doi:10.1016/j.jmb.2004.05.060
J. Mol. Biol. (2004) 341, 93–106
Dynamic Coupling Between the SH2 Domain and Active Site of the COOH Terminal Src Kinase, Csk Lilly Wong1, Scot Lieser1, Barbara Chie-Leon2, Osamu Miyashita3 Brandon Aubol1, Jennifer Shaffer2, Jose` N. Onuchic3 Patricia A. Jennings1, Virgil L. Woods Jr4* and Joseph A. Adams2* 1 Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla CA 92093, USA 2
Department of Pharmacology University of California, San Diego, La Jolla, CA 92093-0506 USA 3 Department of Physics and Center for Theoretical Biological Physics, University of California, San Diego, La Jolla, CA 92093, USA 4 Department of Medicine University of California, San Diego, La Jolla, CA 92093 USA
The SH2 domain is required for high catalytic activity in the COOHterminal Src kinase (Csk). Previous solution studies suggest that a short peptide sequence, the SH2-kinase linker, provides a functional connection between the active site and the distal SH2 domain that could underlie this catalytic phenomenon. Substitutions in Phe183 (tyrosine, alanine, and glycine), a critical hydrophobic residue in the linker, result in large decreases in substrate turnover and large increases in the Km for ATP. Indeed, F183G possesses kinetic parameters that are similar to that for a truncated form of Csk lacking the SH2 domain, suggesting that a single mutation disrupts communication between this domain and the active site. Based on equilibrium and stopped-flow fluorescence experiments, the elevated Km values for the mutants are due to changes in the rates of phosphoryl transfer and not to reduced ATP-binding affinities. Based on hydrogen –deuterium exchange experiments, glycine substitution reduces flexibility in several polypeptide regions in Csk, tyrosine substitution increases flexibility, and alanine substitution leads to mixed effects compared to wild-type. Normal mode analysis indicates that Phe183 and its environment are under strain, a theoretical finding that supports the results of mutations. Overall, the data indicate that domain–domain interactions, controlled through the SH2-kinase linker, provide a dynamic balance within the Csk framework that is ideal for efficient phosphoryl transfer in the active site. q 2004 Elsevier Ltd. All rights reserved.
*Corresponding authors
Keywords: Csk; deuterium exchange; kinase; mass spectrometry; stoppedflow
Introduction Signal transmission at the plasma membrane relies commonly on non-receptor protein tyrosine Abbreviations used: AMPPNP, 50 -adenylylimidodiphosphate; Csk, COOH-terminal Src kinase; DXMS, hydrogen– deuterium exchange mass spectrometry; ESI, electrospray ionization; H –D, hydrogen – deuterium; Mant-ATP, 20 -(30 )-O-(Nmethylanthraniloyl)-ATP; Mant-20 deoxyATP, 30 -O-(Nmethylanthraniloyl)-20 deoxyATP; NMA, normal mode analysis; nrPTK, non-receptor protein tyrosine kinase; poly(Glu4Tyr), random heteropolymer of glutamic acid and tyrosine; SH2, Src homology 2 domain; SH3, Src homology 3 domain; Src, nrPTK from Rous sarcoma virus. E-mail addresses of the corresponding authors:
[email protected];
[email protected]
kinases (nrPTKs), a class of enzymes that utilize phosphotyrosine-binding SH2 domains to link physically to receptor proteins.1,2 The activities of the nrPTKs are influenced greatly by neighboring non-catalytic domains, as revealed through biochemical studies.3 – 5 The orientation of these domains relative to the kinase domain has been investigated using crystallographic methods. In the case of the Src family of nrPTKs, the SH2 domain binds the phosphorylated C terminus of the kinase domain packing tightly onto the large lobe of the kinase domain.6,7 The SH3 domain interacts with the small domain through a prolinerich linker between the SH2 and kinase domains. Deletion of the SH2 or SH3 domain in Src enhances catalytic activity,8 most likely through the perturbation of contacts between the phosphorylated C terminus and the SH2 domain. Other nrPTKs
0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
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appear to utilize different arrangements of the SH2 and SH3 domains, resulting in very different regulatory effects. For Csk, an nrPTK that shares 40% sequence similarity with Src but lacks a phosphorylatable tail segment, the SH2 domain adopts a unique position where it interacts with the small lobe of the kinase domain rather than the large lobe.9 Deletion studies reveal that the SH2 and SH3 domains elevate the activity of the kinase domain in Csk by about 100-fold, with the SH2 domain providing most of this activation.5,10 Thus, while non-catalytic domains are important for down-regulation in Src, they dramatically upregulate function in Csk.11 To understand how the non-catalytic domains in Csk enhance the catalytic efficiency of the kinase domain, it is essential to identify the rate-limiting steps associated with substrate phosphorylation. Using pre-steady-state kinetic methods, we showed that catalytic function in Csk is regulated by slow ADP release and associated conformational changes.12 To provide a molecular basis for these nucleotide-linked effects and to better understand factors that underpin regulation, we previously employed hydrogen– deuterium (H– D) exchange kinetics to isolate short polypeptide regions in Csk that undergo environmental and/ or stability changes upon the binding of ADP and ATP.13 We found that the addition of the nucleotide influences exchange kinetics in the active site (glycine-rich and catalytic loops), as expected, and
Dynamics in Csk
in distal regions in the large lobe of the kinase domain (activation loop and helix aG) and the SH2 domain (helix aA). Curiously, two key polypeptide segments comprising helix aC in the kinase domain and the linker sequence between the kinase and SH2 domains (SH2-kinase linker) are influenced strongly by the nucleotide. On the basis of these findings, we proposed that the SH2kinase linker serves as a conduit for the propagation of these structural effects into the SH2 domain and, thus, provides a functional link between the SH2 domain and catalysis-enhancing events in the active site (Figure 1(A)). The recent X-ray structure of Csk shows that the SH2-kinase linker and helix aC are in close proximity to one another, forming a hydrophobic pocket with p-interactions between Phe183 in the SH2-kinase linker and Phe233 in helix aC (Figure 1(B) and (C)), supporting our model. To demonstrate that the SH2-kinase linker and helix aC are functional components that modulate communication between the SH2 and kinase domains, we investigated the effects of mutants designed to perturb the linkage between the domains on the catalytic mechanism and solution conformation of Csk. Replacement of Phe183 with tyrosine, alanine and glycine causes dramatic decreases in catalytic efficiencies ranging from fivefold to 800-fold. These reductions are due to decreases in substrate turnover rate constants (kcat) and reductions in the apparent binding affinity of
Figure 1. SH2-kinase linker of Csk. (A) Regions in primary structure of Csk affected by ATP binding. Regions are identified using H– D exchange techniques as described.13 Red arrows show potential connectivities among these affected regions. (B) X-ray structure of full-length Csk. The SH2 and SH3 domains flank the small lobe of the kinase domain. The active site lies between the small and large lobes of the kinase domain. (C) Interaction between the SH2kinase linker and helix aC. Phe183 in the SH2-kinase linker and Phe233 in helix aC are labeled.
