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Originally published In Press as doi:10.1074/jbc.M313185200 on April 2, 2004 Originally published In Press as doi:10.1074/jbc.M313185200 on March 31, 2004

J. Biol. Chem., Vol. 279, Issue 28, 29700-29708, July 9, 2004
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Solution Structure and Backbone Dynamics of the Non-receptor Protein-tyrosine Kinase-6 Src Homology 2 Domain*

Eunmi Hong, Joon Shin, Han-Ie Kim, Seung-Taek Lee, and Weontae Lee{ddagger}

From the Department of Biochemistry and Protein Network Research Center, College of Science, Yonsei University, Seoul 120-749, Korea

Received for publication, December 3, 2003 , and in revised form, March 12, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Human protein-tyrosine kinase-6 (PTK6, also known as breast tumor kinase (Brk)) is a member of the non-receptor protein-tyrosine kinase family and is expressed in two-thirds of all breast tumors. To understand the structural basis of PTK6 function, we have determined the solution structure and backbone dynamics of the PTK6-Src homology 2 (SH2) domain using multidimensional NMR spectroscopy. The solution structure clearly indicates that the SH2 domain of human PTK6 contains a consensus {alpha}/{beta}-fold and a Tyr(P) peptide binding surface, which are common to other SH2 domains. However, two of the {alpha}-helices ({alpha}A and {alpha}B) are located on opposite faces of the central {beta}-sheet. In addition, the topological arrangement of a central four-stranded antiparallel {beta}-sheet (strands {beta}A, {beta}B, {beta}C, and {beta}D) differs from that of other Src family members. Backbone dynamics and Tyr(P) peptide titration experiments revealed that the putative ligand binding sites of the PTK6-SH2 domain undergo distinctive internal motions when compared with other regions of the protein. Surface plasmon resonance analysis showed that the Tyr(P) peptide had a dissociation constant of about 60 µM, which is substantially weaker binding than previously reported for Src family members. The solution structure together with data from the ligand binding mode of PTK6-SH2 provides insight into the molecular basis of the autoinhibitory role of PTK6.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Protein-tyrosine kinases (PTKs)1 play an important role as regulators of cellular proliferation, differentiation, and apoptosis. Hyperactivation of PTKs often occurs in tumorigenesis through mutation, rearrangement, or gene amplification. The human PTK6 (also known as breast tumor kinase (Brk)) is a non-receptor protein-tyrosine kinase that was identified from a partial cDNA clone during a survey of PTK mRNAs in human melanocytes (1). It has been found that PTK6 is overexpressed in breast carcinomas (2) and colon tumors (3). Since the expression of PTK6 in human mammary epithelial cells resulted in an activation of the mitogenic response of the cells to epidermal growth factor (4), its expression serves as a probe of the tumor development process. PTK6 is considered to be most closely related to the Frk family proteins; however, it differs from Src family kinases both in lacking an N-terminal myristoylation site (5) and in having low sequence identity with members of this family. In addition, PTK6 showed an evolutionary divergence in the genomic structure (6). Although mouse Sik (a mouse homologue of PTK6) did not phosphorylate GTPase-activating protein-associated p65 protein, a phosphotyrosine residue of GTPase-activating protein-associated p65 interacts with and activates Sik through its Src homology 2 (SH2) domain in mouse keratinocytes (7). Derry et al. (8) reported that PTK6 phosphorylated Sam68, a nuclear RNA-binding protein. Recently a protein, BKS, encoded by cDNA that was isolated using a yeast two-hybrid method was found to contain an N-terminal pleckstrin homology-like domain connected to an SH2 domain, suggesting that BKS protein would be a strong candidate for phosphorylation mediated by the kinase domain of PTK6 (9).

Analysis of the primary structure of PTK6 showed that it is composed of SH3, SH2, and catalytic domains (5, 6). For the case of the non-receptor protein-tyrosine kinases, the SH2 domain is typically involved in negative regulation of kinase activity by binding to a phosphorylated tyrosine residue near to the C terminus. A recent report suggested that the inactive form of Src was further stabilized by interactions between the SH3 domain and a polyproline linker near the kinase domain (10). The SH2 domain has been determined as an independent folded module involved in a number of intracellular signaling pathways (Fig. 1) that binds to specific amino acid sequences in growth factor receptors and other molecules with phosphotyrosine residues. A number of reports have revealed that, in most cases, the SH2 domain adopts a common {alpha}/{beta}-folding motif with two binding pockets for the phosphotyrosine peptide (11-15). The structure of ligand peptide has also been determined as an extended or turnlike conformation, which enables the anchoring of its phosphotyrosyl group to the ligand binding pocket of the SH2 domain.



