Differential Responses of the Backbone and Side-Chain Conformational Dynamics in FKBP12 upon Binding the Transition-State Analog FK506: Implications for Transition-State Stabilization and Target Protein Recognition

J. Mol. Biol. (2009) 387, 233–244

doi:10.1016/j.jmb.2009.01.047

Available online at www.sciencedirect.com

Differential Responses of the Backbone and Side-Chain Conformational Dynamics in FKBP12 upon Binding the Transition-State Analog FK506: Implications for Transition-State Stabilization and Target Protein Recognition Ulrika Brath and Mikael Akke⁎ Division of Biophysical Chemistry, Center for Molecular Protein Science, Lund University, PO Box 124, SE-22100 Lund, Sweden Received 15 November 2008; received in revised form 12 January 2009; accepted 23 January 2009 Available online 30 January 2009

FKBP12 serves a dual role as a peptidyl-prolyl cis–trans isomerase and as a modulator of several cell signaling pathways. The macrolide FK506 is a transition-state analog of the catalyzed reaction and displaces FKBP12 from its natural target proteins. We compared the conformational exchange dynamics of the backbone and methyl-bearing side chains of FKBP12 in the free and FK506-bound states using NMR relaxation-dispersion experiments. Our results show that the free enzyme exchanges between the ground state and an excited state that resembles the ligand-bound state or Michaelis complex. In FK506-bound FKBP12, the backbone is confined to a single conformation, while conformational exchange prevails for many methyl groups. The residual side-chain dynamics in the transition-state analogbound state suggests that the transition-state ensemble involves multiple conformations, a finding that challenges the long-standing concept of conformational restriction in the transition-state complex. Furthermore, exchange between alternative conformations is observed in the bound state for an extended network of methyl groups that includes locations remote from the active site. Several of these locations are known to be important for interactions with cellular target proteins, including calcineurin and the ryanodine receptor, suggesting that the conformational heterogeneity might play a role in the promiscuous binding of FKBP12 to different targets. © 2009 Elsevier Ltd. All rights reserved.

Edited by A. G. Palmer III

Keywords: enzyme catalysis; dynamics; NMR relaxation; transition-state stabilization

Introduction Enzymes play a central role in biology by catalyzing the great majority of biochemical reactions that enable life. Enzymes achieve catalysis by a sequence of steps that enable substrate binding, formation of the transition state, and product release. These separate steps are often associated with distinct basins in the energy landscape of the enzyme such that it moves through a series of conformations along the reaction coordinate. The *Corresponding author. E-mail address: [email protected]. Abbreviations used: TSA, transition-state analog; TβRI, transforming growth factor-β type I receptor.

dramatic catalytic power exhibited by many enzymes is attained by lowering the energy of the transition state relative to the substrate or product ground states. Transition-state stabilization is realized by preferential binding of the transition-state structure over the substrate or product. Consequently, the dynamic interactions between the enzyme and the substrate vary along the reaction coordinate, and it is generally believed that the binding energies are more favorable in the transition state than in the substrate- and product-bound states.1–4 Based on this concept, a long-standing hypothesis posits that the conformational degrees of freedom are preferentially restricted in the transition state.2,3,5–8 The enzyme transition state is a quasi-thermodynamic state with a lifetime on the order of 10− 13 s,

