From the Skirball Institute of Biomolecular Medicine
and Department of Pharmacology, New York University School of Medicine,
New York, New York 10016 and the § Department of
Biochemistry and Molecular Biology, Mount Sinai School of Medicine, New
York, New York 10029
Received for publication, November 8, 2000, and in revised form, December 18, 2000
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ABSTRACT |
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The tyrosine kinase domain of the insulin
receptor is subject to autoinhibition in the unphosphorylated basal
state via steric interactions involving the activation loop. A mutation
in the activation loop designed to relieve autoinhibition, Asp-1161 The insulin receptor is an Previous crystallographic studies of the tyrosine kinase domain of the
insulin receptor (IRKD) have demonstrated that upon autophosphorylation, the kinase A-loop undergoes a major change in
conformation (12, 13). In the crystal structure of the unphosphorylated, low activity form of IRKD (IRKD0P)
without ATP (apo), Tyr-1162 in the A-loop is situated in the active
site, blocking access to peptide substrates (12). In this A-loop
configuration, the beginning (proximal end) of the A-loop interferes
with ATP binding. The crystal structure of the tris-phosphorylated,
activated form of IRKD (IRKD3P) reveals how
autophosphorylation of Tyr-1158, Tyr-1162, and Tyr-1163 stabilizes a
specific A-loop configuration in which the substrate binding sites
(MgATP and peptide) are accessible and the important catalytic residues
are properly positioned (13, 14).
Solution studies of IRKD indicate, however, that in the presence of
millimolar quantities of ATP (as are present in a cell), the A-loop of
unphosphorylated IRKD is in equilibrium between inhibiting,
"gate-closed" conformations, as represented by the apoIRKD0P crystal structure, and "gate-open"
conformations in which Tyr-1162 is displaced from the active site (15).
When the A-loop adopts a gate-open conformation, the kinase is
competent to serve as either enzyme or substrate in a
trans-autophosphorylation reaction. Prior to A-loop
autophosphorylation, gate-open conformations of the A-loop would exist
in which the majority of the A-loop has no particular conformation
(because of lack of phosphotyrosine-mediated interactions), but is
nevertheless disengaged from the active site. After
autophosphorylation, the A-loop is stabilized in the gate-open
conformation observed in the IRKD3P structure.
A detailed understanding of the mechanism by which insulin
triggers the initial autophosphorylation event in the insulin receptor requires a structural description of the basal state (unphosphorylated) kinase with bound substrates (ATP and protein). Ideally, this would be
provided by a crystal structure of IRKD0P with bound ATP
analog and peptide substrate. To date, attempts to obtain crystals of
such a ternary (or binary) complex have been unsuccessful. Steady-state
kinetic studies of IRKD provide a plausible explanation for this
failure: the Km values for ATP and peptide substrate
are elevated prior to autophosphorylation, 0.9 and 2 mM,
respectively, decreasing to 0.04 and 0.05 mM upon autophosphorylation.2 These
data are consistent with the autoinhibitory mechanism suggested by the
apoIRKD0P structure and underscore the inherent difficulty
of loading the kinase with substrates prior to A-loop autophosphorylation.
Recently, a substitution in the A-loop of IRKD, Asp-1161 The lower Km(ATP) for this mutant
affords the possibility of structurally characterizing IRKD with ATP
bound prior to insulin-stimulated A-loop autophosphorylation. Indeed,
crystals of IRKDDA with a bound ATP analog (AMP-PCP) were
readily obtained. Here we present the structure of the binary complex
of IRKDDA with MgAMP-PCP at 2.4-Å resolution. This
structure and the accompanying viscometric and denaturation data
provide insights into the structural rearrangements that occur within
the basal state kinase to promote catalysis.
Protein Production--
The 46-kDa form (residues 953-1355) of
the Asp-1161 Crystallographic Studies--
Crystals of the binary complex of
IRKDDA and MgAMP-PCP were grown at 20 °C by vapor
diffusion in hanging drops containing 2.0 µl of protein solution (9 mg/ml IRKDDA, 1.5 mM AMP-PCP (Sigma), and 4.5 mM MgCl2) and 2.0 µl of reservoir buffer
(18% polyethylene glycol 8000, 100 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 5 mM dithiothreitol). Crystals
belong to the orthorhombic space group
P212121 with unit cell dimensions
a = 57.9 Å, b = 69.6 Å, and
c = 89.3 Å when frozen. There is one molecule in the
asymmetric unit, and the solvent content is 51% (assuming a protein
partial specific volume of 0.74 cm3/g). Crystals were
transferred into a cryosolvent consisting of 30% polyethylene glycol
8000, 100 mM Tris-HCl, pH 7.5, 100 mM NaCl, and
15% ethylene glycol. A data set was collected from a flash-cooled
crystal on a Rigaku RU-200 rotating anode equipped with a Rigaku R-AXIS
IIC image plate detector. Data were processed using DENZO and SCALEPACK
(19). A molecular replacement solution was found with AMoRE (20), using
the structure of IRKD0P (PDB entry 1IRK) (12) as a search
model. Rigid-body, positional, and B-factor refinement and simulated
annealing were carried out using CNS (21) (Table
I). Model building was performed
using O (22).
