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The cellular CT10 regulator of kinase protein (c-CrkII) is a central adapter molecule in signaling pathways that propagate extracellular activation fr...
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Molecular Determinants of a Regulatory Prolyl Isomerization in the Signal Adapter Protein c‑CrkII Philipp A. M. Schmidpeter and Franz X. Schmid* Laboratorium für Biochemie und Bayreuther Zentrum für Molekulare Biowissenschaften, Universität Bayreuth, 95440 Bayreuth, Germany S Supporting Information *

ABSTRACT: The cellular CT10 regulator of kinase protein (c-CrkII) transmits signals from oncogenic tyrosine kinases to cellular targets. Nuclear magnetic resonance studies had suggested that in chicken c-CrkII a native state prolyl cis− trans isomerization is involved in signal propagation. Corresponding evidence for the closely related human cCrkII was not obtained. Here we analyzed the kinetics of folding and substrate binding of the two homologues and found that cis−trans isomerization of Pro238 determines target binding in chicken but not in human c-CrkII. A reciprocal mutational analysis uncovered residues that determine the isomeric state at Pro238 and transmit it to the binding site for downstream target proteins. The transfer of these key residues to human c-CrkII established a regulatory proline switch in this protein, as well. We suggest that Pro238 isomerization extends the lifetime of the signaling-active state of c-CrkII and thereby functions as a long-term molecular storage device.

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thus assumed to act as a molecular switch that controls signal transduction by c-CrkII. The structure of the human c-CrkII protein was determined by NMR spectroscopy, as well,6 but surprisingly, evidence for a cis−trans equilibrium at Pro238 was not obtained. Human and chicken c-CrkII are closely related in sequence and structure, and therefore, it appears to be surprising that they might use different molecular principles to regulate the transition between the active and autoinhibited forms. To determine whether the two c-CrkII proteins differ in their regulatory mechanisms, we examined the role of Pro238 in their function. The analysis of folding and substrate binding revealed that binding of the substrate to the SH3N domain is governed by Pro238 isomerization in the SH3C domain of chicken but not of human c-CrkII. A reciprocal mutational analysis uncovered the residues that are critical for the regulatory link between Pro238 isomerization and substrate binding in chicken c-CrkII and allowed us to create a corresponding proline switch in the human homologue, as well.

he cellular CT10 regulator of kinase protein (c-CrkII) is a central adapter molecule in signaling pathways that propagate extracellular activation from oncogenic tyrosine kinases to cellular targets.1−4 Many human cancers are accompanied by the overexpression of Crk proteins.1,5 cCrkII consists of three domains: the amino-terminal SH2 domain, which interacts with phospho-Tyr-containing proteins, the SH3N domain, which binds to proline-rich ligands, and the SH3C domain, which controls the binding activity of SH3N and an extended linker between the two SH3 domains.6−10 c-CrkII is supposed to exist in two conformations. In the open, active form, the two SH3 domains are disassembled and the substrate binding site on SH3N is accessible to communicate signals to downstream target proteins. In the closed, autoinhibited form, the SH3C domain associates with SH3N and occludes its binding site.6,7 Crk was originally discovered as the oncogene product v-Crk of the chicken CT10 sarcoma virus.1 v-Crk lacks the modulatory SH3C domain, and therefore, it is permanently active and promotes fast tumor growth. The disassembly of the two SH3 domains is thus thought to switch c-CrkII between the closed autoinhibited and the signaling-active conformation.7,9,11 Pro238 is located at the end of the inter-SH3N−SH3C linker, at the beginning of the SH3C domain. In the nuclear magnetic resonance (NMR) structure of chicken c-CrkII, this proline was found in two conformations, cis and trans, and it was suggested that it controls the switching between the active and autoinhibited forms of c-CrkII.7,9,11 In this model, the cis isomer of Pro238 allows SH3N and SH3C to interact with each other to form the autoinhibited state, whereas the trans isomer interferes with domain interaction and opens the substrate binding site of the SH3N domain. Pro238 is © 2014 American Chemical Society

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RESULTS AND DISCUSSION The Folding Kinetics of Human and Chicken c-CrkII Differ. All models for the function of c-CrkII suppose that autoinhibition is mediated by the docking of the two SH3 domains, which closes the substrate binding site of the SH3N domain.6,7,11 Thermodynamic linkage requires that such a Received: January 2, 2014 Accepted: February 26, 2014 Published: February 26, 2014 1145

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docking reaction stabilizes the participating domains and protects them from unfolding. Therefore, we produced the SH3N−SH3C two-domain fragment of human and chicken cCrkII, as well as the individual domains SH3N (residues 126− 199, according to the numbering of chicken c-CrkII) and SH3C (residues 199−305), which also comprises the linker region. For both proteins, SH3N is more stable than SH3C, and the transitions of the two-domain proteins lie between those of the individual domains (Figure 1). Thus, there is no evidence of stabilizing interactions between the SH3N and SH3C domains in the two Crk proteins (Table S1 of the Supporting Information).