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  		FIG. 1.
Sequence alignment for the SH2 domains. The residues with dashes have been determined to be in the ordered regions of secondary structure.

 
It was recently proposed that the C-terminal sequence of PTK6 (PTSpYENPT where pY is phosphotyrosine) might be a self-ligand for the SH2 domain. The solution structure of PTK6-SH2 revealed a distinct binding surface, providing evidence for an interdomain interaction of PTK6. Therefore, the structural data together with the binding mechanism of the ligand peptide derived from the C-terminal sequence of PTK6 would add to our understanding of the biochemical data and the biological activity of PTK6. In this report, we present the high resolution solution structure of the human PTK6-SH2 domain together with its ligand binding mode determined using heteronuclear multidimensional NMR spectroscopy and surface plasmon resonance analysis.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Protein Expression and NMR Sample Preparation—The cDNA segment encoding the PTK6-SH2 (residues 75-174) was amplified using a primer pair (5'-cGGgaTCcGAACCGTGGTTCTTTGGC-3' and 5'-ggaattcaCTCGTGCTTCCGGCAGG-3' where lowercase letters indicate nucleotides that were introduced to generate restriction enzyme sites and a termination codon) by PCR from the full-length PTK6 cDNA (6) and cloned into the BamHI-EcoRI sites of pGEX 4T-3 (Amersham Biosciences), an Escherichia coli expression vector. The recombinant protein contained a glutathione S-transferase (GST) tag encoded from the vector sequence in its N terminus. This vector was used to transform the E. coli strain BL21(DE3)pLysS for expression of the fusion protein. Uniformly 13C/15N- and 15N-labeled protein samples were prepared by growing cells in M9 minimal media containing 15NH4Cl, either with or without 13C6-D-glucose. The purified GST-PTK6-SH2 was digested using bovine thrombin (Amersham Biosciences), and the final purification of PTK6-SH2 was performed using fast protein liquid chromatography with a Superdex 75 HR 10/30 column in 2 mM dithiothreitol, 50 mM potassium phosphate buffer at pH 6.5. The NMR samples were concentrated and transferred to a 5-mm symmetrical microcell (Shigemi). The protein-peptide complex solution was prepared by titrating Tyr(P) peptide into PTK6-SH2 with a final molar ratio of 1:2 (protein:peptide).

Preparation of the Tyr(P) Peptide Sample—The peptide (Ac-SSFTS-pYENPT-COOH) derived from the C-terminal region of PTK6 (residues 442-451) was synthesized commercially (Anygen Co., Kwangju, Korea) by solid-phase Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry (17). After trifluoroacetic acid cleavage, the peptide was eluted by analytical HPLC with the pH value of 3-4. The purity of synthetic peptide was confirmed by preparative HPLC and matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

NMR Spectroscopy—All NMR experiments were performed at 298 K on a Bruker DRX500 or DRX600 or Varian Unity INOVA 500-MHz spectrometer equipped with a triple resonance probe and shielded triple axis gradients. The 1H chemical shifts were referenced to internal sodium 4,4-dimethyl-4-silapentane-1-sulfonate. The 13C and 15N chemical shifts were referenced indirectly using the 1H/13C or 1H/15N frequency ratios of the zero point: 0.101329118 (15N) and 0.251449536 (13C) (18). The 1H-15N HSQC (19-20) experiment was acquired using a uniformly 15N-labeled sample. Pulsed-field gradient techniques with a WATERGATE pulse sequence (21) were used for all H2O experiments, resulting in a good suppression of the solvent signal. An HCCH-TOCSY (22) experiment with 12-ms mixing time and two sets of three-dimensional 15N-edited TOCSY-HSQC (21, 23-25) experiments with 45- and 77.75-ms mixing times were performed. 15N-Edited NOESY-HSQC (21, 24, 25) experiments with mixing times of 150 and 200 ms in 90% H2O, 10% D2O solution and a 13C-edited NOESY experiment (21, 24-26) in D2O solution with a mixing time of 100 ms were collected. Two-dimensional 1H-15N heteronuclear multiple quantum coherence-J (27), three-dimensional HNHA (28), and a series of 1H-15N HSQC experiments were performed. The three-dimensional triple resonance experiments, HNCA (29-31), HN(CO)CA (28, 29), HNCACB (31, 32), CBCA(CO)NH (33), HNCO (29, 32, 34), and HCACO (35), were collected for backbone resonance assignment. All NMR data were processed on a Silicon Graphics Indigo2 work station using NMRPipe/NMRDraw software (Biosym/Molecular Simulations, Inc.). SPARKY 3.95 (37) and XEASY programs (38) were used for spectral analysis.