Viscometric Measurements--
The viscosity dependence for
kcat and
kcat/Km was determined using
sucrose as the microviscogen following published procedures (23, 24).
The solution viscosities were determined using an Ostwald capillary
viscometer maintained at 24 ± 0.1 °C in a
temperature-controlled circulating water bath. The relative viscosity,
Denaturation Experiments--
IRCDDA (46-kDa form of
the mutant) was denatured by increasing concentrations of guanidinium
chloride at 24 °C in 50 mM Tris acetate, pH 7.0, with 1 mM dithiothreitol. The protein concentration was 0.5 µM. The excitation wavelength was 295 nm, and
steady-state emission spectra were collected between 310 and 420 nm at
1-nm increments, using an SLM 4800 spectrofluorimeter operating in the
single-photon counting mode. The centroid of the emission spectrum was
determined after subtraction of a blank spectrum for each guanidinium
chloride concentration, which was obtained from the refractive index
measured with a Bausch and Lomb refractometer. Details regarding
instrument settings and data handling are given in Bishop et
al. (18).
Crystal Structure of IRKDDA in Complex with
MgAMP-PCP--
In the original structure of unphosphorylated (low
activity) IRKD, the A-loop traverses the ATP-binding cleft between the N- and C-terminal lobes of the kinase, and Tyr-1162 in the A-loop is
bound in the active site, hydrogen-bonded to Asp-1132 and Arg-1136 in
the catalytic loop (12). Asp-1161 contributes to the stabilization of
this inhibitory conformation of the A-loop by participating in four
hydrogen bonds (Fig. 1). In the crystal
structure of the Asp-1161
In the IRKDDA structure, the proximal end of the A-loop,
containing the protein kinase-conserved 1150DFG sequence,
is positioned more similarly to that in the activated IRKD3P structure than that in the apoIRKD0P
structure (Fig. 3A). A-loop
residues 1155-1171, which include the three autophosphorylation sites
Tyr-1158, Tyr-1162, and Tyr-1163, have no supporting electron density
in the IRKDDA structure and are presumed to be disordered.
The A-loop becomes ordered again at PTK-conserved Pro-1172, which
adopts the same conformation in the three IRKD structures
(IRKDDA, IRKD0P, and IRKD3P). The
absence of Tyr-1162 in the active site in the binary IRKDDA
structure is consistent with biochemical studies (16) and modeling exercises, which indicate that MgATP and Tyr-1162 cannot bind in the
active site simultaneously, i.e. that
cis-autophosphorylation of Tyr-1162 is not sterically
possible.
In protein kinases, residues in both the N- and C-terminal lobes bind
and thus position ATP for phosphoryl transfer (29). The extent to which
ATP is bound productively depends on the degree of lobe closure, the
relative disposition of the two lobes. A superposition of C-terminal
lobe residues for the three IRKD structures reveals that the degree of
lobe closure for IRKDDA is intermediate between
IRKD0P and IRKD3P (Fig. 3A). From
its position in the IRKD0P structure, the N-terminal lobe
is rotated 11° toward the C-terminal lobe in the IRKDDA
structure. An additional 8° is required for the N-terminal lobe to
reach the position observed in the IRKD3P structure.
Analysis of the changes in backbone
When the N-terminal
The IRKDDA structure suggests that the rotation of
The ATP analog (AMP-PCP) that was co-crystallized with
IRKDDA is bound in the cleft between the two kinase lobes
(Fig. 2) and is ordered throughout, including the
Due to the incomplete rotation of the N-terminal lobe toward the
C-terminal lobe in IRKDDA, AMP-PCP binds to the "roof"
of the cleft (N-terminal residues) but not to the "floor"
(C-terminal residues). Lys-1030 in
Although the A-loop in the IRKDDA structure does not
occlude the peptide binding site as in the IRKD0P
structure, Km(peptide), unlike
Km(ATP), is not decreased in the
Asp-1161 Viscometric Analysis--
The binding and chemical steps
associated with an IRKD-catalyzed phosphorylation reaction are
summarized in Scheme 1 for the experimental conditions where enzyme
(E) is saturated with ATP (T), tyrosyl peptide (Y) binds
with on- and off-rate constants k2 and
k
To identify whether chemistry is the rate-limiting step in steady-state
phosphorylation of the peptide substrate Y-IRS939 by
IRKDDA, the viscosity dependence of
kcat and
kcat/Km(peptide) was determined. The parameters
(kcat)rel and
(kcat/Km)rel are
the ratio of kcat and
kcat/Km, respectively, in the
presence of viscogen versus the control reaction in aqueous buffer without viscogen. If the rate constant for a
diffusion-dependent step is much smaller than for a
diffusion-independent step, then the plot of
(kcat)rel or
(kcat/Km)rel
versus Denaturation Experiments--
Denaturation of IRCDDA
(46-kDa form of the mutant) in guanidinium chloride was monitored using
fluorescence and is presented (Fig. 6) as
the change in centroid of the emission spectrum (defined in Ref. 18).