Figure 1. Stabilities of the human and chicken c-CrkII variants. GdmCl-induced equilibrium unfolding transitions of SH3N (□), SH3C (△), the SH3N−SH3C fragment (●), and the SH3N−SH3C P238A fragment (red circles) of (a) the human protein at 1 μM and (b) the chicken protein at 1 μM at 15 °C in 0.1 M potassium phosphate (pH 7.4). The fluorescence was measured at 330 nm after excitation at 280 nm. The parameters obtained from the analysis according to a twostate model are listed in Table S1 of the Supporting Information.

Figure 2. Unfolding and refolding kinetics of the human and chicken c-CrkII variants. Normalized refolding kinetics of SH3C (black), SH3C P238A (red), and SH3N (dashed line) are shown for (a) the human protein and (b) the chicken protein. Refolding was initiated by stopped flow (SH3C) or manual (SH3N) dilution from 4.0 to 0.5 M GdmCl at 15 °C. The rate constants of unfolding (empty symbols) and refolding (filled symbols) as a function of the GdmCl concentration are given in panel c for the isolated human SH3C domain and in panel e for the human SH3N−SH3C fragment. Data for the P238A variant are colored red. The corresponding data for the chicken proteins are shown in panels d and f, respectively. In panel d, the second refolding branch of chicken SH3C is shown as squares. Squares and triangles in panels e and f depict the rate constants for SH3N and SH3C, respectively. Kinetics were measured at 1 μM protein in 0.1 M potassium phosphate (pH 7.4) at 15 °C by the fluorescence at 330 nm (3 nm bandwidth) after excitation at 280 nm after manual mixing or by the fluorescence above 320 nm after stopped-flow mixing. The rate profiles were analyzed according to a two-state model. The parameters from the analysis are summarized in Table S2 of the Supporting Information.

In the equilibrium transitions of the SH3N−SH3C twodomain proteins (Figure 1), the contributions of the individual domains to the decrease in protein fluorescence could not be discriminated. However, SH3C folds and unfolds at least 10-fold faster than SH3N (Figure 2a,b), and therefore, the two domains could be analyzed individually. The unfolding and refolding kinetics of SH3N (Figure S1 of the Supporting Information) and SH3C (Figure 2c,d) consist of denaturant-dependent conformational folding reactions, which lead to chevron-type rate profiles, and of a slow, denaturant-independent reaction with a rate near 0.01 s−1. It originates probably from prolyl isomerization in SH3N. The folding kinetics reveal a difference between the human and chicken SH3C domains. The refolding of chicken SH3C (Figure 2b,d and Figure S2 of the Supporting Information) shows a second, slower conformational folding phase, which is absent in the refolding of human SH3C (Figure 2a,c). In the SH3N−SH3C fragment, the two domains show the same refolding and unfolding kinetics and the same chevrontype rate profiles as the isolated domains (Figure 2c−f and Figure S1 of the Supporting Information). The two domains of the SH3N−SH3C fragment are thus independent of each other and not coupled in terms of stability and folding kinetics. The second conformational folding reaction of the SH3C domain could not be resolved in the two-domain chicken protein (Figure 2f) because it coincides with the refolding reaction of the SH3N domain. The two-state analysis of the folding kinetics (Table S2 of the Supporting Information) confirms that the domains fold independently and that the human SH3C domain is more stable than chicken SH3C. Evidence of a separate

docking reaction after domain folding could not be obtained from the folding kinetics of the two-domain proteins. Replacement of Pro238 Abolishes the Slow Conformational Folding Reaction of Chicken SH3C. To reveal the contribution of Pro238 to the folding kinetics, we replaced it with Ala in the human and chicken SH3N−SH3C proteins, as well as in the isolated SH3C domains. The P238A substitution left the slowest folding reaction unchanged (Figure 2), suggesting that this reaction originates from Pro residues other than Pro238. It abolished, however, the second conformational folding reaction of chicken SH3C (Figure 2d). The folding kinetics of human SH3C was not affected by the P238A replacement (Figure 2c). We conclude that Pro238 introduces kinetic heterogeneity into the conformational folding of chicken but not human SH3C. In the P238A variant, the Gly237−Ala238 peptide bond is presumably trans, because non-prolyl cis peptide bonds are energetically highly unfavorable.12,13 The unchanged folding kinetics of human SH3C thus suggests that the 237−238 peptide bond is trans in both the 1146