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Dynamics in Csk
Table 1. Steady-state kinetic parameters for wild-type and mutants Parameter
Wild-type
F183Y
F183A
F183G
kcat (min21) Km,ATP (mM) kcat/Km,ATP (mM21 min21) KI,AMPPNP (mM) KI,ADP (mM)
5.1 ^ 0.1 55 ^ 4.5 93 ^ 7.8 40 ^ 10 0.80 ^ 0.20
3.7 ^ 0.4 210 ^ 92 18 ^ 8.1 40 ^ 20 1.4 ^ 0.10
1.4 ^ 0.15 450 ^ 170 3.5 ^ 1.4 67 ^ 15 1.0 ^ 0.20
0.3 ^ 0.06 2400 ^ 850 0.12 ^ 0.049 70 ^ 30 1.1 ^ 0.10
ATP (Km). Fluorescent nucleotide-binding studies reveal that the mutant enzymes bind ATP with the same thermodynamic and kinetic parameters as that for the wild-type enzyme. This unusual finding can be explained by a two-channel mechanism where one enzyme form binds ATP well but does not support efficient substrate turnover. H – D exchange profiles indicate that the mutants induce long-range changes in the Csk framework that affect the active site without influencing the overall folding of the protein. Surprisingly, flexibility within the Csk framework is not correlated linearly with catalytic activity. While F183G appears less flexible in several regions, F183A displays mixed effects and F183Y is more flexible than wild-type Csk. Analysis of the normal modes of Csk reveals that Phe183 and its environment are under strain compared to the remainder of the molecule. The data show that the SH2 domain through the linker sequence enhances substrate phosphorylation by striking a dynamic balance among the domains in the full-length Csk.
were measured under conditions of 3 mM ATP, 5 mg ml21 of poly(Glu4Tyr), and 5 mM free Mn2þ. As shown in Table 1, mutation at Phe183 does not influence significantly the inhibitory constants for the natural product, ADP, nor the non-hydrolyzable ATP analog, AMPPNP. To test the role of Phe233 in helix aC, an alanine mutant was made (F233A) and found to have steady-state kinetic parameters that differ by less than twofold compared to wild-type Csk (Km,ATP ¼ 98ð^13Þ mM and kcat ¼ 4:5ð^0:3Þ min21). Thus, it appears that Phe183 is more important than Phe233 for domain– domain communication. Equilibrium binding of nucleotides
Steady-state kinetic parameters for wild-type and mutants
To determine whether the large effects of mutation on Km,ATP are due to destabilization of the nucleotide in the active site, a fluorescence displacement assay was employed to determine the true thermodynamic affinity of ATP.15,16 The binding of the ATP derivative, Mant-ATP, to several protein kinases leads to large increases in fluorescence emission at 440 nm.15,17 – 19 Likewise, the binding of Mant-ATP to Csk causes an increase in emission intensity at this wavelength (data not shown). As shown in Figure 2, these fluorescence
To evaluate the catalytic role of Phe183 in the SH2-kinase linker, the steady-state kinetic parameters of the mutant and wild-type enzymes were assayed using poly(Glu4Tyr) as a substrate. Initial velocities for the enzymes were measured at varying concentrations of ATP (0.01 – 4 mM) using constant amounts of poly(Glu4Tyr) (5 mg ml21) and free Mn2þ (5 mM). The steadystate kinetic parameters, kcat and Km,ATP, for mutant and wild-type Csk are displayed in Table 1. Although tyrosine replacement has little effect on substrate turnover, kcat is reduced by fourfold and 17-fold for F183A and F183G. The effects of F183A on kcat are similar to a recent report where this mutation caused a fivefold reduction in crude specific activity.14 The Km,ATP values for F183Y, F183A, and F183G are fourfold, eightfold and 44-fold larger, respectively, than that for wild-type Csk (Table 1). None of the mutations had a significant effect on the apparent affinity of the heteropolymer substrate, poly(Glu4Tyr). The Km values for this substrate range from 0.2 mg ml21 to 0.5 mg ml21 for all enzymes. The inhibitory constants for two nucleotides, ADP and AMPPNP,
Figure 2. Fluorescence changes associated with MantATP binding to wild-type Csk in the absence (X) and in the presence (W) of ATP. Relative fluorescence is measured by the difference in the emission intensity of Mant-ATP in the presence and in the absence of Csk and normalized to an endpoint of one at infinite concentration of Mant-ATP. The total concentrations of Csk and ATP are 1 mM and 25 mM. The two curves are fit to hyperbolic functions to provide Kd values of 3.2(^ 0.2) mM and 20(^ 0.8) mM.
