Crystallographic and Solution Studies of an Activation Loop Mutant of the Insulin Receptor Tyrosine Kinase
2001; Elsevier BV; Volume: 276; Issue: 13 Linguagem: Inglês
10.1074/jbc.m010161200
ISSN1083-351X
AutoresJeffrey H. Till, Ararat J. Ablooglu, Mark Frankel, Steven M. Bishop, Ronald A. Kohanski, Stevan R. Hubbard,
Tópico(s)PI3K/AKT/mTOR signaling in cancer
ResumoThe 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 → 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 α-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.1I44 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 → 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 α-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.1I44 protein-tyrosine kinase adenylyl-(β,γ-methylene)-diphosphonate insulin receptor substrate tyrosine kinase domain of the insulin receptor kilobase adenosine 5′-3-O-(thio)triphosphate The insulin receptor is an α2β2heterotetrameric glycoprotein possessing intrinsic protein-tyrosine kinase (PTK)1 activity (1Ullrich A. Bell J.R. Chen E.Y. Herrera R. Petruzzelli L.M. Dull T.J. Gray A. Coussens L. Liao Y.C. Tsubokawa M. Mason A. Seeburg P.H. Grunfeld C. Rosen O.M. Ramachandran J. Nature. 1985; 313: 756-761Crossref PubMed Scopus (1518) Google Scholar,2Ebina Y. Ellis L. Jarnagin K. Edery M. Graf L. Clauser E. Ou J.H. Masiarz F. Kan Y.W. Goldfine I.D. Roth R.A. Rutter W.J. Cell. 1985; 40: 747-758Abstract Full Text PDF PubMed Scopus (971) Google Scholar). Upon insulin binding to the α subunits, the insulin receptor undergoes a poorly characterized conformational change that results in autophosphorylation of specific tyrosine residues in the cytoplasmic portion of the β subunits. Three regions in the β subunits are sites of autophosphorylation: the juxtamembrane region, the activation loop (A-loop) within the tyrosine kinase domain, and the C-terminal tail (3Tornqvist H.E. Pierce M.W. Frackelton A.R. Nemenoff R.A. Avruch J. J. Biol. Chem. 1987; 262: 10212-10219Abstract Full Text PDF PubMed Google Scholar, 4Tavare J.M. O'Brien R.M. Siddle K. Denton R.M. Biochem. J. 1988; 253: 783-788Crossref PubMed Scopus (62) Google Scholar, 5White M.F. Shoelson S.E. Keutmann H. Kahn C.R. J. Biol. Chem. 1988; 263: 2969-2980Abstract Full Text PDF PubMed Google Scholar, 6Kohanski R.A. Biochemistry. 1993; 32: 5773-5780Crossref PubMed Scopus (43) Google Scholar). Autophosphorylation of tyrosine residues stimulates receptor catalytic activity (7Rosen O.M. Herrera R. Olowe Y. Petruzzelli L.M. Cobb M.H. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3237-3240Crossref PubMed Scopus (304) Google Scholar, 8Ellis L. Clauser E. Morgan D.O. Edery M. Roth R.A. Rutter W.J. Cell. 1986; 45: 721-732Abstract Full Text PDF PubMed Scopus (696) Google Scholar) and creates recruitment sites for downstream signaling molecules such as the insulin receptor substrate (IRS) proteins (9White M.F. Yenush L. Curr. Top. Microbiol. Immunol. 1998; 228: 179-208Crossref PubMed Google Scholar) and Shc (10Skolnik E.Y. Batzer A. Li N. Lee C.H. Lowenstein E. Mohammadi M. Margolis B. Schlessinger J. Science. 1993; 260: 1953-1955Crossref PubMed Scopus (504) Google Scholar, 11Pronk G.J. McGlade J. Pelicci G. Pawson T. Bos J.L. J. Biol. Chem. 1993; 268: 5748-5753Abstract Full Text PDF PubMed Google Scholar). 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 (12Hubbard S.R. Wei L. Ellis L. Hendrickson W.A. Nature. 1994; 372: 746-754Crossref PubMed Scopus (957) Google Scholar, 13Hubbard S.R. EMBO J. 1997; 16: 5572-5581Crossref PubMed Scopus (779) Google Scholar). 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 (12Hubbard S.R. Wei L. Ellis L. Hendrickson W.A. Nature. 1994; 372: 746-754Crossref PubMed Scopus (957) Google Scholar). 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 (13Hubbard S.R. EMBO J. 1997; 16: 5572-5581Crossref PubMed Scopus (779) Google Scholar, 14Hubbard S.R. Prog. Biophys. Mol. Biol. 1999; 71: 343-358Crossref PubMed Scopus (157) Google Scholar). 