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Pro238 form and the Ala238 form. In chicken SH3C, the cis and trans forms of Pro238 coexist and give rise to two conformational folding phases. The second, slower phase that is abolished by the P238A substitution is thus tentatively assigned to the conformational folding of the form with a cis Pro238. It shows an amplitude of only 10−20% (Figure S2b of the Supporting Information) because the trans form is favored in unfolded proteins. SH3C Interferes with Binding of the Substrate to SH3N in Chicken but Not in Human c-CrkII. The guanine nucleotide exchange factor C3G (Crk SH3 domain-binding guanine nucleotide releasing factor) regulates cell migration and adhesion. It is a cellular target of active c-CrkII,2,14−17 and a proline-rich peptide derived from C3G has been used to examine substrate binding in functional studies of c-CrkII. Binding of this C3G peptide to the SH3N domain increases the fluorescence of this domain.7,14,18 The binding kinetics of the human and chicken proteins differ. The binding of the C3G peptide to the chicken SH3N− SH3C fragment is ∼4-fold slower than binding to the human SH3N−SH3C fragment (Figure 3a). Panels b and c of Figure 3 show the apparent rates of binding to the SH3N−SH3C fragment and to the isolated SH3N domains of both human and chicken c-CrkII at 15 °C as a function of peptide concentration. The rates of association (kon) and dissociation (koff), as derived from the slopes and intercepts, respectively, of these plots are listed in Table 1. In the isolated SH3N domains of both species, the binding sites are fully exposed, and they bind rapidly to the C3G peptide. For the human protein, the binding kinetics (Figure 3b) and the affinity (Table 1) are virtually unaffected by the SH3C domain (in the SH3N−SH3C protein). For the chicken protein, the rate of association with the C3G peptide is decreased 4-fold (Figure 3c) and the affinity becomes 4-fold lower when the SH3C domain is present. A significant fraction of the fast association reaction is complete within the dead time of stopped-flow mixing at 15 °C (Figure 3a). Therefore, kinetic experiments were also performed at 4.5 °C, where binding is slower. The results (Figure S3 and Table S3 of the Supporting Information) confirm the data obtained at 15 °C for the human protein. For the chicken SH3N−SH3C protein, biphasic kinetics were found. The fast reaction shows a rate that is the same as that observed for the isolated SH3N domain; the slow reaction was 7-fold slower, and the koff/kon ratio was increased 8-fold (Table S3 of the Supporting Information). This indicates that the chicken SH3N−SH3C protein consists of a mixture of a high-affinity form and a low-affinity form. We assume that this mixture exists at 15 °C, as well, but the two reactions could not be separated, because part of the fast reaction occurs during the dead time of stopped-flow mixing. The P238A substitution left the association rate of the human protein almost unchanged (Figure 3b); however, for the chicken protein, it led to a >3-fold increase in the observed rate at 15 °C (Figure 3c), and at 4.5 °C, it abolished the slow binding reaction (Figure S3 of the Supporting Information). Binding experiments were also performed with full-length cCrkII (Figure 3d). For the human full-length protein and its P238A variant, the association kinetics with the C3G peptide were virtually identical. For the chicken full-length protein, the association was 3-fold retarded, relative to that of the isolated SH3N domain, and this retardation was relieved by the P238A substitution as in the SH3N−SH3C protein (Figure 3d). Apparently, the SH3N−SH3C fragment provides an adequate

Figure 3. Kinetics of binding of the C3G peptide to SH3N. (a) Representative kinetics for the association of 6.0 μM C3G peptide with the human (black) or chicken (red) SH3N−SH3C fragment. The lines represent exponential fits to the data. (b and c) The apparent rate constants of association between the C3G peptide and SH3N (□), the SH3N−SH3C fragment (■), and the SH3N−SH3C P238A fragment (red squares) are shown for the (b) human and (c) chicken c-CrkII variants. (d) Data for full-length human (□) and chicken (■) c-CrkII and their P238A variants (red). The kinetics were followed after stopped-flow mixing of 0.5 μM protein in 0.1 M potassium phosphate (pH 7.4) with increasing concentrations of C3G peptide by the fluorescence above 320 nm (excitation at 295 nm). The apparent rate constants (λ) were derived from fitting exponential functions to the kinetic traces. The lines represent linear regression to the λ values to obtain the kon parameters from the slope and koff from the intercept. These parameters and the resulting dissociation constant (KD = koff/ kon) are listed in Table 1.