Structure Calculations—The solution structures were calculated by the distance geometry and dynamic simulated annealing (SA) procedures with the CNS 1.0 program on an Silicon Graphics Indigo2 work station. Structural calculations were carried out following methods published previously (39-41). The target functions for the molecular dynamics and energy minimization consisted of the covalent structure, van der Waals repulsion, NOE, and torsion angle terms. All restraints for the structure calculations were derived from the distance, torsion angle, and hydrogen bond information. Based on cross-peak intensities of NOESY spectra, distance constraints were classified as strong, medium, and weak corresponding to the upper distance bound of 2.7, 3.3, and 5.0 Å, respectively. Appropriate pseudoatom corrections were also applied to non-stereospecifically assigned methylene, methyl groups, and ring protons (42). Hydrogen bond constraints were used by defining two upper limit distances for each hydrogen bond: rN(i)-O(j) < 3.3 Å and rNH(i)-O(j) < 2.3 Å). Angle constraints for the {phi} torsion angles were set to -120 ± 50° for 3JHNa > 8 Hz and -60 ± 35° for 3JHNa < 6 Hz, respectively. The 20 lowest energy structures (SAk) from 50 simulated annealing conformers were selected for further structural analysis. None of the 20 SAk structures had any constraint violation greater than 0.5Å for distance and 3° for angle constraints. Structural analysis of the final 20 SAk structures was done using the MOLMOL (36) program.

Backbone Dynamics—NMR pulse sequences for backbone dynamic experiments were used as reported previously (43). To eliminate the effects of cross-correlation between 15N-1H dipolar and 15N chemical shift anisotropy relaxation mechanisms, 1H 180° pulses were inserted during the relaxation times (44-46). Longitudinal (T1) and transverse (T2) relaxation data for the backbone amide 15N nuclei were recorded as 2048 x 64 data sets with 64 scans per point using 1 s of relaxation recovery time. Seven different relaxation times for T1 and T2 experiments were recorded with T1 = 5, 65, 145, 246, 366, 527, and 757 ms and T2 = 8.3, 25.1, 41.8, 58.6, 75.3, 108.8, and 142.3 ms, respectively. For the estimation of noise levels, duplicate spectra were recorded for values of T1 = 246 ms and T2 = 56.8 ms. The steady-state heteronuclear NOE (43, 47) experiment was obtained using a relaxation delay of 5 s. All data were processed on Silicon Graphics Indigo2 work stations using the NMRPipe software. The standard errors of T1 and T2 values were calculated from the non-linear least square fit. The overall tumbling motion of the SH2 domain was calculated from the T1/T2 ratios of N-H groups using the program Quadric Diffusion 1.11 (A. G. Palmer, Columbia University).

Surface Plasmon Resonance Analysis—Surface plasmon resonance measurements were performed by the BIACORE biosensor and BIAe-valuation version 3.1 interactive software (Biocore) (48, 49). GSTPTK6-SH2 was immobilized at a flow rate of 10 µl/min using phosphate-buffered saline buffer solution (pH 6.5 with 2 mM dithiothreitol). Equal volumes of 0.1 M N-hydroxysuccinimide and 0.4 M N-ethyl-N'-(3-diethylaminopropyl)carbodiimide were mixed and injected into a CM5 sensor chip to activate the carboxymethylated dextran surface. The volume was adjusted to achieve immobilization levels of GST-PTK6-SH2, making 6000 resonance units. After injection of GST-PTK6-SH2, the residual N-hydroxysuccinimide esters were deactivated by 25 µl of ethanolamine (1 M, pH 8.5). The Tyr(P) peptide at a concentration of 5.2-166.8 µM was injected into the immobilized sensor chip of GSTPTK6-SH2 at a flow rate of 30 µl/min.