The data are compared with denaturation profiles from unphosphorylated
and phosphorylated wild-type IRCD taken from previous work (18). As
before, there are three transitions in the denaturation profile.
Denaturation over transition I follows the same pattern observed for
phosphorylated wild-type IRCD, and denaturation over transition III
follows the same pattern as observed for unphosphorylated wild-type
IRCD. This indicates that the Asp-1161
The conformational free energy in the unphosphorylated, gate-open
mutant (IRCDDA) was calculated from these results:
Structural Studies--
With few exceptions, phosphorylation of
the A-loop in PTKs is one of the key regulatory mechanisms by which
catalytic activity is increased (33). Crystallographic studies of PTKs
and the related protein-serine/threonine kinases in their
unphosphorylated (basal) and phosphorylated (activated) forms have
provided a wealth of structural data for understanding this regulatory
mechanism. The A-loop in the structures of basal state PTKs, including
IRKD (12), the fibroblast growth factor receptor tyrosine kinase (34),
Src (35-37), Hck (38, 39), and Abl (40), exhibit a wide range of
conformations. In the IRKD and Abl structures, one of the tyrosines in
the A-loop (Tyr-1162 in IRKD, Tyr-393 in Abl) is bound in the active
site, mimicking an exogenous tyrosine substrate. In the Src (37) and
Hck (39) structures, the corresponding tyrosine in the A-loop, Tyr-416,
does not mimic a tyrosine substrate but nevertheless contributes to the
obstruction of the peptide substrate binding site. In contrast, in the
structure of the unphosphorylated fibroblast growth factor receptor
kinase (34), the A-loop tyrosines do not sterically occlude the peptide
binding site, yet the configuration at the distal end of the A-loop
hinders peptide binding.
The ATP binding sites in the crystal structures of basal state PTKs
also display various degrees of accessibility. In the apoIRKD0P structure (12), the proximal end of the A-loop
passes through the ATP binding cleft. This is probably true for Abl as
well, although the Abl structure contains a bound ATP-competitive
inhibitor (40), and therefore the course of the A-loop in the absence of inhibitor is not known. In the Src (37) and fibroblast growth factor
receptor kinase (34) structures, in which an A-loop tyrosine is not
mimicking a tyrosine substrate, an ATP analog is bound in the
nucleotide binding site. However, in these two structures, the ATP
analog is not bound productively by residues from both the N- and
C-terminal lobes of the kinase, as in the ternary IRKD3P
structure (13).
Solution studies indicate that at millimolar concentrations of ATP
present in cells, gate-open A-loop conformations are favored in basal
state IRKD (15). Previous attempts to co-crystallize IRKD0P
with an ATP analog have been unsuccessful, probably because of the high
Km(ATP). The present crystal structure
is of an A-loop mutant of IRKD (Asp-1161
Prior to phosphoryl transfer, ATP is bound between the N- and
C-terminal lobes of protein kinases, interacting with residues in both
lobes (29). In the structure of IRKDDA with bound ATP
analog, the nucleotide interacts with the N-terminal but not the
C-terminal lobe, and the degree of lobe closure is intermediate between
the IRKD0P and IRKD3P forms (Fig.
3A). In IRKDDA, lobe closure is hindered by
Phe-1151 of the protein kinase-conserved DFG motif at the proximal end
of the A-loop. Rather than tucking underneath
The IRKDDA structure indicates that proper positioning of
conserved Phe-1151 is critical for the downward and inward rotation of
Upon autophosphorylation, the conformation of the A-loop is stabilized
by short Integration of Solution and Structural Studies--
The
IRKDDA crystal structure is the third structure of the
kinase domain of the insulin receptor, each of which represents a different conformational state. For each structure, we also have available kinetic parameters (Fig. 5)2 and conformational
free energies (Fig. 6 and Ref. 18). Each type of analysis (structural,
kinetic, and thermodynamic) shows that IRKDDA is
intermediate between basal IRKD0P and fully activated
IRKD3P and together provide further insights into the
mechanism by which A-loop conformation regulates this kinase.
