Crystallographic and Solution Studies of an Activation Loop Mutant of the Insulin Receptor Tyrosine Kinase

INSIGHTS INTO KINASE MECHANISM*

Jeffrey H. TillDagger , Ararat J. Ablooglu§, Mark Frankel§, Steven M. Bishop§, Ronald A. Kohanski§||, and Stevan R. HubbardDagger **

From the Dagger  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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 right-arrow 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 alpha -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

The insulin receptor is an alpha 2beta 2 heterotetrameric glycoprotein possessing intrinsic protein-tyrosine kinase (PTK)1 activity (1, 2). Upon insulin binding to the alpha  subunits, the insulin receptor undergoes a poorly characterized conformational change that results in autophosphorylation of specific tyrosine residues in the cytoplasmic portion of the beta  subunits. Three regions in the beta  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).

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 right-arrow 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.

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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Protein Production-- The 46-kDa form (residues 953-1355) of the Asp-1161 right-arrow 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 right-arrow Ser and Tyr-984 right-arrow 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.

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).

                              
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Table I
X-ray data collection and refinement statistics

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, eta 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 ATPgamma S to show the absence of viscosity effects in the IRKDDA-catalyzed reaction because phosphoryltransferase reactions with ATPgamma S are usually chemistry-limited rather than diffusion step-limited (26-28). The control reactions were done at 1 mM ATPgamma 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

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).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 right-arrow 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 alpha D, and Gln-1208 is in the loop between alpha F and alpha G (Fig. 2).


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Fig. 2.   Ribbon diagram of the IRKDDA structure. beta  strands (numbered) are shown in cyan and alpha  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).

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.


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Fig. 3.   Lobe closure and alpha C rotation coupled to Phe-1151. A, comparison of the positions of the nucleotide binding loop (N-loop), A-loop (containing Phe-1151), and alpha 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 Calpha 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 alpha C in the IRKDDA (green) and IRKD3P (orange) structures. The N-terminal beta  sheet (alpha C not included) of the two structures were superimposed (0.3 Å root mean square deviation for 37 Calpha atoms). The arrows indicate the two rotational operations that bring alpha C into its IRKD3P position from its IRKDDA position.

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 phi , psi  torsion angles shows that the hinge points for the N-terminal lobe rotation are at Arg-1061, before beta -strand 4 (beta 4), and Met-1079, in the segment linking the N- and C-terminal lobes.

When the N-terminal beta  sheet is superimposed for the three IRKD structures, alpha -helix C (alpha C) in the IRKDDA structure is observed to be in essentially the same position with respect to the beta  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), alpha C undergoes an independent (from the beta  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 alpha C, which mainly occur through phi , psi  changes at Phe-1054 and Thr-1055 at the base of the helix, are necessary to position protein kinase-conserved Glu-1047 (in alpha C) proximal to conserved Lys-1030 (in beta 3). Lys-1030 coordinates the alpha - and beta -phosphates of ATP in an active protein kinase configuration (30, 31).

The IRKDDA structure suggests that the rotation of alpha 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 alpha C (Fig. 4A). This pocket is composed of residues from alpha C (Glu-1047, Val-1050, Met-1051), from the alpha C-beta 4 loop (Phe-1054, Val-1059), from alpha E (Leu-1123), and from beta 8 (Ile-1148). In contrast, the side chain of Phe-1151 in the IRKDDA structure points upward toward alpha 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, alpha 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 Cgamma atom of Phe-1151) is shown in mesh representation (gray).

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 gamma -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 gamma -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 beta - and gamma -phosphates.

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 beta 3 is hydrogen-bonded to the alpha -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 beta - and gamma -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.

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 right-arrow 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 beta  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

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-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'.


E · <UP>T</UP>+<UP>Y</UP> <LIM><OP><ARROW>⇄</ARROW></OP><LL>k<SUB>−2</SUB></LL><UL>k<SUB>2</SUB></UL></LIM> E · <UP>T</UP> · <UP>Y</UP> <LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB>3</SUB></UL></LIM> E · <UP>D</UP> · <UP>pY</UP> <LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB>4′</SUB></UL></LIM> E+<UP>D</UP>+<UP>pY</UP>

<UP><SC>Scheme</SC> 1</UP>
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).
k<SUB><UP>cat</UP></SUB>=k<SUB>3</SUB> · k<SUB>4′</SUB>/(k<SUB>3</SUB>+k<SUB>4′</SUB>) (Eq. 1)

k<SUB><UP>cat</UP></SUB>/K<SUB>m (<UP>peptide</UP>)</SUB>=k<SUB>2</SUB> · k<SUB>3</SUB>/(k<SUB>−2</SUB>+k<SUB>3</SUB>) (Eq. 2)
The principle behind viscometric analysis is that increased solution viscosity will affect the diffusion-dependent steps of substrate binding (k2 and k-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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Fig. 5.   Viscosity dependence of the IRKDDA-catalyzed reaction. Plots of (kcat)rel (filled circles) and (kcat/Km)rel (open circles) versus relative viscosity eta 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 ATPgamma 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).

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 eta 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 eta rel was 0.8 ± 0.1, and the slope for (kcat/Km)rel versus eta 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

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 right-arrow 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.


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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.

The conformational free energy in the unphosphorylated, gate-open mutant (IRCDDA) was calculated from these results: Delta GH2O = -2.8 kcal/mol over transition I and -8.5 kcal/mol over transition III, for a net free energy of unfolding Delta GH2O,net -11.3 kcal/mol. Compared with values published previously for the basal and activated state wild-type kinase (Delta GH2O,net -14.1 and -10.2 kcal/mol, respectively) (18), the overall difference in free energy (Delta Delta 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 right-arrow 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

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 right-arrow 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.

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 alpha 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).

The IRKDDA structure indicates that proper positioning of conserved Phe-1151 is critical for the downward and inward rotation of alpha C and suggests that binding of ATP to the basal state kinase is not sufficient to induce the alpha C transition. In the IRKD3P structure, conserved Glu-1047 in alpha C is proximal to conserved Lys-1030 in beta 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.

Upon autophosphorylation, the conformation of the A-loop is stabilized by short beta -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 alpha 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.

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 alpha 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.

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 beta  sheet and alpha 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.

We envision that peptide binding begins at the P-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 alpha 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.

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 alpha 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 eta rel); k3 = kcat·k4'/(k4' - kcat).

    ABBREVIATIONS

The abbreviations used are: PTK, protein-tyrosine kinase; AMP-PCP, adenylyl-(beta ,gamma -methylene)-diphosphonate; IRS, insulin receptor substrate; IRKD, tyrosine kinase domain of the insulin receptor; kb, kilobase; ATPgamma S, adenosine 5'-3-O-(thio)triphosphate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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