Modeling of the PTK6-SH2·Tyr(P) Peptide Complex—The energy-minimized average structure of PTK6-SH2 was used as a template to generate a model of the protein·peptide complex. The template structures of the peptides were derived from the p56lck-peptide (Protein Data Bank accession code 1LCJ [PDB] ) and Grb2-peptide (Protein Data Bank accession code 1QG1 [PDB] ) structure. The initial structure of the PTK6-SH2·Tyr(P) complex was further optimized using consistent valence force field on the DISCOVER3 module. To obtain the lowest energy conformation, restrained molecular dynamics was performed for 250 ps at 298 K. The lowest energy conformers were sampled every 100 steps during the molecular dynamic calculation. The restrained energy minimization procedure using the Broyden-Fletcher-Goldfard method was performed for the final structure until its energy gradient deviation decreased to 0.001 kcal·mol-1.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Resonance Assignments and Secondary Structures—The backbone resonance assignments were completed by data from HNCACB, CBCA(CO)NH, HNCO, and HCACO spectra. For the assignment of the residues preceding proline, HCACO data proved useful (50). Most of the side chain assignments were accomplished by three-dimensional HCCH-TOCSY and 15N-edited TOCSY-HSQC experiments. The secondary structures were determined from the chemical shift indices (51), NOEs, 3JHN{alpha} coupling constant, and slowly exchanging amide hydrogen information (Fig. 2). PTK6-SH2 is composed of two {alpha}-helices, six {beta}-strands, and seven flexible loops.



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  		FIG. 2.
Summary of sequential and medium range NOEs, amide proton exchange, and 3JHN{alpha} coupling constant data. The amino acid sequence for the SH2 domain of PTK6 is shown, and the strength of the observed NOEs is classified by the thickness of the lines. Coupling constant data are represented with a {square} for 3JHNa greater than 8 Hz and a {blacksquare} for 3JHNa less than 4 Hz. kex indicates slowly exchanging amide protons in the HSQC exchange experiment. CSI, chemical shift indices.

 
Three-dimensional Structure of PTK6-SH2—A total of 1532 distance constraints, including 250 intraresidue, 374 sequential, 271 medium range, and 637 long range constraints, were derived from NOESY spectra. For 23 backbone hydrogen bonds, 46 corresponding interatomic distance constraints were used for the structure calculation. Fig. 3 summarizes backbone-backbone, side chain-side chain, and backbone-side chain NOEs. The structural statistics associated with the 20 SAk structures are listed in Table I. The final structures are well converged with a root mean square deviation of 0.29 Å for secondary structural regions and 0.78 Å for all backbone heavy atoms, respectively. The PTK6-SH2 fold consists of a central four-stranded {beta}-sheet ({beta}A, {beta}B, {beta}C, and {beta}D), two {alpha}-helices ({alpha}A and {alpha}B), and a small {beta}-sheet ({beta}E and {beta}F) near the second helix. The structure of PTK6-SH2 is relatively rigid, and a central {beta}-barrel is well defined (Fig. 4). The topology of PTK6-SH2 is similar to that of other SH2 domains (52); however, it shows some differences from a canonical SH2 domain. A spatial rearrangement of the secondary structures especially in the central {beta}-barrel region due to different length of {beta}-strands was clearly observed (Fig. 5). In addition, the C-terminal {beta}-strand ({beta}G), which is involved in not only {beta}-barrel formation but also in ligand binding, is not observed in PTK6-SH2. This implies that PTK6-SH2 might have a unique binding feature for its specific ligand peptide.



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  		FIG. 3.
Inter-residue NOE contacts for PTK6-SH2. Backbone-backbone (solid boxes) and side chain-side chain (shaded boxes) (A) and backbone-side chain (B) NOEs are displayed.