Results
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Dynamics in Csk
changes measured with and without ATP present increase in a hyperbolic manner with the total concentration of Mant-ATP, providing a Kd (Kd,Mant-ATP) of 3.2 mM. Similar hyperbolic responses are observed with the mutant proteins and the corresponding dissociation constants are displayed along with wild-type Csk in Table 2. Overall, no significant change in Kd,Mant-ATP is observed as a function of mutation compared to the wild-type protein. To measure the Kd values of the natural nucleotides, titration curves were obtained in the presence of fixed amounts of ATP and ADP. As shown in Figure 2, the addition of 25 mM ATP increases the Kd,Mant-ATP from 3.2 to an apparent value of 20 mM. This apparent Kd (appKd,Mant-ATP) can be then used to calculate the Kd for ATP using equation (1): ½N app Kd,Mant – ATP ¼ Kd,Mant – ATP 1 þ ð1Þ Kd where [N] is the fixed concentration of the natural nucleotide and Kd is its dissociation constant. The Kd values for ADP (Kd,ADP) and ATP (Kd,ATP) were determined using this technique for wild-type Csk and the mutants and are displayed in Table 2. Unlike the large effects on Km,ATP (Table 1), mutation does not alter significantly the dissociation constants for ATP and ADP relative to wild-type. Furthermore, the addition of the Mant group does not reduce the affinity of ATP. Mant-ATP association kinetics: relaxation methods To evaluate how a stable enzyme – nucleotide complex is attained, we monitored the kinetics of Mant-ATP binding using stopped-flow fluorescence spectroscopy. In relaxation experiments, equal amounts of Csk and Mant-ATP are mixed in the stopped-flow instrument and fluorescence enhancements above 400 nm are monitored as a function of time. As shown in Figure 3(A), the association of Mant-ATP (5 mM) with wild-type Csk (0.5 mM) is associated with a rapid increase in fluorescence emission. The data are best fit by a double-exponential function with rate constants of 69 s21 and 15 s21 (residuals not shown). The amplitude of the fast phase is approximately tenfold greater than that for the second. As shown in the inset in Figure 3(A), the rate of the fast phase increases with total concentration of Mant-ATP,
Table 2. Mant-ATP and natural nucleotide dissociation constants for wild-type and mutants Parameter Kd,Mant-ATP (mM) Kd,ATP (mM) Kd,ADP (mM)
Wildtype
F183Y
F183A
F183G
3.2 ^ 0.23 4.8 ^ 0.60 1.4 ^ 0.18
2.6 ^ 0.10 4.4 ^ .070 1.8 ^ 0.26
2.9 ^ 0.20 5.5 ^ 0.76 1.1 ^ 0.28
3.3 ^ 0.72 5.4 ^ 0.33 1.0 ^ 0.11
Figure 3. Stopped-flow kinetic binding of Mant-ATP to wild-type Csk. (A) Association kinetics. Equal amounts of Csk (0.5 mM) and Mant-ATP (5 mM) are mixed and fluorescence changes above 400 nm are monitored. The data are fit to a double-exponential function to obtain rate constants of 69(^ 1) s21 and 15(^4) s21. The amplitudes for the fast and slow phases are 174(^5) mV and 17(^3) mV. The inset displays the effects of total concentration of Mant-ATP (2.5– 25 mM) on the two phases. (B) Trapping kinetics. Csk (1 mM) is pre-equilibrated with Mant-ATP (30 mM) in one syringe and then mixed with an equal volume of excess ATP (2 mM). The fluorescence change is fit to a double-exponential function with rate constants of 25(^ 1) s21 and 7(^ 1) s21. The amplitudes for the fast and slow phases are 124(^ 4) mV and 28(^1) mV.
whereas the slow phase does not vary significantly over this concentration range. The slope of the linear plot is the association rate constant ðkon Þ for Mant-ATP, whereas the y-intercept is the observed dissociation rate constant (obskoff). Similar time and ligand-dependent effects are observed with the mutants (data not shown). The values of kon and obs koff for the wild-type and mutant forms of Csk are listed in Table 3. The slow phases did not change significantly as a function of Mant-ATP concentration over the range 2.5– 25 mM and the average values (l2) are shown in Table 3. The slow phase for F183Y was too small to characterize reliably. Overall, the mutations do not affect the apparent association kinetics for the fluorescent nucleotide significantly. The ligand effects for Mant-ATP binding to wild-type Csk is consistent with a kinetic
97
Dynamics in Csk
Table 3. Stopped-flow kinetic binding parameters for Mant-ATP Parameter kon (mM21 s21) obs koff (s21) l2 (s21) trap koff (s21)
Method
Wild-type
F183Y
F183A
F183G
Relaxation Relaxation Relaxation Trap
4.9 ^ 0.3 45 ^ 4 20 ^ 5 25 ^ 1 7^4
5.0 ^ 0.20 29 ^ 3
5.0 ^ 0.2 29 ^ 3 20 ^ 10 21 ^ 2 8^5
4.6 ^ 0.5 39 ^ 9 15 ^ 5 24 ^ 1 8^2
mechanism where more than one form of the binary enzyme – nucleotide complex is populated. In this mechanism, shown in Scheme 1, two kinetic transients for ligand binding may be observed with the fast phase varying in a linear manner and the slow phase varying in a hyperbolic manner with the total ligand concentration.20 The slope of the linear phase defines the association rate constant, kon , and the maximum rate of the second phase represents the sum of the forward ðkf Þ and reverse ðkr Þ rate constants for the conformational change separating E·L and E·Lp ðl2 ¼ kf þ kr Þ. For wildtype Csk, kon is 5 mM21 s21 and kf þ kr is 20 s21 (Table 3). Furthermore, the intercept for the linear phase represents the sum of the dissociation rate constant (koff) and the forward and reverse rate constant for the conformational change ðobs koff ¼ koff þ kf þ kr Þ.20 Thus, koff can be calculated from the difference in the intercept and the maximum value for the second phase (for wild-type, koff ¼ 25 s21). Using these relationships, the dissociation constant for the formation of the initial encounter complex, E·L, is approximately 5 mM (koff/kon ¼ 25 s21/5 mM21 s21). We can then estimate the equilibrium constant for the conformational using the change ðKc ¼ kf =kr ¼ ½ELp =½ELÞ relationship Kd;Mant-ATP ¼ koff =kon =ð1 þ Kc Þ, and the experimentally determined values for Kd,Mant-ATP and koff =kon . The calculated value of 0.6 for Kc implies that E·Lp; is less populated than E·L for wild-type and, at high concentrations of MantATP, only one-third of the total protein is in the isomerized form. A similar distribution of E·L and E·Lp is calculated for F183G ðKc ¼ 0:6Þ. For F183Y, however, this calculation cannot be applied owing to a poorly defined l2 phase. For F183A, koff =kon is slightly less than Kd,Mant-ATP, making a determination of Kc impossible. In general, when koff =kon and Kd,Mant-ATP are close in value, it is difficult to define Kc with adequate precision. Mant-20 deoxyATP binding studies Since Mant-ATP is a mixture of two isomers differentiated by either 20 or 30 positioning of the mant group on the ribose ring,21,22 the binding of
Scheme 1. Kinetic mechanism for ligand binding.