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-closedȁ conformations, as represented by the apoIRKD0P crystal structure, and "gate-openȁ conformations in which Tyr-1162 is displaced from the active site (15Frankel M. Bishop S.M. Ablooglu A.J. Han Y.P. Kohanski R.A. Protein Sci. 1999; 8: 2158-2165Crossref PubMed Scopus (20) Google Scholar). When the A-loop adopts a gate-open conformation, the kinase is competent to serve as either enzyme or substrate in atrans-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. 2Ablooglu, A. J. and Kohanski, R. A. (2001) Biochemistry 40, 504–513.2Ablooglu, A. J. and Kohanski, R. A. (2001) Biochemistry 40, 504–513. 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 → Ala (IRKDDA), has been introduced that dramatically alters the A-loop equilibrium in the unphosphorylated kinase. 3M. Frankel, A. J. Ablooglu, J. W. Leone, E. Rusinova, J. B. A. Ross, R. L. Heinrikson, and R. A. Kohanski, submitted for publication.3M. Frankel, A. J. Ablooglu, J. W. Leone, E. Rusinova, J. B. A. Ross, R. L. Heinrikson, and R. A. Kohanski, submitted for publication. 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 theKm(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. The 46-kDa form (residues 953–1355) of the Asp-1161 → 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 theXhoI-StuI sites of pALTER-IRK vector, which includes the point mutations Cys-981 → Ser and Tyr-984 → Phe described previously (16Wei L. Hubbard S.R. Hendrickson W.A. Ellis L. J. Biol. Chem. 1995; 270: 8122-8130Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). The fragment swap was verified withNheI digestion; this restriction enzyme site was introduced previously to replace a StuI site by a silent mutation at Ala-1048 and Ser-1049 (17Cann A.D. Kohanski R.A. Biochemistry. 1997; 36: 7681-7689Crossref PubMed Scopus (22) Google Scholar). 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 withBamHI. This was inserted into theBamHI-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 (18Bishop S.M. Ross J.B. Kohanski R.A. Biochemistry. 1999; 38: 3079-3089Crossref PubMed Scopus (19) Google Scholar).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. 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 dimensionsa = 57.9 Å, b = 69.6 Å, andc = 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 (19Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38526) Google Scholar). A molecular replacement solution was found with AMoRE (20Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5028) Google Scholar), using the structure of IRKD0P (PDB entry 1IRK) (12Hubbard S.R. Wei L. Ellis L. Hendrickson W.A. Nature. 1994; 372: 746-754Crossref PubMed Scopus (957) Google Scholar) as a search model. Rigid-body, positional, and B-factor refinement and simulated annealing were carried out using CNS (21Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16957) Google Scholar) (TableI). Model building was performed using O (22Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13009) Google Scholar).Table IX-ray data collection and refinement statisticsData collectionResolution (Å)30.0–2.4Observations76712Unique reflections14608Completeness (%)99.0 (93.5)1-aValue in parentheses is for the highest resolution shell (2.5–2.4 Å).Rsym1-bRsym = 100 × Σ‖I − 〈I〉‖/Σ I.(%)6.4 (22.3)1-aValue in parentheses is for the highest resolution shell (2.5–2.4 Å).Refinement1-cAtomic model includes 2205 protein atoms, 31 AMP-PCP atoms, 1 Mg2+ ion, and 141 water molecules.Resolution (Å)30.0–2.4Reflections13915Rcryst1-d/RRcryst = 100 × Σ∥Fo‖ − ‖Fc∥/Σ‖Fo‖, whereFo and Fc are the observed and calculated structure factors, respectively (Fo > 0ς). Rfree was determined from 5% of the data.free(%)21.2/26.4Root mean square deviationsBond lengths (Å)0.006Bond angles (°)1.3B-factors1-eFor bonded protein atoms.(Å2)2.0Average B-factors (Å2)All atoms25.1Protein24.9AMP-PCP28.5Water26.91-a Value in parentheses is for the highest resolution shell (2.5–2.4 Å).1-b Rsym = 100 × Σ‖I − 〈I〉‖/Σ I.1-c Atomic model includes 2205 protein atoms, 31 AMP-PCP atoms, 1 Mg2+ ion, and 141 water molecules.1-d Rcryst = 100 × Σ∥Fo‖ − ‖Fc∥/Σ‖Fo‖, whereFo and Fc are the observed and calculated structure factors, respectively (Fo > 0ς). Rfree was determined from 5% of the data.1-e For bonded protein atoms. Open table in a new tab The viscosity dependence forkcat andkcat/Km was determined using sucrose as the microviscogen following published procedures (23Blacklow S.C. Raines R.T. Lim W.A. Zamore P.D. Knowles J.R. Biochemistry. 