 


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  		TABLE I
Structural statistics for the 20 final simulated annealing structures of PTK6 SH2 domain

 



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  		FIG. 4.
Final simulated annealing structure of PTK6-SH2. Stereoview of superposition of the final 20 SAk structures of the SH2 domain over the energy-minimized average structure (SAkr) is displayed.

 



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  		FIG. 5.
Richarson plots of PTK6-SH2 and Src-SH2. The structure of PTK6-SH2 (A) is compared with that of Src-SH2 (B). This figure was generated using MOLSCRIPT. The secondary structural elements were labeled according to the nomenclature of Eck et al. (52). The color scheme is as follows: {alpha}-helices are in red, {beta}-strands are in dark blue, and loops are in gray. C, the electrostatic charge distribution of PTK6-SH2. Electrostatic surfaces are shown in red (negative potential), in blue (positive potential), and in white (neutral).

 
Backbone Dynamics—Of the 96 backbone amide protons, the relaxation parameters for 81 residues have been reliably quantified. The experimental T1, T2, and steady-state 15N-{1H} heteronuclear NOE data are summarized in Fig. 6. The average values of T1 and T2 were determined to be 365.9 and 131.6 ms, respectively. The 15N-{1H} heteronuclear NOEs for most residues were observed to be 0.8-1.0. The profile of heteronuclear NOEs shows that the secondary structural regions are relatively rigid. The overall rotational correlation time ({tau}m) determined from the T1 and T2 values and NMR structure is 6.5 ns (Fig. 7). The calculated order parameters (S2) were in the range 0.7-0.95; however, small order parameters were found for the residues in the loops and the terminus of the secondary structural regions. The effective correlation time for internal motions, {tau}e, was determined as 55 ps. Interestingly most residues of the protein involved in Tyr(P) peptide binding exhibited smaller 15N-{1H} steady-state heteronuclear NOEs and order parameters as well as longer internal correlation times than those of residues in the structured regions, implying that the ligand binding site could be intrinsically dynamic in the absence of its ligand molecule (Figs. 6 and 7).



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  		FIG. 6.
Plots of 15N relaxation data as a function of residue number for PTK6-SH2. Measured values of spin-lattice relaxation time (T1) are shown in A, values of spin-spin relaxation time (T2) are shown in B, and heteronuclear NOE (XNOE) data are shown in C. The {alpha}-helical regions are represented by black bars, {beta}-strands are represented by white bars, and loops are represented by gray bars.

 



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  		FIG. 7.
Plots of the optimized model-free and extended model-free parameters as a function of residue number for PTK6-SH2. Optimized values of the generalized order parameter (S2) are shown in A, values of effective correlation time ({tau}e) are shown in B, and exchange parameters (Rex) are shown in C, values of the effective correlation time for internal motions on a slow time scale ({tau}s) are shown in D, and values of the generalized order parameter for the fast motion (Sf2) are shown in E. The parameters and their uncertainties are shown as a function of the protein sequence.

 
Chemical Shift Change upon Ligand Binding—The ligand binding pocket was predicted from the backbone dynamics together with sequence alignment data. To determine residues involved in ligand binding, a phosphorylated peptide derived from the C-terminal region (spanning residues 442-451) of PTK6 was used for NMR titration experiments. It has been observed that the autophosphorylated oligopeptide at Tyr-447 of PTK6, but not the unphosphorylated peptide, was successfully bound to PTK6-SH2 (16). A series of 1H-15N HSQC spectra of PTK6-SH2 were collected with different concentrations of phosphopeptide. Fig. 8A shows a superposition of the 1H-15N HSQC spectra of free PTK6-SH2 and peptide-bound PTK6-SH2 showing the chemical shift perturbations upon ligand binding more graphically. Most of the observed chemical shift perturbations occurred at residues involved in phosphopeptide binding. Chemical shift changes ({Delta}{delta}tot) between free SH2 and complexed SH2 were calculated using the equation,

(Eq. 1)