21 ^ 2 6^4
Mant-20 deoxyATP was monitored to assess whether the observed fluorescence changes in Figure 3 are isomer-specific. In the stopped-flow instrument, the lowest concentration of Mant20 deoxyATP (2 mM) generated a fluorescence increase with an observed rate constant in excess of 200 s21 (data not shown), suggesting that the off rate of this nucleotide is faster than that for Mant-ATP. In equilibrium binding studies, only a lower limit of 50 mM for the Mant-20 deoxyATP Kd could be measured. Given the poor binding properties of Mant-20 deoxyATP, we conclude that the observed fluorescence changes in equilibrium and stopped-flow studies for Mant-ATP arise largely from the binding of the 20 isomer of MantATP. Mant-ATP dissociation kinetics: trapping methods To provide further evidence that two forms of Csk can bind nucleotide (Scheme 1), we measured the dissociation kinetics for Mant-ATP to wildtype and mutant forms using a stopped-flow trapping experiment. In this experiment, Csk (1 mM) is pre-equilbrated with Mant-ATP (30 mM) in one syringe and then mixed 1 : 1 (v/v) with excess ATP (2 mM) from the second syringe. As shown in Figure 3(B), the addition of excess ATP to the Csk : Mant-ATP complex leads to a time-dependent decrease in fluorescence that is best fit by a double-exponential function with rate constants of 25 s21 and 7 s21. Increasing the ATP by 50% does not change these rate fits, indicating that the kinetic transient reflects the dissociation kinetics of Mant-ATP from Csk (data not shown). Similar time-dependent fluorescence changes occur with the mutants, and the individual rate constants for all proteins are listed in Table 3. There is no convincing evidence that the faster phase in these trap experiments reflects the dissociation rate constant for the poorly bound 30 Mant isomer. The release rate for this isomer is expected to be more than tenfold greater than the faster trap phase on the basis of the observed association rate for Mant-20 deoxyATP at the lowest concentration (. 200 s21 at 2 mM). Therefore, the presence of biphasic dissociation kinetics imply that two forms of the Mant-ATP : Csk complex are populated in solution for both wild-type and mutants, a result consistent with the relaxation kinetics.
98
Deuterium incorporation kinetics To determine whether mutation in the SH2kinase linker impacts solution conformation, we measured the incorporation kinetics of solvent deuterium into probes within the Csk structure using DXMS methods.13 Similar pepsin digestion patterns ($ 90% identical) were observed for the mutants and wild-type Csk, greatly facilitating data interpretation in H –D exchange experiments. For the deuterium incorporation studies, the average mass of each probe was calculated by integrating over the full envelope of peaks. Deuterium incorporation into the Csk probes was followed as a function of time and the extent of incorporation was assessed at various time-points by converting the mass of each probe into a number of in-exchanged deuterons.13 This method detects the overall mass change for each probe without assigning the priority of amide exchange within each peptide probe. Figure 4 illustrates the time-dependent incorporation of deuterium into several probes. In many instances, mutation in the SH2-kinase linker has no effect on the timedependent incorporation of deuterium relative to wild-type (e.g. F183G in Figure 4(B) and (C); F183Y in Figure 4(D) and (E)). Probes that fall into this category have no significant structural effect in those regions. By comparison, the mutants alter deuterium incorporation relative to wild-type in several probes (e.g. F183Y in Figure 4(A) –(C) and (F); F183A in Figure 4(C); F183G in Figure 4(A), (D) –(F))†. These probes correspond to regions where changes in backbone flexibility occur as a function of mutation. To illustrate the regions influenced by mutation, the available probes suitable for analysis were displayed on the Csk X-ray structure (Figure 5) and coded by color to define regions that are unaffected (yellow), more dynamic (red), or less dynamic (blue) relative to wild-type. Using this criterion, replacement of Phe183 with tyrosine, alanine, and glycine can be compared readily. For example, the most conservative mutation, F183Y, elicits changes in the SH3 and SH2 domains, the glycine-rich loop, the activation loop, and helices aG/H and aI/J in the large lobe of the kinase domain. Probes in these regions show increases in the rate of deuterium exchange compared to wildtype Csk corresponding to increases in flexibility in these regions. This mutant also has a probe in the SH3 domain that shows a decrease in the rate of deuterium exchange and, therefore, a decrease in flexibility in that region. F183A contains probes in both the SH3 domain and the small lobe of the kinase domain that show a decrease in the rate of
† In general, a plot of deuterons versus time for a mutant is considered different from that for wild-type if the mass of the probe differs from wild-type by 0.2 deuteron or more for, at least, two time-points and the plot defines a kinetic trend in the data.
Dynamics in Csk
deuterium exchange and a decrease in flexibility in these regions. This mutant contains probes in the kinase domain (glycine-rich loop, helix aH, and helix aI/J) that experience an increase in the rate of exchange and an increase in flexibility. On average, F183A has fewer regions colored red than F183Y. F183G displays changes in the SH3 domain, the SH2 domain (helix aA), and the kinase domain (catalytic loop, helices aC, aG/H, and aI/J). Unlike F183Y and F183A, all of these probes show a decrease in the rate of deuterium exchange, implying that the affected regions experience reduced flexibility compared to wild-type. Overall, these data demonstrate that mutation of Phe-183 in the SH2-kinase linker influences the structural flexibility of the protein in solution, although each mutant appears well folded, since major regions in all three domains experience exchange patterns that are indistinguishable from those for wild-type (yellow regions in Figure 5). Computational analysis Normal mode analysis (NMA) is a computational method that captures dynamic properties of biological systems. Here, we used NMA to examine the dynamic properties of Csk. Although NMA is based on the harmonic approximation, the resulting normal modes are able to capture the preferential dynamic direction of the protein molecules. Among normal modes inherent to the protein, a few of the lowest-frequency normal modes are often representative of the large conformational rearrangements.23 With Csk, the lowest-frequency normal mode shows large correlated displacements of all three domains with correlated motions between the SH2 and kinase domains (Figure 6(A)). Effects of conformational fluctuation on the local environment around each of the residues were quantified using strain energy analysis.24 This analysis reveals that Phe183 is under high strain along the lowest-frequency normal mode, as seen in Figure 6(B), which shows the strain energy associated with conformational fluctuations for Csk. High strain energy indicates that the local environment of the residue is correlated to the global dynamics of the protein. Most of the strain is localized in the small lobe of the kinase domain, with a few effects in the large lobe of the kinase and SH2 and SH3 domains.