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234 making it virtually impossible to be observed by any experimental method presently available. Kinetic isotope effects offer a route towards mapping the physicochemical features of the transition state in terms of the substrate structure and substrate– enzyme interactions,8 but they do not enable structural characterization of the surrounding enzyme matrix. Non-reactive ligands that mimic the transition-state structure of the substrate, denoted as transition-state analogs (TSAs), are generally found to bind strongly to the enzyme, as expected based on the concept of transition-state stabilization.1–3,8 The high affinity and long-term stability of TSA–enzyme complexes have enabled detailed structural studies of these transition-state mimics by X-ray crystallography and NMR spectroscopy. Enzyme dynamics related to catalysis and substrate recognition often take place on microsecondto-millisecond timescales, which are amenable to characterization by NMR relaxation-dispersion experiments.9–11 This powerful method can provide detailed information on the energy landscape in terms of the rates of the conformational exchange (kinetics), as well as the populations of the exchanging states (thermodynamics) and the chemical-shift differences (structure) between these states. In this study, we addressed the dynamics of substrate binding and the conformational restriction in the transition state by comparing the conformational exchange dynamics of the free and TSA-bound states of the peptidyl-prolyl cis–trans isomerase FK506 binding protein (FKBP12). FKBP12 belongs to a group of peptidyl-prolyl cis– trans isomerases that plays a critical role in the cellular folding machinery by catalyzing the cis– trans isomerization of peptidyl-prolyl linkages in proteins, thereby reducing the transient accumulation of misfolded species.12,13 FKBP12 comprises 107 residues that form a globular structure composed of six β-strands (β1–β6), one α-helix (α1), two short

Conformational Exchange in FKBP12

helical turns, and several loops (see Fig. 1a). The isomerase activity of FKBP12 is inhibited by the drug FK506 (Fig. 1b), which binds to the enzyme with strong affinity, Kd = 0.4 nM.16–20 FK506 is a 23membered macrolide naturally produced by the bacterium Streptomyces tsukubaensis.21 The strong binding of FK506 to FKBP12 has been rationalized by its similarity to the transition-state structure of natural peptide substrates. The transition state of the peptidyl-prolyl cis–trans isomerization reaction is defined by a 90°ω dihedral angle of the peptidyl-prolyl bond such that the carbonyl of the peptide bond is rotated into a plane approximately orthogonal to the prolyl ring.18 The transition-state structure of FKBP12 stabilizes the twisted peptide moiety by compensating the loss of amide resonance in the substrate with favorable binding energy.17,22,23 The twisted peptide epitope is highly similar to the ketocarbonyl of FK506, which lies in a plane orthogonal to the pipecolinyl ring. In the FKBP12-bound state, the pipecolinyl ring of FK506 mimics the transition-state structure of the proline ring of natural protein substrates.19,24 The transition-state analogy is further emphasized by the similar interactions formed in the Michaelis (FKBP12-protein) and FKBP12–FK506 complexes. The peptide substrate specificity of FKBP12 is increased when the residue preceding the proline changes to a more hydrophobic character, indicating that hydrophobic interactions are important for transition-state stabilization,16 as also observed in the FKBP12–FK506 complex. Indeed, the residues preferred by FKBP12 for catalysis could be predicted on the basis of the structure of FK506,17 which underscores the strong correspondence between FK506 and a twisted peptide substrate in the transition state. Thus, FK506 represents a TSA of natural protein substrates.18 FK506 is administered in the clinic as an immunosuppressant. The FKBP12–FK506 complex suppresses

Fig. 1. The structure of FKBP12 in the free and FK506-bound states. (a) The structure of free FKBP12 (PDB ID 2ppn).14 β-Strands and α-helices are shown in blue and cyan, respectively. Residues in loops and turns are highlighted in red. (b) The FKBP12–FK506 complex (PDB ID 1fkj).15 FK506 is shown in yellow stick representation.

Conformational Exchange in FKBP12

the cellular immune response by binding to calcineurin, thereby inhibiting T-lymphocyte activation.25 FKBP12 has also been implicated in other cellular activities involving natural targets: it regulates the cell cycle by stabilizing the inactive form of the transforming growth factor-β type I receptor (TβRI);26–28 it forms an inhibitory ternary complex with Smad7 and the activated activin type I receptor to regulate the duration of the activin signal;29 it controls the Ca2+release activity of the ryanodine receptors (RyR1 and RyR3) by binding directly to these;30,31 and it modulates Ca2+ release via the inositol 1,4,5-trisphosphate receptor,32 presumably by forming ternary complexes with FK506–calcineurin and rapamycin– mTOR/FRAP.33 Thus, FKBP12 exhibits a degree of promiscuous target recognition that involves accommodating several binding motifs, both in the FK506 binding pocket and in more distal regions on the surface. In this study, we took the FK506-bound state to represent the transition state of FKBP12 and addressed the question whether formation of the transition state is related to changes in the conformational degrees of freedom of the enzyme. We monitored the exchange dynamics between alternative conformations by NMR relaxation-dispersion methods that probe the backbone and methylbearing side chains in both the free and TSAbound states. Our results demonstrate that free FKBP12 populates both the major open conformation and a minor closed conformation that resembles the Michaelis complex. Furthermore, our data suggest that the backbone of FKBP12 is locked in a single conformation in the transition state, as expected from the tight interactions with the TSA. By contrast, the methyl-bearing side chains show increased flexibility and form an expanded set of dynamic sites in the TSA-bound state compared