1988; 27: 1158-1167Crossref PubMed Scopus (245) Google Scholar, 24Wang C. Lee T.R. Lawrence D.S. Adams J.A. Biochemistry. 1996; 35: 1533-1539Crossref PubMed Scopus (25) Google Scholar). 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, ηrel, is the ratio of the viscosity in the presenceversus the absence of viscogen and was determined as described by Adams and Taylor (25Adams J.A. Taylor S.S. Biochemistry. 1992; 31: 8516-8522Crossref PubMed Scopus (159) Google Scholar). The standard buffer was 50 mm Tris acetate, pH 7.0. Viscosity dependences ofkcat were done using 0.1 μmIRKDDA (unphosphorylated), 0.14–2.7 mmY-IRS939 peptide substrate (REETGSEYMNMDLG), and constant 1 mm ATP and then extrapolated to saturating substrate by fitting the observed kcatversuspeptide concentration to a rectangular hyperbola. To determine the viscosity dependences ofkcat/Km(peptide), measurements were done at 1 mm ATP and 0.14 mmY-IRS939. A control reaction measuring kcat was done using ATPγS to show the absence of viscosity effects in the IRKDDA-catalyzed reaction because phosphoryltransferase reactions with ATPγS are usually chemistry-limited rather than diffusion step-limited (26Chlebowski J.F. Coleman J.E. J. Biol. Chem. 1974; 249: 7192-7202Abstract Full Text PDF PubMed Google Scholar, 27Staubs P.A. Reichart D.R. Saltiel A.R. Milarski K.L. Maegawa H. Berhanu P. Olefsky J.M. Seely B.L. J. Biol. Chem. 1994; 269: 27186-27192Abstract Full Text PDF PubMed Google Scholar, 28Grace M.R. Walsh C.T. Cole P.A. Biochemistry. 1997; 36: 1874-1881Crossref PubMed Scopus (61) Google Scholar). The control reactions were done at 1 mm ATPγS, 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, withKm(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 (24Wang C. Lee T.R. Lawrence D.S. Adams J.A. Biochemistry. 1996; 35: 1533-1539Crossref PubMed Scopus (25) Google Scholar, 25Adams J.A. Taylor S.S. Biochemistry. 1992; 31: 8516-8522Crossref PubMed Scopus (159) Google Scholar), and for wild-type IRKD by Ablooglu and Kohanski.2 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. (18Bishop S.M. Ross J.B. Kohanski R.A. Biochemistry. 1999; 38: 3079-3089Crossref PubMed Scopus (19) Google Scholar). 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 (12Hubbard S.R. Wei L. Ellis L. Hendrickson W.A. Nature. 1994; 372: 746-754Crossref PubMed Scopus (957) Google Scholar). 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 → 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 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 apoIRKD0Pstructure (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 IRKDDAstructure is consistent with biochemical studies (16Wei L. Hubbard S.R. Hendrickson W.A. Ellis L. J. Biol. Chem. 1995; 270: 8122-8130Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) and modeling exercises, which indicate that MgATP and Tyr-1162 cannot bind in the active site simultaneously, i.e. thatcis-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 (29Taylor S.S. Radzio-Andzelm E. Structure. 1994; 2: 345-355Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). 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 IRKDDAstructure. 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 φ, ψ torsion angles shows that the hinge points for the N-terminal lobe rotation are at Arg-1061, before β-strand 4 (β4), and Met-1079, in the segment linking the N- and C-terminal lobes. When the N-terminal β sheet is superimposed for the three IRKD structures, α-helix C (αC) in the IRKDDA structure is observed to be in essentially the same position with respect to the β 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), αC undergoes an independent (from the β 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 αC, which mainly occur through φ, ψ changes at Phe-1054 and Thr-1055 at the base of the helix, are necessary to position protein kinase-conserved Glu-1047 (in αC) proximal to conserved Lys-1030 (in β3). Lys-1030 coordinates the α- and β-phosphates of ATP in an active protein kinase configuration (30Zheng J. Trafny E.A. Knighton D.R. Xuong N.H. Taylor S.S. Ten Eyck L.F. Sowadski J.M. Acta Crystallogr. Sect. D Biol. Crystallogr. 