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  		FIG. 8.
Chemical shift perturbations of 1HN and 15N resonances of PTK6-SH2 upon binding the Tyr(P) peptide ligand (Ac-SSFTSpYENPT-COOH). A, superimposed two-dimensional 1H-15N HSQC spectra of PTK6-SH2 both without peptide (red) and with peptide (blue). To assess chemical shift perturbations of PTK6-SH2 upon ligand binding, the titration of 1-2 molar eq. of the peptide to protein solution was performed. The residues are labeled on the spectrum. B, the combined chemical shift difference ({Delta}{delta}tot) for each residue was calculated according Equation 1. Deviations between the free and peptide-bound forms were plotted as a function of residue number. C, mapping of the effect of the ligand binding onto the PTK6-SH2 structure. Colors indicate the degree of chemical shift perturbation. The residues with 0.4-0.8 ppm are colored green; residues with >0.8 ppm are colored red.

 
where {delta}i is the chemical shift of nucleus i, and Wi denotes its weight factor (WHN = 1, WN = 0.2) (53).

A single resonance peak was observed for each residue of PTK6-SH2 during titration, showing that the PTK6-SH2·Tyr(P) peptide is in fast exchange on the NMR time scale (Fig. 8A). The chemical shift perturbations upon Tyr(P) peptide binding were plotted as a function of residue number (Fig. 8B). The model structure of PTK6-SH2·Tyr(P) peptide agrees well with this data, showing that the peptide ligand interacts mainly with residues comprising a continuous patch of both hydrophobic and hydrophilic surfaces, which are {alpha}A (Arg-85 and Val-89), {beta}B (Arg-105), BC loop (Glu-108), {beta}D (His-126, Tyr-127, and Lys-128), DE loop (Ala-133), {beta}E (Leu-139), EF loop (Glu-141), {alpha}B (Tyr-154), and BG loop (Leu-160 and Leu-164) (Fig. 8C).

Surface Plasmon Resonance Analysis—The binding affinity of the Tyr(P) peptide to the SH2 domain was measured by surface plasmon resonance spectroscopy using GST-PTK6-SH2. Fig. 9 illustrates a surface plasmon resonance sensorgram for the ligand peptide binding mode of PTK6-SH2. A rapid response curve indicates that the binding of peptide occurs with a fast association rate (kon) and dissociation rate (koff). Based on surface plasmon resonance data, an apparent dissociation constant (Kd = kon/koff) for the interaction between PTK6-SH2 and Tyr(P) peptide is 60 µM. Since the relative affinities for various SH2-Tyr(P) peptide interactions have been reported as Kd values from 10 µM to 0.1 nM, the binding affinity of Tyr(P) peptide to PTK6-SH2 is lower than those of previously reported phosphopeptide-SH2 binding (48). However, this weak binding is clearly supported by previous biological data showing that the enzyme activity of PTK6 increased about 1.4-fold with 1 mM Tyr(P) peptide, and no activity change has been observed within the micromolar range of peptide concentration (16).



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  		FIG. 9.
Surface plasmon resonance sensorgrams showing the interaction between PTK6-SH2 and Tyr(P) peptide. The peptide binding profile of peptide to GST-PTK6-SH2 immobilized on a carboxymethyl dextran surface. The superimposed curves are derived from experiments for peptide concentrations of 5.2, 10.4, 20.8, 41.7, 83.4, and 166.8 µM (from bottom to top). The observed signals are represented as a plot of the resonance units versus time.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Although the molecular topology of PTK6-SH2 is similar to that of the SH2 domains, the spatial arrangement of a central four-stranded antiparallel {beta}-sheet (strands {beta}A, {beta}B, {beta}C, and {beta}D) clearly differs due to the missing {beta}G strand, which is commonly found in most SH2 structures. The structural superposition with other SH2 domains of p56lck, Hck, and Blk clearly supports the differences; the pairwise backbone (N, C{alpha}, and C') root mean square deviations for these structures are 2.016 Å (p56lck), 2.196 Å (Hck), and 3.271 Å (Blk), respectively. Several notable structural differences were clearly observed in the ordered secondary structural regions, and high root mean square deviation values in the BC, EF, and BG loops between PTK6-SH2 and other SH2 domains were also observed. For example, a dramatic difference was found in loops; PTK6-SH2 has a shorter CD loop and longer AB loop than those of the canonical SH2 domains (Fig. 5A), suggesting that the structural rearrangement of the central {beta}-barrel due to different loop length might enable its ligand specificity for PTK6 signaling.