235 with the free state. Notably, several of the methyl groups that sample alternative conformations in the FK506–FKBP12 complex are located in regions that have been implicated in the binding to protein targets, including calcineurin, mTOR/FRAP, and RyR1.

Results We performed off-resonance rotating-frame (R1ρ) relaxation measurements to investigate the conformational dynamics on the microsecond timescale in the free and FK506-bound states of FKBP12. The dynamics were probed using the backbone amides and side-chain methyl groups by 15N and 13C R1ρ experiments,34–36 respectively. The 13C R1ρ experiments were carried out on a uniformly 13C-enriched and partially randomly 2H-enriched sample. The uniform 13C labeling largely excludes leucine methyl groups from accurate interpretation of exchange parameters due to complications arising from the Hartmann–Hahn type of coherence transfer within the 13C spin system of this residue type.36,37 Reliable 13 C R1ρ relaxation data were obtained for only three leucine methyl groups, L74δ1, L74δ2, and L103δ1, in the FK506-bound state. In the fast-exchange limit, the R1ρ experiment yields the exchange correlation time (τex), which is equal to the inverse of the exchange rate (τex = 1/kex), and the product of the populations of the two exchanging substates and the square of the chemical-shift difference between these (denoted as Φex = pApBΔω2).38,39 A conservative (lower) estimate of the chemical-shift difference between substates A and B is obtained as the minimal value, 1/2 |Δω|min = 2Φex , which is calculated by setting pA = pB. Figure 2 shows representative relaxationdispersion data, including the relatively poorly

Fig. 2. Representative 15N and 13C relaxation-dispersion curves shown for residues A81 (a–d) and I91 (e–h). Data are shown for both free (a, c, e, and g) and FK506-bound (b, d, f, and h) FKBP12. 15N dispersions for A81 (a and b) and I91 (e and f) demonstrate that the backbone conformational dynamics is quenched in the presence of FK506. The 13C dispersions (A81, c and d; I91, g and h) illustrate the differential response of the side chains to binding of FK506 with prevailing (A81 13 β C ) and eliminated (I91 13Cδ1) dispersion steps.

236 defined dispersion curve obtained for A81 13Cβ in the FK506-bound state [panel (d)], which nonetheless yields a statistically significant fit to the twostate conformational exchange model, albeit with a large relative uncertainty (56%) in the estimated value of Φex. Backbone dynamics in the free state Significant exchange contributions to backbone N relaxation dispersions were observed for 23 residues in the free state (Fig. 3a; Table 1). Data could not be obtained for the 7 prolines (in positions 9, 16, 45, 78, 88, 92, and 93) and 9 other residues (1, 3, 6, 35, 46, 82, 84, 86, and 89) that suffered from overlap or extensive broadening of their 1H–15N cross-peaks. The complete set of 23 dispersion curves could be fitted to a common two-state exchange process with a correlation time of τex = 116 ± 10 μs. Since the exchange can be described by a global two-state 1/2 process, the residue-specific values of Φex can be 15