Discussion The catalytic activities of nrPTKs are commonly influenced by neighboring SH2 and SH3 domains that lie on the same polypeptide chain as the kinase domain.25 To establish an experimental connection between catalytic phenomena in the active site of the kinase domain and the neighboring SH2 and SH3 domains in Csk, we previously utilized H – D exchange studies to show that structural information from the nucleotide pocket
Dynamics in Csk
99
Figure 4. Time-dependent incorporation of deuterium into several probes in wild-type and mutant forms of Csk. The probes are colored for identification: black, wild-type; red, F183Y; green, F183A; and blue, F183G. The total number of deuterons is plotted against time (minutes) for the following peptide fragments: (A) A6-C14 (SH3 domain), (B) S139E143 (SH2 domain), (C) L195-M210 (glycine-rich loop), (D) T241-L248 (near helix aC), (E) F310-N319 (catalytic loop), and (F) W377-Y403 (helix aG). Data were recorded over 360 minutes of incubation with 2H2O and only relevant timeframes are displayed for comparison. Replicate studies established that the measurements of deuterium incorporation performed with the automated exchange data acquisition apparatus had standard deviations of 2.5% or less (data not shown).
travels across the protein framework to regions in the SH2 domain (e.g. helix aA) through helix aC and the SH2-kinase linker (Figure 1(A)). The recent X-ray structure of the apoenzyme form of Csk now provides a molecular template describing how these two polypeptide regions interact on an atomic level (Figure 1(B) and (C)). A phenylalanine residue at the interface of the SH2-kinase linker
and helix aC appears to couple these important structural regions. The origin of these long-range effects induced by the nucleotide in the pocket is likely to involve a conserved lysine residue that contacts directly the phosphate groups of ATP, based on other protein kinase structures.26,27 This lysine participates in an electrostatic interaction with a conserved glutamate residue in helix aC.9
100
Dynamics in Csk
Figure 5. H– D exchange probes for Phe183 mutants mapped onto the X-ray of Csk. Probes in yellow display the same deuterium incorporation profiles as that for wild-type and are, thus, unaffected by mutation at Phe183. Probes in blue are less dynamic and display slower deuterium incorporation relative to wild-type. Probes in red are more dynamic and display faster deuterium incorporation relative to wild-type. These structures are rotated by 1808 relative to the structure in Figure 1(B). The SH2 domain is now on the right-hand side and the SH3 domain is on the left-hand side of the central kinase domain.
Here, we investigate how the SH2-kinase linker, through a key hydrophobic interaction with this helix, influences the catalytic mechanism and solution conformation of Csk. Functional disconnection of the non-catalytic and kinase domains Although Phe183 does not make any direct contact with the active site (Figure 1(B)), replacement of this residue has profound effects on catalytic activity. Indeed, the glycine substitution (F183G) lowers catalytic efficiency by nearly 1000fold (i.e. 2kcat =Km;ATP ; Table 1). This position appears to be exquisitely sensitive to the size of the side-chain, since tyrosine and alanine substitutions produce intermediate reductions in kcat =Km;ATP (fivefold and 30-fold). The observed decreases in catalytic efficiency are due to both reductions in the apparent affinities of ATP
ðKm;ATP Þ and the turnover numbers ðkcat Þ for the mutants. Nonetheless, on the basis of two observations, these impressive changes are not thought to be the result of unfolded or misfolded protein. First, the mutations lower catalytic efficiency partly by affecting Km,ATP. Mutations that destabilize structure and cause unfolding are expected to affect exclusively kcat, a steady-state kinetic parameter that, unlike Km,ATP, is determined from the concentration of native protein. Second, the H – D exchange results indicate that mutation does not destabilize large protein regions (Figures 4 and 5). Thus, mutation of a critical interdomain residue can have long-range effects on the kinase active site without altering tertiary structure significantly. These results suggest that a previous report of low activity for F183A is not the result of improper protein folding in the mammalian expression system, rather a change in critical cross-talk pathways.14 Finally, since the catalytic
Figure 6. Computational analysis of Csk with Phe183 shown in cyan. (A) Csk dynamics obtained from NMA. The displacements of Ca atoms along the lowest-frequency normal mode are shown as arrows. Csk is colored by secondary structure: helices (pink), strands (yellow), and loops (light blue). (B) Strain energy due to conformational fluctuations. The structure is colored according to the dynamics of the lowest frequency mode: residues in blue are under no strain, residues in red are under high strain, and residues in white are intermediate. The Figure was prepared with VMD35 and Raster3D.36
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Dynamics in Csk
efficiency of F183G is similar to that for a previously published Csk deletion lacking the SH2 domain,3 we conclude that a single-residue substitution functionally disconnects the noncatalytic domain and the kinase domain. These results support our model for interdomain communication derived from previous H –D exchange experiments (Figure 1(A)). Domain interactions do not influence nucleotide stability in the active site Although replacement of Phe183 increases the Km for ATP greatly, equilibrium and kinetic studies suggest that the mutations impact neither the relative ground state energy of the enzyme – nucleotide complex compared to free enzyme nor the mechanism of ATP binding. Both ATP and ADP bind equally well to wild-type Csk and the mutants, based on fluorescence displacement assays (Table 2). The concentration effects for Mant-ATP association are consistent with a mechanism where the initial encounter complex (E·L) proceeds through a conformational change to an altered, but weakly populated, species, E·Lp (Scheme 1). The relative population of these species in F183G is equivalent to that for wildtype. Whether the mechanism for ATP binding is similar to that for Mant-ATP, an unnatural nucleotide, is not certain. While it is conceivable that the presence of the methylanthraniloyl fluorophore on the ribose ring may influence the binding mechanism, the similar binding affinities of ATP and Mant-ATP (Table 2) suggest that the additional reporter group may not have a profound influence when attached at the 20 position. However, the poor binding of Mant-20 deoxyATP indicates that the Mant group is not accommodated well in the nucleotide pocket when attached to the 30 hydroxyl group. Overall, the data indicate that disruptive mutations in the SH2-kinase linker do not impact the affinity of ATP and, considering the above caveat, may not significantly alter the mechanism by where the nucleotide gains access to the pocket. Kinetic versus thermodynamic affinities in wild-type Csk The substrate affinity for an enzyme can be expressed either in terms of Km , an apparent dissociation constant measured from the substrate dependence on initial velocity, or in terms of Kd , a thermodynamic dissociation constant measured in the absence of catalysis. These values may be nonequivalent, an outcome that provides critical information on the catalytic mechanism of protein
Scheme 2. Kinetic mechanism for substrate phosphorylation.