A surface charge distribution of PTK6-SH2 calculated with the Delphi program suggests that a cluster of positively charged surface residues would be a ligand binding site for the negatively charged phosphotyrosine peptide, providing a strongly favorable electrostatic interaction between protein and ligand molecules. A continuous stretch of the ligand binding surface between BC and {beta}G loop was also observed (Fig. 5B). Since it is well known that one of the major interactions between Tyr(P) peptide and SH2 domain is a hydrophobic interaction, it is expected that the hydrophobic patch of PTK6-SH2 should play a critical role in ligand binding. Fig. 5B clearly shows that the hydrophobic amino acids (Leu-139 and Leu-160) of PTK6-SH2 contact the proline residue of the Tyr(P) peptide, which is the Tyr(P) +3 position of the ligand. The addition of Tyr(P) peptide to PTK6-SH2 also supports the structural data based on chemical shift perturbation of the residues involved in Tyr(P) peptide binding. For example, several charged residues in {alpha}A (Arg-85) and {beta}D (His-126 and Lys-128) undergo large chemical shift perturbation upon ligand binding (Fig. 10). A significant change of the NMR resonance intensities was also observed for residues Val-89 ({alpha}A) and Tyr-127 and Ile-129 ({beta}D). Most of the residues in the BC, EF, and BG loops experience chemical shift perturbations upon ligand binding.



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  		FIG. 10.
Ligand binding pocket and a structure of Tyr(P) peptide·PTK6-SH2 complex generated by Insight II program (Biosym/Molecular Simulations, Inc.). A, protein surface representation of PTK6-SH2. The residues involved in Tyr(P) peptide binding are displayed. B, the structure of PTK6-SH2·Tyr(P) peptide complex. The structure was generated from solution structure of PTK6-SH2 (dark blue), and the ligand structure was derived from Tyr(P) peptide·p56lck-SH2 and Tyr(P) peptide·Grb2-SH2 complexes.

 
Recently Qiu and Miller (16) suggested that the phosphorylation of Tyr-447 of PTK6 might play an important role in its negative regulation of the enzyme activity. They have shown that a single mutation at Tyr-447 increased SH2 domain accessibility to a synthetic ligand and resulted in 2.5-fold higher catalytic activity than that of the wild type. In this study, Tyr(P) peptide derived from the C-terminal region of PTK6 including Tyr(P)-447 successfully bound to the SH2 domain, supporting the autoinhibitory role of PTK6 protein. The apparent dissociation constant (Kd) between Tyr(P) peptide and PTK6-SH2 was determined as 60 µM, which is much weaker than that of other SH2 domains. However, this might be explained by the fact that the local concentration of the phosphorylated C-terminal region of PTK6 is high in vivo because the actual binding will occur as an intramolecular interaction. Our data indicate that the structural motif of PTK6-SH2 would be of importance for specific recognition of ligand because the binding of phosphorylated C-terminal region to SH2 domain would be highly specific for the optimum autoregulation of PTK6 activity. In addition, it could be possible that the enzymatic activity of PTK6 would be tightly regulated through an autophosphorylation process at Tyr-447. Although a recent report suggests that PTK6 phosphorylates two other proteins, Sam68 and BKS in vivo (54), the downstream signaling of PTK6 is still not known in detail. Further structural study of PTK6 together with its interacting molecules will elucidate the signaling network and negative autoregulation process of this family of proteins at the atomic scale.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 1RJA) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This study was supported by the Ministry of Science and Technology of Korea/the Korea Science and Engineering Foundation through the National Research Laboratory program of the Ministry of Science and Tecnology National Research Development Program (Grant M1-0203-00-0020) (to W. L.) and through the Protein Network Research Center at Yonsei University (to W. L. and S.-T. L.) and in part by the Brain Korea 21 Project. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Biochemistry, College of Science, Yonsei University, 134 Shinchon-Dong, Seodaemoon-Gu, Seoul 120-749, Korea. Tel.: 82-2-2123-2706; Fax: 82-2-363-2706; E-mail: wlee{at}spin.yonsei.ac.kr.

1 The abbreviations used are: PTK, protein-tyrosine kinase; SH, Src homology; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; TOCSY, total correlated spectroscopy; HSQC, heteronuclear single quantum coherence; SA, simulated annealing; GST, glutathione S-transferase; HPLC, high performance liquid chromatography. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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