model for studying substrate binding and the interaction between the two SH3 domains of c-CrkII. In combination, the folding and peptide binding experiments indicate that human c-CrkII is a homogeneous species. It exists in an open conformation, and its SH3N domain binds to a substrate peptide equally well in the presence and absence of the SH3C domain. The chicken protein is heterogeneous. It consists of an open form that binds to the C3G peptide with high affinity and of a closed form in which the affinity for the C3G peptide is not lost but decreased ∼4-fold. The low-affinity 1147

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Table 1. Kinetics of Association of Human and Chicken SH3N−SH3C Fragments with the C3G Peptidea protein human

chicken

isolated SH3N SH3N−SH3C P238A I239F V272M I239F V272M P238A I239F V272M c-CrkII c-CrkII P238A isolated SH3N SH3N−SH3C P238A F239I M272V F239I M272V c-CrkII c-CrkII P238A

kon (μM−1 s−1) 106 90 72 57 68 11.7 65 57 54 71 16 54 82 52 55 21 58

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

6 4 3 2 1 0.6 5 3 2 4 3 2 4 2 2 1 4

koff (s−1)

koff/kon (μM)

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.47 0.31 0.66 0.75 0.28 3.31 0.39 0.62 0.54 0.70 2.58 0.91 0.36 0.43 0.77 1.70 0.60

50 28 47 43 19 39 25 36 29 50 42 50 30 22 43 36 35

26 18 16 8 5 4 12 16 10 22 16 10 21 11 10 5 20

Figure 4. Effects of cross-variations on the folding and function of cCrkII. Rate constants of unfolding (empty symbols) and refolding (filled symbols) as a function of GdmCl concentration are given for the isolated SH3C domains from human (a) and chicken (b). For the cross variants with substitutions at positions 238 and 272, data are colored red. Data for the second conformational folding reaction are shown as squares. All experiments were performed at a final protein concentration of 1.0 μM in 0.1 M potassium phosphate (pH 7.4) at 15 °C. The fluorescence above 320 nm (excitation at 280 nm) was monitored after stopped-flow mixing. The dependencies of the rate constants on GdmCl concentration were analyzed according to a twostate model. Parameters from the analysis are summarized in Table S2 of the Supporting Information. (c) Binding of the C3G peptide to the human SH3N−SH3C fragment (■) and its I239F (○) and V272M (△) variants. (d) Binding of the C3G peptide to the chicken SH3N− SH3C fragment (■) and its F239I (○) and M272V (△) variants. Protein (0.5 μM) in 0.1 M potassium phosphate (pH 7.4) was mixed at 15 °C with increasing concentrations of the peptide in a stoppedflow device. The fluorescence was excited at 295 nm and recorded above 320 nm. The lines represent linear regression to the rate constants. The values for kon and koff are listed in Table 1.

a kon and koff were derived from the slopes and the ordinate intercepts of plots such as those shown in Figures 3−5. The standard deviations from the analysis are given. Data were reproduced in independent experiments. The kinetics were followed by the fluorescence above 320 nm (excitation at 295 nm) in 0.1 M potassium phosphate (pH 7.4) at 15 °C.

state originates most likely from the form with the cis isomer of Pro238. The Residue after Pro238 Is Important for cis−trans Isomerization in SH3C and for Binding of the Substrate to SH3N. Pro238 is followed by Ile in human c-CrkII and by Phe in chicken c-CrkII. To examine the role of this residue, we analyzed the reciprocal variants I239F in the human protein and F239I in the chicken protein. Chicken SH3C is stabilized by the F239I substitution (Figure S4b of the Supporting Information), and its unfolding is decelerated (Figures S5d and S6b of the Supporting Information); however, the second, slower conformational folding reaction is still present. The reciprocal I239F replacement destabilizes human SH3C (Figure S4a of the Supporting Information), accelerates its unfolding, and renders refolding biphasic, as observed for the wild-type form of the chicken SH3C domain (Figures S5c and S6a of the Supporting Information). The nature of the amino acid following Pro238 thus accounts for the differences in the stability and in part for the differences in the folding kinetics between the SH3C domains of human and chicken c-CrkII. In the chicken two-domain protein, the F239I substitution increased the rate of association between the SH3N domain and the C3G peptide to the rate observed for the isolated SH3N domain (Figure 4d, Figure S7b of the Supporting Information, and Table 1), apparently by abolishing the inhibitory interdomain interactions. The reciprocal I239F substitution in the human protein could, however, not induce a domain-closed low-affinity conformation as in the wild-type form of the chicken protein. The rate of association with the C3G peptide decreased by only 30% (Figure 4c and Figure S7a of the Supporting Information) and was still ∼3-fold higher than the association rate observed for the chicken protein (Table 1). Met272 Is Essential for Converting the Isomeric State of Pro238 into a Functional Signal. The results obtained with the cross variants at the position following Pro238 were