Every catalytic cycle comprises binding and chemical steps and
conformational changes. These may be discrete or overlapping, depending
on the relative rate constants for individual steps. For IRKD these
steps were summarized in Scheme 1 (see "Results"). Comparing
reactions catalyzed by the unphosphorylated forms, IRKDDA
and IRKD0P, the dissociation constants for peptide
substrate are nearly the same, but the rate constants for the chemical
step (k3) differ by almost 50-fold. Peptide
binding equilibrates prior to the chemical step in the
IRKD0P-catalyzed reaction2 but not in the
IRKDDA reaction (see "Results"). The smaller rate
constant for k3 in the IRKD0P
versus IRKDDA reaction indicates a higher free
energy barrier to phosphoryl transfer for IRKD0P, making it
50 times less likely that a ternary complex will convert to a
transition state complex. This barrier in IRKD0P may arise
structurally if the A-loop reconfiguration were less advanced than
observed in the IRKDDA·AMP-PCP binary complex. The
conformational change in the A-loop needed to complete cleft closure
and
When IRKDDA and IRKD3P are compared,
k3 is virtually the same for both. The same
k3 indicates that an equivalent free energy
barrier exists between the respective ternary and transition state
complexes. This suggests that the ternary IRKDDA complex
should resemble the ternary IRKD3P complex (13), with
repositioning of the N-terminal
We envision that peptide binding begins at the P
These and previous studies from our laboratories cited here show that a
definable set of conformational changes are required for substrate
binding and product formation on the insulin receptor kinase. Among
these conformational changes, we have now identified a critical role
for Phe-1151 of the conserved DFG motif in closure of the catalytic
cleft and Ala, substantially increases the ability of the unphosphorylated kinase
to bind ATP. The crystal structure of this mutant in complex with an
ATP analog has been determined at 2.4-Å resolution. The structure
shows that the active site is unobstructed, but the end of the
activation loop is disordered and therefore the binding site for
peptide substrates is not fully formed. In addition, Phe-1151 of the
protein kinase-conserved DFG motif, at the beginning of the activation
loop, hinders closure of the catalytic cleft and proper positioning of
-helix C for catalysis. These results, together with viscometric
kinetic measurements, suggest that peptide substrate binding induces a
reconfiguration of the unphosphorylated activation loop prior to the
catalytic step. The crystallographic and solution studies provide new
insights into the mechanism by which the activation loop controls
phosphoryl transfer as catalyzed by the insulin receptor.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
2
heterotetrameric glycoprotein possessing intrinsic protein-tyrosine
kinase (PTK)1 activity (1,
2). Upon insulin binding to the
subunits, the insulin receptor
undergoes a poorly characterized conformational change that results in
autophosphorylation of specific tyrosine residues in the cytoplasmic
portion of the
subunits. Three regions in the
subunits are
sites of autophosphorylation: the juxtamembrane region, the activation
loop (A-loop) within the tyrosine kinase domain, and the C-terminal
tail (3-6). Autophosphorylation of tyrosine residues stimulates
receptor catalytic activity (7, 8) and creates recruitment sites for
downstream signaling molecules such as the insulin receptor substrate
(IRS) proteins (9) and Shc (10, 11).
Ala
(IRKDDA), has been introduced that dramatically alters the
A-loop equilibrium in the unphosphorylated
kinase.3 This particular
substitution was motivated by the apoIRKD0P crystal
structure in which the Asp-1161 side chain participates in several
hydrogen bonds that stabilize the gate-closed configuration of the
A-loop (Fig. 1). Steady-state kinetic experiments demonstrate that the
Km(ATP) in the basal state is ~10-fold
lower for this mutant than for wild-type IRKD, suggesting that the
A-loop equilibrium is shifted toward gate-open
conformations.3 Interestingly, this mutation does not
affect Km(peptide) in the
unphosphorylated state, which remains high (several millimolar). The
kinetic properties of IRKDDA after autophosphorylation are
indistinguishable from those of the wild-type kinase.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Ala mutant cytoplasmic domain of the insulin receptor,
IRCDDA, was generated and purified as
described.3 This form of the kinase was used for
denaturation studies. To generate the 35-kDa form (residues 978-1283)
of the mutant (IRKDDA) used in the crystallographic and
viscometric studies, the 0.65-kb XhoI-StuI
fragment from pX-D1161A-IRCD was inserted into the
XhoI-StuI sites of pALTER-IRK vector, which
includes the point mutations Cys-981
Ser and Tyr-984
Phe
described previously (16). The fragment swap was verified with
NheI digestion; this restriction enzyme site was introduced
previously to replace a StuI site by a silent mutation at
Ala-1048 and Ser-1049 (17). The resulting pALTER-IRKDDA was
digested with HindIII, filled in with the large Klenow
fragment of DNA polymerase, and a 0.9-kb fragment was released with
BamHI. This was inserted into the
BamHI-SmaI sites of the baculovirus expression
vector pVL1393 (PharMingen), producing pVL1393-IRKDDA, and
the recombinant virus was generated using a Baculogold kit (PharMingen); the mutation was reconfirmed in
pVL1393-IRKDDA by DNA sequencing. Proteins were expressed
and purified as described (18).3 The absence of A-loop
phosphorylation in each form (46 kDa and 35 kDa) of the mutant was
determined by endoproteinase Lys-C digestion and peptide mapping by
reverse-phase high performance liquid chromatography.