kinases.28 For wild-type Csk, the Km for ATP is higher than the Kd of the nucleotide by approximately tenfold (Tables 1 and 2). To understand the source of this difference, we consider the simple phosphorylation mechanism in Scheme 2. Here, the Km for ATP at high concentrations of substrate can be related to the individual rate constants by equation (2): Km ¼
k4 k21 þ k3 £ k1 k3 þ k4
ð2Þ
For wild-type Csk, the Km expression can be simplified to: Km ¼ k4 ðk21 þ k3 Þ=k1 k3 since it has been shown using pre-steady-state kinetic experiments that k3 . k4 .12 While a direct measure of k21 is unavailable, based on the release rate for Mant-ATP (7 s21; Table 3) and lower limit on the phosphoryl transfer rate (. 100 s21),12 it reasonable to assume that k21 , k3 . Under these constraints, the Km expression further reduces to Km ¼ k4 =k1 . Thus, the Km for ATP can be higher than Kd , since Kd is not expressed in the Km term under the kinetic parameters of the wild-type mechanism. Csk utilizes a two-channel catalytic mechanism While the mechanism in Scheme 2 can explain the variance between Km,ATP and Kd,ATP for wildtype Csk, it cannot explain the large increases in Km,ATP when Kd,ATP is low and unaffected for the mutants. To overcome this dilemma, a second form of Csk is introduced into the mechanism in Scheme 3 (starred complexes). While the upper channel is largely unproductive owing to a slow phosphoryl transfer rate, the enzyme in this pathway still possesses good ATP-binding properties. In contrast, while the lower channel is highly productive owing to a fast phosphoryl transfer step ðk3 . k4 Þ, ATP-binding affinity could be comparatively weaker ðk21 =k1 . p k21 =p k1 Þ. Given the unaffected binding parameters for ATP and ADP, it is reasonable to assume that the large reductions in kcat for the mutants are the results of reduced rates of phosphoryl transfer. Thus, the elevated Km values for the mutants can arise from reduced values of k3 so that Km,ATP approximates k21 =k1 rather than k4 =k1 as in wild-type. An unaffected, low value for Kd,ATP is maintained within the
Scheme 3. Dual channel mechanism for substrate phosphorylation.
102
mutants because the upper channel binds the nucleotide with high affinity but does not participate appreciably in catalysis. While the two-channel mechanism in Scheme 3 adequately accounts for the unusual steady-state kinetic effects of mutation, three independent observations provide further support for this model. (1) Csk has been crystallized in two distinct forms that differ by rotation of the SH2 domain about the SH2-kinase linker.9 The authors of that report suggest that only one of these forms is catalytically viable. (2) The amplitude of the presteady-state “burst” phase for Csk using a peptide substrate does not exceed 20% of the total protein concentration.12 This finding suggests that two forms of the enzyme exist in solution. The weakly populated form is highly active and generates a rapid burst of phophorylated material in the first 100 ms of the reaction, while the more populated form would be undetectable in the pre-steadystate owing to slow phosphoryl transfer. (3) The stopped-flow binding studies presented here show two forms of Csk bound to Mant-ATP (Figure 3). The equilibrium distribution of these two forms is approximately the same as the distribution of the active and inactive forms in the pre-steady-state. Finally, it is very likely that other kinases utilize a multi-channel catalytic mechanism. For example, the burst amplitude for the tyrosine kinase Her-2 is similar in value to that for Csk, suggesting that only one of the two enzyme forms is productive.18 Long-range effects modulated by the SH2-kinase linker In a previous report, we showed that a helix in the SH2 domain (aA) and a linker region separating the SH2 and kinase domains of Csk undergo dynamic changes upon the binding of ATP.13 These studies suggest that occupancy of the active site induces changes in Csk that emanate from the nucleotide pocket to distal regions in the SH2 domain through the SH2-kinase linker (Figure 1). The kinetic data presented here now supply evidence for this pathway and indicate that Phe183 in the linker provides a functional connection for this inter-domain communication. To determine whether mutations in this important linker residue can influence solution dynamics, deuterium incorporation experiments were performed (Figure 4). Generally, substitutions at Phe183 lead to both increases and decreases in H – D exchange in several polypeptide regions. Most notably, mutation causes changes in the SH2 domain in two mutants (F183G and F183Y) and changes in the large lobe in all mutants studied. Both of these regions are sensitive to ATP binding, based on the results of a previous H – D exchange study.13 In addition to these common regions, mutation alters exchange in the SH3 domain in all three mutants. These effects occur far from the site of mutation, reflecting alterations in structural dynamics within the Csk framework. While it is
Dynamics in Csk
not possible to decipher the molecular nature of these effects at this time, their detection indicates that the linker is important for communicating structural information within the Csk framework. Furthermore, while Phe183 plays a significant role, other residues in the SH2-kinase linker could play a cooperative role in the communication pathway. The H –D exchange and kinetic results indicate that Phe183 is located near a critical region for interdomain regulation. In a recent study, adenylate kinase was used in NMA to illustrate global structural transformations in biological machines.24 To understand the global forces within the Csk framework that might account for the effects of mutation, we used NMA to determine the role of physical mechanics in conformational dynamics. The amplitudes of fluctuation of atoms along the lowest-frequency normal mode are shown by arrows in Figure 6(A). This mode involves large correlated motions between the SH2 and kinase domains. Although Phe183 does not show a large displacement along this mode, it is located at a hinge region between the flexible domains (i.e. a critical region for the conformational dynamics of the protein) and mutation in this site could largely influence the dynamic properties of the molecule, as was proposed in a study of the ribosome.29 Analysis from strain energy reveals that Phe183 is under high strain along the lowest-frequency normal mode, which indicates that mutation of this residue could affect the mode (Figure 6(B)). Interestingly, this mode induces strain in regions where changes in the rates of H – D exchange have been observed (e.g. glycine-rich loop, helix aC, and activation loop), thus, further supporting long-range communications between Phe183 and the affected regions. Similar effects are seen for other low-frequency normal modes. SH2-kinase linker