puzzling. In the chicken protein, the F239I substitution did not change the complex refolding kinetics but apparently abolished the closed low-affinity state. In the human protein, the reciprocal I239F replacement produced the cis form of the Gly237−Pro238 peptide bond and thus complex refolding kinetics as in the chicken protein, but it did not lead to the closed form with the lowered affinity for the C3G peptide. Human and chicken SH3C differ also at position 272, which is occupied by Val and Met, respectively. The V272M substitution left the stability (Figure S4a of the Supporting Information) of the human SH3N−SH3C protein and of its isolated SH3C domain unchanged, as expected for a surfaceexposed position. Refolding became biphasic, as observed for the human I239F variant and for the chicken protein (Figure S5e of the Supporting Information), but it still bound to the C3G peptide, almost as well as the wild-type protein (Figure 4c and Figure S7a of the Supporting Information). Similar to the I239F substitution, the V272M substitution led to an increase in the level of the cis conformation at Pro238, but not to an increase in the level of the low-affinity form in the human protein. We then combined the I239F and V272M substitutions in the SH3N−SH3C human two-domain protein (Figures S4c and S6c of the Supporting Information) as well as in the isolated SH3C domain. Again, refolding was biphasic as in the singly 1148

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virtually all molecules. In the chicken protein, Pro238 exists as a mixture of cis and trans forms. In the closed cis form, the affinity for the C3G peptide is low. The molecular communication of the isomeric state of Pro238 with the domain interface involves Phe239 and Met272. Substitution of either residue interrupts this path in the chicken protein. The reciprocal introduction of both these residues into the human SH3N−SH3C protein is necessary and sufficient to establish this communication and to create the proline-controlled low-affinity form in this protein, as well. C3G Peptide Binding Is Linked Energetically with Pro238 cis−trans Isomerization. Molecular communication is a bidirectional process. If the C3G peptide binds preferentially to SH3N when Pro238 in SH3C is trans, then peptide binding should lead to an increase in the level of the trans species via the same communication path. A slow binding reaction should thus be observed and rate-limited by a shift in the cis−trans equilibrium at Pro238 toward trans. Additional slow binding of the C3G peptide was indeed observed for the chicken protein (Figure 5c). It showed a rate of 0.01 s−1, typical of prolyl isomerization.19 This reaction was absent in the variants P238A, F239I, M272V, and F239I M272V (Figure 5c and Figure S8 of the Supporting Information). All of them are known to interrupt the communication path between Pro238 and the domain interface. In the human SH3N−SH3C protein, the I239F V272M double substitution induced such a slow proline-coupled binding reaction (Figure 5c). This confirms the bidirectional communication path between Pro238 in SH3C and binding of C3G to SH3N and the essential roles of Phe239 and Met272 in the transfer of information along this path. The Activated Form Is an Intermediate on the Folding Pathway of Chicken c-CrkII. When the isolated chicken SH3N domain is refolded in the presence of the AEDANSlabeled C3G peptide, most molecules reach the native state and bind to the labeled peptide within the dead time of manual mixing (cf. Figure S1 of the Supporting Information), which leads to an increase in the efficiency of energy transfer from SH3N to the AEDANS label of the peptide. A further slow increase in the efficiency of energy transfer is monitored, as well, because a small fraction of SH3N molecules contain nonnative prolines and thus refold and bind slowly (Figure 5d and Figure S1 of the Supporting Information). In the SH3N−SH3C chicken two-domain protein, this increase is overcompensated by a decrease in AEDANS fluorescence (Figure 5d), indicative of a slow peptide displacement reaction. In the unfolded SH3N−SH3C protein, Pro238 is largely trans, and the rapid conformational folding thus leads to the open form with trans Pro238 and high affinity for the C3G peptide. Subsequently, a part of Pro238 in SH3C isomerizes to the cis form, because this isomer is favored in the folded form of the chicken protein. As a consequence, the affinity of SH3N for the AEDANS-labeled C3G peptide and thus the efficiency of energy transfer decrease. The P238A substitution arrests the SH3N−SH3C protein in the trans state, and the affinity for the peptide binding remains as high as for the isolated SH3N domain (Figure 5d). This confirms that in fact the trans Pro238 isomer is the bindingactive form and that isomerization to the cis form induces the low-affinity form and thus autoinhibition. Conclusion. The c-CrkII signal adapter proteins from human and chicken are closely related, with an overall level of sequence identity of 85% and 14 amino acid differences in the SH3N−SH3C fragment. Both contain a proline (Pro238) at a