X-ray data collection and refinement statistics
rel, is the ratio of the viscosity in the presence
versus the absence of viscogen and was determined as
described by Adams and Taylor (25). The standard buffer was 50 mM Tris acetate, pH 7.0. Viscosity dependences of
kcat were done using 0.1 µM
IRKDDA (unphosphorylated), 0.14-2.7 mM
Y-IRS939 peptide substrate (REETGSEYMNMDLG), and constant 1 mM ATP and then extrapolated to saturating substrate by
fitting the observed kcat versus
peptide concentration to a rectangular hyperbola. To determine the
viscosity dependences of
kcat/Km(peptide),
measurements were done at 1 mM ATP and 0.14 mM
Y-IRS939. A control reaction measuring kcat was
done using ATP
S to show the absence of viscosity effects in the
IRKDDA-catalyzed reaction because phosphoryltransferase
reactions with ATP
S are usually chemistry-limited rather than
diffusion step-limited (26-28). The control reactions were done at 1 mM ATP
S, 0.1-2.0 mM Y-IRS939, and 0.4 µM IRKDDA, and the data were extrapolated to
saturating peptide substrate by fit to a hyperbolic equation, with
Km(peptide) = 2.8 mM.3 All reactions were done twice in
triplicate. The calculation of stepwise rate constants for
IRKDDA was done according to Adams and co-workers (24, 25),
and for wild-type IRKD by Ablooglu and Kohanski.2
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Ala mutant IRKD in complex with MgAMP-PCP
(Fig. 2), the A-loop adopts a
conformation in which the active site is unobstructed (gate-open),
consistent with solution studies measuring the accessibility of the
active site.3
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Fig. 1.
Interactions of Asp-1161 in the
apoIRKD0P structure (12). The A-loop is shown in
blue, and selected side chains are shown in stick
representation. The remainder of the structure is represented as a
molecular surface (gray). Carbon atoms are colored
orange, nitrogen atoms blue, and oxygen atoms
red. Hydrogen bonds are shown as dashed black
lines. Asp-1161 and Tyr-1162 are in the A-loop, Asp-1132 and
Arg-1136 are in the catalytic loop, Lys-1085 is in D, and Gln-1208
is in the loop between
F and
G (Fig. 2).
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Fig. 2.
Ribbon diagram of the
IRKDDA structure. strands (numbered)
are shown in cyan and
helices (lettered) in
red and yellow. The nucleotide analog AMP-PCP is
shown in ball and stick representation (black).
The dashed gray line indicates the portion of the A-loop
that is disordered (residues 1155-1171).
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Fig. 3.
Lobe closure and
C rotation coupled to Phe-1151. A,
comparison of the positions of the nucleotide binding loop (N-loop),
A-loop (containing Phe-1151), and
C in the IRKD0P
(blue) (12), IRKD3P (orange) (12),
and IRKDDA (green) structures. Residues in the
C-terminal lobe were superimposed (0.8 Å (IRKDDA
versus IRKD0P) and 0.4 Å (IRKDDA
versus IRKD3P) root mean square deviation for
182 C
atoms). The view in the right panel is ~90°
from the view in the left panel. B, schematic
illustration of the coupling between the position of Phe-1151 and the
position of
C in the IRKDDA (green) and
IRKD3P (orange) structures. The N-terminal
sheet (
C not included) of the two structures were superimposed (0.3 Å root mean square deviation for 37 C
atoms). The arrows
indicate the two rotational operations that bring
C into its
IRKD3P position from its IRKDDA position.