maintains a dynamic balance in Csk The H –D exchange data presented here show that a single residue substitution can impact regions distal from the immediate locus of the mutation. The observed changes provide an interesting example of how domain– domain interactions control flexibility at specific points in the kinase scaffold. In Figure 5, we summarize the effects of mutation on key probes throughout the Csk structure and express these differences in terms of changes in flexibility compared to wildtype (red, increased flexibility; blue, decreased flexibility). F183G possesses both the lowest catalytic activity and the lowest level of flexibility, suggesting a possible correlation between poor catalysis and reduced dynamics. Replacement of Phe with a less disruptive residue Ala (F183A) leads to a mutant with greater flexibility and activity compared to F183G. While these findings suggest that further increases in flexibility beyond that observed in the Ala mutation may cause more increases in catalytic efficiency, replacement of Phe
103
Dynamics in Csk
with Tyr (F183Y) has the opposite effect. Indeed, the subtlest mutation, F183Y, leads to increases in flexibility beyond that of wild-type, yet the catalytic parameters for this mutant are lower (Table 1). Thus, the biophysical data show a nonlinear correspondence between catalytic efficiency and structural dynamics. We believe that these findings suggest that Csk requires a discrete level of flexibility for optimum catalysis, and that the SH2 domain through the linker provides this appropriate dynamic balance. Adaptor proteins such as Cbp bind to the SH2 domain and up-regulate Csk function.30,31 The data presented here suggest that this biomolecular interaction may occur through a transmission of dynamic effects from the SH2 domain to the active site of the kinase domain. It will be interesting to see whether Cbp and other adaptors proteins alter the flexibility of these species or the distribution of active and inactive conformers in the two-channel catalytic mechanism.
Materials and Methods Materials Adenosine diphosphate (ADP), adenosine triphosphate (ATP), 50 -adenylyl-imidodiphosphate (AMPPNP), poly(Glu4Tyr), thrombin, glutathione, glutathione immobilized on 4% (w/v) beaded agrose, and Mops were obtained from Sigma. MnCl2, EDTA, formic acid and liquid scintillant were obtained from Fisher. Micro Bio-Spin chromatography columns were obtained from Bio Rad. 2H2O (99% deuterium) was purchased from ISOTEC Inc. H-2H-Phe-Pro-Arg-chloro-methylketone trifluoroacetate salt (p-PACK) was obtained from Bachem. The fluorescent probe 20 -(30 )-O-(N-methylanthraniloyl)ATP trisodium salt (Mant-ATP) was purchased from Molecular Probes. [g-32P]ATP was obtained from NEN Products. Mant-20 deoxyATP was synthesized from N-methylisatoic anhydride and 20 -deoxyATP and purified as described.21 Enzyme production A cDNA containing human Csk in the pGEX-ST vector was a generous gift from Dr Gongqin Sun and was used as a template to generate mutant Csk constructs: F183G, F183A, and F183Y. Mutations were introduced directly into the cDNA using the QuickChange site-directed mutagenesis method (Stratagene). Mutations were detected by restriction enzyme digestion and the nucleotide sequences were confirmed by doublestranded sequencing of the relevant regions. Mutant proteins were expressed in Escherichia coli as GST-fusion proteins and purified as described.12 Briefly, fusion proteins were cleaved with thrombin at 25 8C for 90 minutes followed by treatment with p-PACK for 30 minutes to inactivate thrombin. Cleaved products were separated using a Q-Sepharose column (Amersham Biosciences) with a 50 – 300 mM KCl gradient. The Csk proteins were concentrated and dialyzed into two separate buffers, one containing 25 mM potassium phosphate (pH 7.5) and the other containing 50 mM Tris – HCl (pH 7.5) and 50 mM NaCl. The proteins were stored at
280 8C in small aliquots and used in experiments without glycerol. Radiochemical assay Steady-state kinetic parameters were measured using poly(Glu4Tyr) as a substrate in 50 mM Mops (pH 7.0). The enzyme was first pre-equilibrated with [g-32P]ATP (600– 1000 cpm pmol21) and 5 mM free Mn2þ for two minutes before reaction initiation with poly(Glu4Tyr). Initial velocities were determined after two minute reaction periods at 23 8C. The reactions (20 ml) were quenched with 40 ml of 75 mM EDTA. A portion (50 ml) of each reaction was then applied to a Micro Bio-Spin Chromatography Column. The column was centrifuged at 4200 rpm for two minutes and the collected flowthrough containing phosphorlyated poly(Glu4Tyr) was then counted on the 32P channel in liquid scintillant. Control experiments were performed to determine the background phosphorylation (i.e. phosphorylation of poly(Glu4Tyr) in the presence of quench). The specific activity of [g-32P]ATP was determined by measuring the total counts of the reaction mixture. The time-dependent concentration of phosphorylated poly(Glu4Tyr) was then determined by considering the total counts per minute (CPM) of the flow-through, the specific activity of the reaction mixture, and the background phosphorylation of the flow-through. Equilibrium fluorescence measurements Fluorescence experiments were performed on a Fluoromax-2 (Jobin Yvon-SPEX, Instruments S. A., Inc. Edison, NJ) spectrofluorimeter equipped with a circulating waterbath. Mant-ATP binding was detected by a fluorescence increase at 440 nm in the presence of Csk (0.5 mM) at an excitation wavelength of 293 nm (excitation and emission slit widths were set at 1 nm). Mant-ATP titrations were carried out using a 1 ml cuvet in 50 mM Mops (pH 7.0). Microliter amounts of MantATP were titrated into a 1 ml sample from a 5 mM stock solution and emissions spectra were monitored between 300 nm and 500 nm. A control titration lacking Csk was carried out for each Mant-ATP titration in the presence of the protein. To measure ADP and ATP dissociation constants, Mant-ATP titrations were carried out, as described above, in the presence of 10 mM ADP and 25 mM ATP. DXMS instrumentation Equipment configuration for these studies has been described, except for the following changes†.13In-line filters (0.5 mm PEEK filter end fitting, Upchurch cat. no. A-428X) were placed on each side of the pepsin column and just before the C18 column (0.5 mm PEEK filter frit, Upchurch cat. no. A-735X) to minimize column fouling and carryover of aggregated material. Temperature control was maintained by storing valves, tubing, columns, and autosampler within a refrigerator maintained at 2.8 8C, with columns also immersed in ice. † Woods V. Jr (2001). Methods for the high-resolution identification of solvent-accessible amide hydrogens in polypeptides or proteins and for the characterization of the fine structure of protein binding sites. In US Patent #6,291,189. Carta Proteonomics, Inc., USA.
