 10.1074/jbc.M011543200
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ABBREVIATIONS |
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The abbreviations used are: C-subunit, catalytic subunit of PKA; PKA, protein kinase A; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; MS, mass spectrometry; H-D, hydrogen-deuterium; mant, N-methylanthraniloyl; Mops, 3-(N-morpholino)propanesulfonic acid; HPLC, high pressure liquid chromatography; pH*, pH values uncorrected for the isotope effect.
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REFERENCES |
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