,
torsion angles shows that
the hinge points for the N-terminal lobe rotation are at Arg-1061,
before
-strand 4 (
4), and Met-1079, in the segment linking the N-
and C-terminal lobes.
sheet is superimposed for the three IRKD
structures,
-helix C (
C) in the IRKDDA structure is
observed to be in essentially the same position with respect to the
sheet as it is in the IRKD0P structure. Thus, in the
transition from the autoinhibited, gate-closed conformation of the
A-loop (IRKD0P) to a gate-open conformation with bound ATP
(IRKDDA), the entire N-terminal lobe rotates as a rigid
body. However in the transition to the activated state
(IRKD3P),
C undergoes an independent (from the
sheet) motion that entails a 12° rotation toward the C-terminal lobe
and a 28° rotation about the helical axis (Fig. 3B). These
movements of
C, which mainly occur through
,
changes at
Phe-1054 and Thr-1055 at the base of the helix, are necessary to
position protein kinase-conserved Glu-1047 (in
C) proximal to
conserved Lys-1030 (in
3). Lys-1030 coordinates the
- and
-phosphates of ATP in an active protein kinase configuration (30,
31).
C
required for a properly configured active site relies on the precise
positioning of Phe-1151 in the DFG motif. Although the position of
Phe-1151 in the IRKDDA structure is roughly similar to that
in the IRKD3P structure, there are critical differences. In
the IRKD3P structure, Phe-1151 is buried deep in a
hydrophobic pocket underneath
C (Fig.
4A). This pocket is composed
of residues from
C (Glu-1047, Val-1050, Met-1051), from the
C-
4 loop (Phe-1054, Val-1059), from
E (Leu-1123), and from
8 (Ile-1148). In contrast, the side chain of Phe-1151 in the
IRKDDA structure points upward toward
C and is situated
in a shallow hydrophobic pocket comprising the same residues as above
(some with different side-chain rotamers) and additionally Phe-1128 in
the segment preceding the catalytic loop (Fig. 4B). With
Phe-1151 in this position,
C is sterically hindered from undergoing
the movements that bring Glu-1047 into the active site. Moreover, conserved Asp-1150, which coordinates Mg2+, is pulled back
from the active site vis à vis its position in the
IRKD3P structure (Fig. 3B).
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Fig. 4.
Hydrophobic environment of
Phe-1151. Stereo view of the hydrophobic binding pocket for
Phe-1151 in IRKD3P (12) (A) and
IRKDDA (B). Selected side chains are shown in
stick representation with carbon atoms colored
green (orange for Phe-1151), oxygen atoms
red, and sulfur atoms yellow. The backbone
representation (worm) is colored blue, and the
molecular surface in the pocket (within 6.5 Å of the C atom of
Phe-1151) is shown in mesh representation
(gray).
-phosphate. The
conformation of AMP-PCP in the IRKDDA structure is
different from the conformation of AMP-PNP observed in the ternary
IRKD3P structure and closely resembles the conformation of
ATP and AMP-PNP in crystal structures of cyclic
AMP-dependent protein kinase (30, 31). In the ternary
IRKD3P structure, the
-phosphate of AMP-PNP is swung
away from the hydroxyl group of the tyrosine substrate in the active
site, presumably due to the imperfect fit with nitrogen rather than
oxygen as the bridging atom between the
- and
-phosphates.
3 is hydrogen-bonded to the
-phosphate, but the ribose hydroxyl groups are not within
hydrogen-bonding distance to Asp-1083 in the C-terminal lobe, as in
IRKD3P. Moreover, only one Mg2+ ion is evident
in the IRKDDA structure, coordinated by Asp-1150 and the
- and
-phosphates of AMP-PCP. Because of the retracted position
of Asp-1150, the coordination of this Mg2+ is weak: Mg-O
distances
2.4 Å. Due to the lack of lobe closure and the
consequent positioning of AMP-PCP at the roof of the cleft, the second
Mg2+ ion present in the IRKD3P structure,
coordinated by Asn-1137 of the catalytic loop (C-terminal lobe), is
absent in the binary IRKDDA structure.
Ala mutant.3 In the structure of ternary
IRKD3P, residues 1169-1171 at the distal end of the A-loop
are hydrogen-bonded via main-chain atoms to peptide substrate residues
P+1 through P+3 (P0 is the acceptor
tyrosine), forming two short, antiparallel
strands (13). In
addition, the side chains of Leu-1170 and Leu-1171 are constituents of
the binding pockets for the P0 and P+3 side
chains, respectively. Thus, the disorder in the IRKDDA
A-loop at the distal end results in a peptide binding site that is not
fully formed, which is reflected in the high
Km(peptide). In contrast, in the
gate-open conformation stabilized by A-loop autophosphorylation, the
distal end of the A-loop is ordered even in the absence of peptide
substrate.4
2, the rate constant for the chemical step is
given by k3, and the net rate constant for
release of products ADP (D) and phosphotyrosyl peptide (pY) is given by
k4'.