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Mass spectrometric analyses were carried out with a Finnigan LCQ electrospray ion trap type mass spectrometer (Thermo Finnigan) with capillary temperature at 200 8C or an electrospray Micromass (Manchester, U.K.) Q-TOF mass spectrometer with cone voltage at 60 V, capillary voltage at 3200 V, source block temperature at 80 8C and desolvation temperature at 250 8C.
Dynamics in Csk
complete exchange of the backbone amide protons for deuterons. To assess the amount of label lost during the experiment (i.e. back exchange), the deuterated sample was treated with quench solution and analyzed by ESIMS. This control provides, for each peptide, the maximal experimental mass that relates to a fully deuterated peptide. Stopped-flow fluorescence measurements
Operational procedures—DXMS Protein samples were handled as described.13 However, in these experiments, digested protein samples (100 ml, stored at 280 8C) were melted individually by hand in melting ice over 1.5 minutes (keeping the temperature at 0 8C) and then injected (95 ml) into the solvent stream flowing over the pepsin column. Digestion optimization was determined previously for wild-type Csk,13 and applied to the mutants. To identify pepsingenerated peptides present in the LC eluate for each Csk form, spectra were acquired on the Q-TOF mass spectrometer in a “double-play” data acquisition mode consisting of four sequential scan events performed repeatedly over the 32 minutes of chromatographic elution: (1) an initial MS1 scan (mass range 200– 2000 m/z) followed by; (2) collision-induced dissociation (CID) on the most intense ion present in the spectrum of the preceding MS1 scan, with collection of spectral data on the generated fragments (MS2); (3) CID performed on the second most prevalent ion in (1) with MS2 data acquisition; and (4) CID performed on the third most prevalent ion in (1) with MS2 data acquisition. The three parent ions subjected to CID in each cycle are placed on an exclusion list lasting 30 seconds. The double play data set was then analyzed employing the Sequest software program (Thermo Finnigan Inc) to identify the sequence of the dynamically selected parent peptide ions. This tentative peptide identification was verified by visual confirmation of the parent ion charge state presumed by the Sequest program for each peptide. This set of peptides was then further examined to determine if the “quality” of the measured isotopic envelope of peptides was sufficient to allow accurate measurement of the geometric centroid of isotopic envelopes on deuterated samples. Deuterium exchange experiments Samples for H – D exchange were prepared as described.13 Briefly, all exchange mixtures for wild-type and mutant Csk contained 9.8 nM protein, 18 mM Mops, 45 mM NaCl, and 0.9 mM DTT with a final pH of 7.0 and percentage 2H2O of 90%. Deuterium exchange was initiated by the addition of 40 ml of protein solution in H2O to 360 ml of buffer solution in 2H2O on ice. At designated time intervals, 40 ml aliquots were removed and added to a 60 ml quench solution containing 0.8% formic acid and 0.8 M GuHCl. Quenched samples were flash-frozen in liquid nitrogen and placed in solid CO2 prior to electron spray ionization mass spectroscopy (ESI-MS) analysis. The in-exchange and back-exchange controls were performed as described.13 In-exchange under quench conditions was measured by adding a protein solution directly to a mixture of quench and 2 H2O solutions. This sample corresponds to time-point zero. The back-exchange was determined by incubating wild-type and mutant Csk in 2H2O containing 0.5% (v/v) formic acid overnight at 25 8C. This allows for the
All transient kinetic measurements were made using an Applied Photophysics stopped-flow spectrometer. Samples were mixed 1 : 1 (v/v) in the instrument cuvet using two identical 2.5 ml syringes. The excitation wavelength was 290 nm and fluorescence emission was measured using a 420 nm cut-on filter. The instrument collected a total of 400 datum points in each experiment and, in some cases, the data were separated with 200 points for the short time-frames and 200 points for the longer time-frames. For data analysis, the average of five to ten individual traces was used. The fluorescence data were recorded in units of millivolts (mV). All fluorescence measurements were made in buffers containing 50 mM Mops (pH 7.0) and 5 mM free Mn2þ. Normal mode analysis Normal mode analysis was performed on the active form of Csk32 using the simplified energy function, Tirion potential,33 in conjunction with the RTB method.34 In NMA, the potential energy is approximated as a harmonic function around a minimum energy conformation, therefore allowing the dynamic properties of biological systems to be described as a set of independent harmonic oscillators (i.e. the normal modes). The strain energy was calculated as described.24
Acknowledgements This work was supported by NIH (GM68168) and NSF grants (111068) to J.A.A., NSF grants PHY-0216576, 0225630 and MCB-0084797 to J.O., an NIH grant (CA099835) to V.L.W., an NIH grant (DK55541) to P.A.J. and by grants from the University of California BioStar and LSI programs, grants S97-90, S99-44, L98-30 to V.L.W., with the matching corporate sponsor for these grants being ExSAR Corporation, Monmouth Junction, NJ. V.L.W. has an equity interest in ExSAR Corporation. L.W. was supported by an NIH/NCI NRSA (T32 CA09523) grant and S.L was supported by the Heme and Blood Proteins training grant. We thank Dr Gongqin Sun for the gift of the wild-type Csk plasmid. The authors thank the W.M. Keck foundation for computational support and Dr Peter Wolynes for discussions on the development of strain energy analysis.
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Edited by I. Wilson (Received 10 February 2004; received in revised form 26 May 2004; accepted 26 May 2004)