substituted variants and as in the chicken protein (Figure 4a), pointing to cis−trans heterogeneity at Pro238. Remarkably, the I239F V272M variant bound much more slowly to the C3G peptide (Figure 5a), and the rate of association was reduced 9-

Figure 5. Energetic linkage between C3G peptide binding and prolyl isomerization in the SH3N−SH3C fragment. (a) Representative kinetics of association between 6.0 μM C3G peptide and 0.5 μM human (black) and chicken (blue) SH3N−SH3C protein and the I239F V272M variant of the human protein (red). Exponential functions, fit to the data, are shown, as well. (b) Apparent rate constants of association between 0.5 μM SH3N−SH3C protein and increasing concentrations of the C3G peptide of the chicken protein (□), its P238A variant (empty red squares), and the I239F V272M (■) and P238A I239F V272M (filled red squares) variants of human SH3N−SH3C protein. The lines represent linear regressions to the λ values to obtain kon from the slope and koff from the intercept (Table 1). The dashed line for the human SH3N−SH3C protein was taken from Figure 3b. (c) Slow association reaction of 4.0 μM C3G peptide with 1.0 μM SH3N−SH3C protein for the human SH3N−SH3C protein (gray) and its I239F V272M (red) and P238A I239F V272M (blue) variants and for the chicken SH3N−SH3C protein (black) and its P238A variant (blue). The fluorescence at 336 nm (excitation at 295 nm) was monitored. (d) Binding of 2 μM AEDANS-labeled C3G peptide during the refolding of 1.0 μM chicken SH3N−SH3C protein (black), isolated SH3N (gray), and the P238A variant (red). The transfer of energy from Trp in SH3N to the AEDANS moiety at the Cterminus of the labeled C3G peptide was measured at 500 nm after excitation at 280 nm. The proteins were denatured in 3.0 M GdmCl in the presence of C3G-AEDANS for 1 h prior to the measurement of refolding and binding. All experiments were performed in 0.1 M potassium phosphate (pH 7.4) at 15 °C.

fold to a value lower than the rate observed for the chicken SH3N−SH3C protein (Figure 5b). Evidently, the two I239F and V272M substitutions in combination were sufficient to induce a closed, low-affinity form in the human SH3N−SH3C protein. When, in this variant, the P238A substitution was introduced, the closed form was abolished again, and the rate of association with the C3G peptide increased almost to the value observed for the wild-type protein (Figure 5b and Figure S7d of the Supporting Information). This completes the evidence that Pro238 in the SH3C domain of c-CrkII regulates the binding of the C3G peptide to the SH3N domain. In the human protein, Pro238 is predominantly trans, and therefore, the binding site is open in 1149

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position where the SH3N−SH3C interdomain linker leads into the SH3C domain (Figure 6a). Only the chicken protein uses