The stepwise rate constants present in the steady-state kinetic
parameters were derived using Cleland's method (32) and were
established from the viscosity dependence of
kcat and
kcat/Km(peptide) (Fig. 5).
(Eq. 1)
The principle behind viscometric analysis is that increased
solution viscosity will affect the diffusion-dependent
steps of substrate binding (k2 and
k
(Eq. 2)
2) and product release
(k4') but not the chemical step
(k3), because the latter does not involve solute
(substrate) exchange between the bulk phase and the enzyme's active
site (23).
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[in a new window]
Fig. 5.
Viscosity dependence of the
IRKDDA-catalyzed reaction. Plots of
(kcat)rel (filled
circles) and
(kcat/Km)rel
(open circles) versus relative viscosity
rel are shown using ATP and Y-IRS939 as substrates and
sucrose as the viscogen. The control experiment for a chemically
limited reaction was done using ATP
S and Y-IRS939, and
(kcat)rel was determined
(squares). Error bars show 1 S.D. Data were fit
by linear regression (solid lines for
(kcat)rel and dotted
lines for
(kcat/Km)rel).
rel will have zero slope. For
IRKDDA, both global parameters were sensitive to changes in
viscosity, increasing linearly with increasing viscogen (Fig. 5). The
slope for (kcat)rel
versus
rel was 0.8 ± 0.1, and the slope
for (kcat/Km)rel
versus
rel was 1.2 ± 0.2. For
IRKDDA-catalyzed reactions without viscogen,
kcat = 9.6 ± 0.4 s
1 and
kcat/Km(peptide) = 4.2 ± 0.7 × 103
M
1 s
1.
From these values, we calculate k3 = 59 s
1 and k4' = 14 s
1.5 Because
these rate constants differ by only 4-fold, the steady-state rate
constant of the reaction (kcat) is partially
limited by chemistry and partially by product release (Equation 1).
These are approximately the same values of k3
and k4' determined for the activated kinase,
IRKD3P: 46 s
1 and 11 s
1,
respectively.2
Ala substitution, while
releasing the A-loop from the gate-closed conformation, does not
otherwise alter the intrinsic conformational flexibilities within the
A-loop or the two kinase lobes.
View larger version (15K):
[in a new window]
Fig. 6.
Denaturation of IRCDDA.
Denaturation of IRCDDA (46-kDa form) was done with
increasing concentrations of guanidinium chloride (GdmCl)
and monitored by changes in the fluorescence emission spectrum
(CES, centroid of the emission spectrum). Circles
are data for IRCDDA. The best-fit lines from denaturation
of the wild-type, basal kinase (dashed lines), and activated
kinase (solid lines) were taken from Bishop et
al. (18), where the three transitions of denaturation (Roman
numerals) are described in detail.
GH2O =
2.8 kcal/mol over
transition I and
8.5 kcal/mol over transition III, for a net free
energy of unfolding
GH2O,net =
11.3 kcal/mol. Compared with values published previously for the
basal and activated state wild-type kinase
(
GH2O,net =
14.1 and
10.2 kcal/mol, respectively) (18), the overall difference
in free energy (
GH2O,net) is
2.8 kcal/mol less inherent conformational stability in
IRCDDA compared with the unphosphorylated wild-type kinase,
mostly from transition I. The mutant showed 1.1 kcal/mol greater
stability than the phosphorylated wild-type kinase, mostly from
transition III. Therefore, the Asp-1161
Ala mutation in the A-loop
results in a kinase that has a conformational stability intermediate
between the basal state and the activated state.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Ala) that results in an
approximate 10-fold reduction in Km(ATP)
(the fold change in Km(ATP) also
reflects the change in affinity). In this structure, an ATP analog is
bound in the nucleotide-binding site, and the majority of the A-loop,
including the three autophosphorylation sites, is disordered
(i.e. assumes multiple conformational states). Even though
the A-loop is largely disordered, the active site is clearly unobstructed. Thus, the IRKDDA structure and the
apoIRKD0P structure (12) are representative of gate-open
and gate-closed conformations, respectively, of the unphosphorylated kinase.
C into a deep
hydrophobic pocket, as observed in the IRKD3P structure,
Phe-1151 is situated in a shallower pocket nearby (Fig. 4B).
The distal end of the A-loop, which will serve as a platform for
peptide substrate binding (13), is disordered in the IRKDDA
structure, providing a rationale for the elevated
Km(peptide) for this mutant (see below).
C and suggests that binding of ATP to the basal state kinase is not
sufficient to induce the
C transition. In the IRKD3P
structure, conserved Glu-1047 in
C is proximal to conserved Lys-1030
in
3, orienting the lysine side chain for ATP (phosphate) binding
(Fig. 3B). The optimal position (for catalysis) of conserved Asp-1150, which coordinates an essential Mg2+ ion, is also
dependent on the proper positioning of neighboring Phe-1151.