1993; 49: 362-365Crossref PubMed Google Scholar,31Bossemeyer D. Engh R.A. Kinzel V. Ponstingl H. Huber R. EMBO J. 1993; 12: 849-859Crossref PubMed Scopus (372) Google Scholar). The IRKDDA structure suggests that the rotation of αC 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 αC (Fig.4A). This pocket is composed of residues from αC (Glu-1047, Val-1050, Met-1051), from the αC-β4 loop (Phe-1054, Val-1059), from αE (Leu-1123), and from β8 (Ile-1148). In contrast, the side chain of Phe-1151 in the IRKDDA structure points upward toward αC 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, αC 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). 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 γ-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 (30Zheng J. Trafny E.A. Knighton D.R. Xuong N.H. Taylor S.S. Ten Eyck L.F. Sowadski J.M. Acta Crystallogr. Sect. D Biol. Crystallogr. 1993; 49: 362-365Crossref PubMed Google Scholar, 31Bossemeyer D. Engh R.A. Kinzel V. Ponstingl H. Huber R. EMBO J. 1993; 12: 849-859Crossref PubMed Scopus (372) Google Scholar). In the ternary IRKD3P structure, the γ-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 β- and γ-phosphates. Due to the incomplete rotation of the N-terminal lobe toward the C-terminal lobe in IRKDDA, AMP-PCP binds to the "roofȁ of the cleft (N-terminal residues) but not to the "floorȁ (C-terminal residues). Lys-1030 in β3 is hydrogen-bonded to the α-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 β- and γ-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 IRKD0Pstructure, Km(peptide), unlikeKm(ATP), is not decreased in the Asp-1161 → 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 β strands (13Hubbard S.R. EMBO J. 1997; 16: 5572-5581Crossref PubMed Scopus (779) Google Scholar). 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 IRKDDAA-loop at the distal end results in a peptide binding site that is not fully formed, which is reflected in the highKm(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. 4J. H. Till and S. R. Hubbard, unpublished data. 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 andk−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 byk4′. E·T+Y⇄k−2k2E·T·Y→k3E·D·pY→k4′E+D+pYSCHEME1The stepwise rate constants present in the steady-state kinetic parameters were derived using Cleland's method (32Cleland W.W. Biochemistry. 1975; 14: 3220-3224Crossref PubMed Scopus (301) Google Scholar) and were established from the viscosity dependence ofkcat andkcat/Km(peptide)(Fig. 5).kcat=k3·k4′/(k3+k4′)Equation 1 kcat/Km(peptide)=k2·k3/(k−2+k3)Equation 2 The principle behind viscometric analysis is that increased solution viscosity will affect the diffusion-dependent steps of substrate binding (k2 andk−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 (23Blacklow S.C. Raines R.T. Lim W.A. Zamore P.D. Knowles J.R. Biochemistry. 1988; 27: 1158-1167Crossref PubMed Scopus (245) Google Scholar). To identify whether chemistry is the rate-limiting step in steady-state phosphorylation of the peptide substrate Y-IRS939 by IRKDDA, the viscosity dependence ofkcat andkcat/Km(peptide)was determined. The parameters (kcat)rel and (kcat/Km)rel are the ratio of kcat andkcat/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)relversus ηrel 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)relversus ηrel was 0.8 ± 0.1, and the slope for (kcat/Km)relversus ηrel was 1.2 ± 0.2. For IRKDDA-catalyzed reactions without viscogen,kcat = 9.6 ± 0.4 s−1 andkcat/Km(peptide)= 4.2 ± 0.7 × 103m−1 s−1. From these values, we calculate k3 = 59 s−1 and k4′ = 14 s−1. 5These parameters were calculated fromk4′ = kcat/(slope from (kcat)relversusηrel); k3 =kcat·k4′/(k4′− kcat). 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 k3and k4′ determined for the activated kinase, IRKD3P: 46 s−1 and 11 s−1, respectively.2 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. 18Bishop S.M. Ross J.B. Kohanski R.A. Biochemistry. 1999; 38: 3079-3089Crossref PubMed Scopus (19) Google Scholar). The data are compared with denaturation profiles from unphosphorylated and phosphorylated wild-type IRCD taken from previous work (18Bishop S.M. Ross J.B. Kohanski R.A. Biochemistry. 1999; 38: 3079-3
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