mutual stabilization of the two domains in the SH3N−SH3C protein was not detected in the folding studies. It also explains why in murine c-CrkII stabilizing domain interactions were not found.18 The divergent behavior of the human and chicken proteins could be traced back to only two positions in the sequence. In the chicken SH3N−SH3C protein, the replacement of the residue after Pro238 (Phe239) with Ile, as in the human protein, or of Met272 with Val relieved the conformational constraints that maintained the Gly237−Pro238 peptide bond in the cis conformation and thus increased the affinity for the C3G peptide. In the human SH3N−SH3C protein, the individual reverse mutations (I239F and V272M) induced trans → cis switching at Pro238 in the SH3C domain, but they were unable to propagate the cis Pro238 signal to the SH3N domain. The introduction of both residues, Phe238 and Met272, was necessary and sufficient to create the interdomain signal transfer pathway in the human protein. The proline switch could thus be established in the purified protein in vitro, and therefore, it is unlikely that auxiliary proteins are necessary to communicate the isomeric state of Pro238 within c-CrkII from the SH3C domain to the SH3N domain. At present, it remains unclear why the use of a slow proline timer is restricted to the chicken isoform of c-CrkII. Interestingly, a statistical analysis of the amino acid preferences around prolines that can occur in alternative conformations in folded proteins23 indicated that Phe (as in chicken c-CrkII) is favored and Ile (as in human c-CrkII) disfavored after such prolines. The linker between SH3N and SH3C is ∼40 residues long and consists of two parts. The first half is flexible and presumably unordered. The second half (residues 222−236) is well-ordered,6,7 and in the forms with cis or trans Pro238, it interacts differently with the SH3C domain.7,9 It remains a challenge to unravel how this difference in packing is translated into a changed accessibility of the substrate binding site of the SH3N domain and how this is steered by Phe239 and Met272. Prolyl isomerization is an intrinsically slow reaction, and its critical role for protein folding is well-documented.19,24,25 More recently, prolyl isomerizations have also been identified as slow molecular switches or timers in regulatory proteins.7,21,26−31 It is largely unknown how such switches are triggered. In the infection protein of phage fd, binding to a bacterial pilus creates a signal that is propagated via the unfolding of an interdomain hinge to a proline, and the switching rate is set by the local sequence around this proline.21,22 In c-CrkII, it remains to be elucidated how the cis−trans isomerization at Pro238 is initiated, how the cis−trans equilibrium is shifted, and how the lifetime of the open form is regulated. In human and chicken c-CrkII, Tyr222 can be phosphorylated by kinases such as Abl, and this phosphorylation might influence Pro238 isomerization, as well. Tyr222 phosphorylation was suggested to regulate the binding activity of the SH3N domain of human c-CrkII.2,6,7 Chicken c-CrkII is switched by Pro238 isomerization not between an “on” state and an “off” state but between a highaffinity state when in trans and a low-affinity state when in cis, similar to an allosteric transition between a relaxed R and a tense T state (Figure 6b). The interdomain communication between the substrate (effector) binding site on the SH3N domain and the proline switch in the SH3Cdomain might serve as a feed-forward regulation with a built-in memory effect, because it is coupled with a slow prolyl isomerization, which prolongs the lifetime of the relaxed high-affinity state. When the

Figure 6. Proposed binding mechanism of the chicken SH3N−SH3C protein. (a) The backbone structure of chicken SH3C is given for the different conformational states with Pro238 in the cis (dark red, Protein Data Bank entry 2L3P) or trans (dark green, Protein Data Bank entry 2L3Q) conformation, as determined by NMR spectroscopy.7,9 Pro238, Phe239, and Met272 are shown in bright colors in ball-and-stick form. (b) P238 isomerization switches the chicken SH3N−SH3C protein between the high-affinity (trans) and low-affinity (cis) forms. The protein is largely in the cis form, and effector binding shifts the equilibrium toward the trans form. The functionally important residues Pro238, Phe239, and Met272 are colored red.

this proline as a switch or molecular timer, with a trans Pro238 in the open, signaling-active form and a cis Pro238 in the closed, less active state (Figure 6). The cis and trans isomers of Pro238 coexist in the folded protein, and the cis form predominates in the SH3N−SH3C two-domain protein, which agrees with the conformational heterogeneity detected by NMR spectroscopy.7,9 For human c-CrkII, we detected only the trans form of Pro238 in the analysis of its folding kinetics, and consequently, only the high-affinity form was found in substrate binding experiments. The trans form of chicken c-CrkII bound to the C3G peptide with the same high affinity as the human protein. In the cis form, the affinity for the substrate peptide was decreased but not abolished. The trans → cis isomerization at Pro238 is not followed by tight domain association and complete inhibition of substrate binding, as observed previously for a proline switch in a phage infection protein.20−22 The cis Pro238 form of c-CrkII is thus not a fully autoinhibited state but a low-affinity state. The lack of a tight domain docking reaction explains why a 1150

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2.0 μM AEDANS-labeled C3G, and 0.06 M GdmCl. The donor fluorescence was excited at 280 nm (3 nm bandwidth), and the AEDANS fluorescence was monitored at 500 nm (10 nm bandwidth).

concentration of an effector such as C3G increases and remains high, the conformation of c-CrkII is shifted toward this highaffinity and signaling-active trans form. Even when the concentration of the effector protein has decreased again, the high-affinity form continues to exist for an extended time, because the reversal to the low-affinity form is delayed by Pro238 trans → cis re-isomerization. Thus, Pro238 might extend the lifetime of the highly active state and act as a molecular long-term capacitor in the function of c-CrkII. Possibly, this proline capacitor is regulated by a prolyl isomerase under physiological conditions.