-strand interactions between the A-loop and other
C-terminal lobe residues and by electrostatic interactions involving
the phosphoryl groups of Tyr(P)-1162 and Tyr(P)-1163 (13). In
this configuration, the proximal and distal ends of the A-loop are
"pulled taut," which favors the buried position of Phe-1151 beneath
C (Figs. 3A and 4A). Prior to
autophosphorylation, the IRKDDA structure suggests that
peptide substrate binding to the distal end of the A-loop is sufficient
to reconfigure, at least transiently, the A-loop for catalysis.
C rotation in the IRKD0P basal state would occur
after peptide binding has equilibrated. If the conformational change is
folded into the chemical step in this way, it could yield the 50-fold
smaller rate constant in the basal state IRKD0P catalytic cycle.
sheet and
C accomplished in the
ternary IRKDDA complex prior to the actual phosphoryl
transfer event. A similar ternary complex for IRKDDA and
IRKD3P would require a conformational change at the
proximal end of the IRKDDA A-loop from the position
observed in the binary complex (Fig. 3). The kinetics suggest that this
might be accomplished through peptide binding, which is slower to
IRKDDA than to IRKD3P and reaches equilibrium
before the chemical step for the latter but not the former (see
"Results").2 These kinetic features of unphosphorylated
IRKDDA could be explained by a substrate-induced
conformational change in the A-loop that occurs coincidentally with
peptide substrate binding.
1 and
P0 sites, which are fully open in the IRKDDA
structure, and proceeds until the P+1 and P+3
residues become seated properly. The peptide substrate is captured by
the faster chemical step of the catalytic cycle
(k3 > k
2), and thus
binding does not equilibrate before the phosphoryl transfer occurs.
Backbone hydrogen bonding and hydrophobic interactions involving the
P+1 and P+3 side chains could impose order on
Gly-1169-Leu-1171 at the distal end of the A-loop, and peptide would
be bound as observed in the ternary IRKD3P structure (13).
This peptide-induced ordering of residues at the distal end of the
A-loop would trigger a concomitant structural rearrangement at the
proximal end, burying Phe-1151 underneath
C and bringing Lys-1030,
Glu-1047, and Asp-1150 into the proper alignment prior to the chemical
step. We suggest that in the transition state of each IRKD-catalyzed
reaction (including the basal state reaction), the A-loop conformation
at the proximal and distal ends is essentially the same, with the final
configuring of the A-loop occurring after substrate binding
for IRKD0P, during substrate binding for
IRKDDA, and before substrate binding for
IRKD3P.
C rotation.
![]() |
ACKNOWLEDGEMENT |
---|
We thank N. Covino for technical support.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants DK52916 (to S. R. H.) and DK50074 (to R. A. K.). X-ray equipment at the Skirball Institute is partially supported by grants from the Kresge Foundation and the Hyde and Watson Foundation.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.
The atomic coordinates and the structure factors (code 1I44) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
¶ Present address: Dept. of Pharmaceutical R & D, Genentech Inc., South San Francisco, CA 94080.
To whom correspondence may be addressed: Dept. of Biochemistry
and Molecular Biology, Mount Sinai School of Medicine, New York, NY
10029. Tel.: 212-241-7288; Fax: 212-996-7214; E-mail: Ronald.Kohanski@mssm.edu.
** To whom correspondence may be addressed: Skirball Institute of Biomolecular Medicine, New York University School of Medicine, 540 First Ave., New York, NY 10016. Tel.: 212-263-8938; Fax: 212-263-8951; E-mail: hubbard@tallis.med.nyu.edu.
Published, JBC Papers in Press, December 21, 2000, DOI 10.1074/jbc.M010161200
2 Ablooglu, A. J. and Kohanski, R. A. (2001) Biochemistry 40, 504-513.
3 M. Frankel, A. J. Ablooglu, J. W. Leone, E. Rusinova, J. B. A. Ross, R. L. Heinrikson, and R. A. Kohanski, submitted for publication.
4 J. H. Till and S. R. Hubbard, unpublished data.
5
These parameters were calculated from
k4' = kcat/(slope from
(kcat)rel versus
rel); k3 = kcat·k4'/(k4'
kcat).
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
PTK, protein-tyrosine kinase;
AMP-PCP, adenylyl-(,
-methylene)-diphosphonate;
IRS, insulin receptor
substrate;
IRKD, tyrosine kinase domain of the insulin receptor;
kb, kilobase;
ATP
S, adenosine
5'-3-O-(thio)triphosphate.
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