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AUTHOR INFORMATION

* Supporting Information S

Supporting methods, tables, and figures. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author

*E-mail: [email protected]. Notes

METHODS

The authors declare no competing financial interest.

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Cloning and expression of the c-CrkII variants and the C3G-derived peptide are described in the Supporting Information. Equilibrium Unfolding Transitions. GdmCl-induced unfolding transitions were measured with 1.0 μM protein in 0.1 M potassium phosphate (pH 7.4) at 15 °C with a Jasco FP-6500 fluorescence spectrophotometer (Jasco, Tokyo, Japan). All samples were incubated at 15 °C for at least 1 h prior to measurement to allow the protein to reach the unfolding equilibrium. Fluorescence was detected at 330 nm (3 nm bandwidth) after excitation at 280 nm (3 nm bandwidth) in 1 cm cells. The GdmCl concentration of each sample was calculated from the refractive index. Analysis was performed according to a twostate model.3 Kinetics of Unfolding and Refolding. The unfolding and refolding kinetics were measured using a DX17MV stopped-flow spectrophotometer (Applied Photophysics, Leatherhead, U.K.) by the change in fluorescence above 320 nm or after manual mixing using a Jasco FP-6500 fluorescence spectrophotometer by the fluorescence at 330 nm (3 nm bandwidth) in a 1 cm cell. Excitation was always at 280 nm. All measurements were performed at 15 °C with 1.0 μM protein in 0.1 M potassium phosphate (pH 7.4) and varying GdmCl concentrations. In the stopped-flow experiments, native or denatured (in 4.0 M GdmCl) protein was diluted 11-fold, and the kinetics of unfolding or refolding were measured at least eight times under identical conditions and averaged. For manual mixing, the native or denatured (4.0 M GdmCl) protein was diluted 20-fold in the cuvette. The final GdmCl concentrations were calculated from the refractive index32 of the samples. All kinetics were analyzed by fitting exponential functions to the data. Association Kinetics. The association reaction between the C3G peptide and c-CrkII variants was measured under pseudo-first-order conditions by monitoring the increase in fluorescence upon binding to SH3N in the DX17MV stopped-flow spectrophotometer. All measurements were performed in 0.1 M potassium phosphate (pH 7.4) at 15 or 4.5 °C. The solution containing the c-CrkII variant was mixed with an equal volume of solutions with increasing concentrations of the peptide. The final concentration of the c-CrkII variant was 0.5 μM. The increase in fluorescence was monitored above 320 nm. Excitation was at 295 nm to avoid contributions of the Tyr residues of the C3G peptide to the observed fluorescence. Kinetics were measured at least eight times under identical conditions, averaged, and analyzed by fitting exponential functions to the fluorescence time courses. To monitor the slow binding reaction, 1.0 μM protein was equilibrated in the cuvette at 15 °C while being stirred constantly. Association was initiated by diluting the peptide to a final concentration of 4 μM in the cuvette. The protein fluorescence was measured at 336 nm (5 nm bandwidth) after excitation at 295 nm (3 nm bandwidth). Binding during refolding of the c-CrkII variants was monitored by following the transfer of energy from Trp in SH3N to the AEDANSlabeled C3G peptide. Trp fluorescence was not used as a probe, because it changes upon both folding and C3G peptide binding. The cCrkII variants were denatured in 3.0 M GdmCl in the presence of the labeled peptide for at least 1 h. Refolding was initiated by diluting the protein/peptide mixture 50-fold with 0.1 M potassium phosphate (pH 7.4) at 15 °C, leading to final concentrations of 1.0 μM c-CrkII variant,

ACKNOWLEDGMENTS We thank F. Hög and R. P. Jakob for initial experiments with cCrkII, J. Koch for many discussions, and L. Ries, O. Stemmann, and S. Moniot for critical comments on the manuscript.

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ABBREVIATIONS c-CrkII, cellular CT10 regulator of kinase protein; SH2 and SH3, Src homology domains 2 and 3, respectively; SH3N and SH3C, N- and C-terminal SH3 domains of c-CrkII, respectively; C3G, Crk SH3 domain-binding guanine nucleotide releasing factor; AEDANS, 5-{[(acetylamino)ethyl]amino}naphthalene1-sulfonic acid; GdmCl, guanidinium chloride; λ, apparent rate constant of a reaction; kon and koff, rate constants of association and dissociation, respectively

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REFERENCES

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