Conformational Exchange in FKBP12

directly compared between different residues to indicate the relative magnitude of the chemicalshift differences between the exchanging substates (Fig. 3a). The minimal chemical-shift differences [|ΔωexF(15N)|min] range between 0.5 and 2.0 ppm. For comparison, the chemical-shift changes upon binding FK506 [|ΔωF–B(15N)|] are b 2.3 ppm. The exchanging residues are located primarily in the sequence stretches 26–44, 53–57, and 75–98, which surround the active site (Fig. 3a). The dynamic regions map to those that show the largest structural change upon binding FK506 (Fig. 3g) and the largest chemical-shift changes upon binding of FK506 or peptide (Fig. 3c and e), directly suggesting that the free protein samples a minor conformation similar to the ligand-bound state. To this extent, linear regres2 sion of Φex versus ΔωF–B may be expected to yield pApB as the slope of the fitted line [see Eq. (2)].35 However, residue-by-residue comparison of Φex and 2 does not reveal any correlation between these ΔωF–B data sets, as discussed further below. Fig. 3. Comparison of chemical-shift differences between exchanging conformations in free FKBP12 (|ΔωexF|min) and in FK506-bound FKBP12 (|ΔωexB|min) and between free and FK506- or peptide-bound FKBP12 (|ΔωF–B|). The structure of free FKBP12 (PDB ID 2ppn)14 is color-coded using a continuous scale from yellow (low values) to red (high values). In panels (a) to (f), the color codes for the chemicalshift difference normalized by the maximum shift change observed upon FK506 binding: gray indicates residues that have flat relaxation-dispersion profiles (a, b, and f) or do not show chemical-shift changes upon binding (c–e) and those for which data could not be obtained (prolines, overlapped cross-peaks, or extensive line broadening; a–f). In panels (g) and (h), the color codes for the coordinate RMSD of the backbone nitrogen atoms (g) or the side-chain methyl carbon atoms (h) between the crystal structures of the free (PDB ID 2ppn) and FK506-bound (PDB ID 1fkj)15 states. (a) |ΔωexF (15 N)|min/|ΔωF–B(15 N)|max. |ΔωexF(15 N)|min ranges between 0.5 and 2.0 ppm. (b) |ΔωexF(13C)|min/|ΔωF–B (13C)|max.36 |ΔωexF(13C)|min ranges between 0.2 and 0.9 ppm. (c) |ΔωF–B(15N)|/|ΔωF–B(15N)|max, comparing the free and FK506-bound states. (d) |ΔωF–B(13C)|/|ΔωF–B (13C)|max. (e) |ΔωF–B(15N, pep)|/|ΔωF–B(15N)|max, comparing the free and partially peptide-bound states, where the latter chemical shifts are measured in the presence of less than saturating amounts of peptide (b 50% saturation). (f) |ΔωexB(13C)|min/|ΔωF–B(13C)|max, color-coded onto the structure of the FKBP12–FK506 complex. |ΔωexB(13C)|min ranges between 0.2 and 0.5 ppm. The maximum chemical-shift differences between the spectra of free and bound FKBP12 are |ΔωF–B(15N)|max = 2.3 ppm and |ΔωF–B(13C)|max = 2.0 ppm. Data are shown only for values of |ΔωF–B| equal to or greater than the smallest detected value of |ΔωexF|min, which are 0.37 ppm (c) and 0.15 ppm (d); in panel (e), data are shown for |ΔωF–B| ≥ 0.10 ppm. |ΔωexF|min and |ΔωexB|min are the minimum chemical-shift differences for each residue as calculated from Φex and equal populations of the exchanging conformations [|Δωex|min = (Φex/pApB)1/2 = 2Φ1/2 ex ]. In panels (b), (d), and (f), all methyl-bearing side-chains are shown in stick representation and both the backbone and methyl groups are color-coded according to the chemicalshift difference. (g) Coordinate RMSD of the backbone N atoms. (h) Coordinate RMSD of the methyl C atoms.

Conformational Exchange in FKBP12

237

Table 1. Exchange parameters for backbone free FKBP12

15

N spins in

Residue

Φex (103 s− 2)

R1 (s− 1)

R2,0 (s− 1)

Χ2v

Rex(ωeff→0) (s− 1)

R18 Y26 T27 E31 F36 S38 S39 D41 K44 Q53 V55 R57 A64 T75 D79 A81 G83 T85 I90 I91 A95 L97 V98

8.2 ± 1.0 31 ± 5 22 ± 3 9.3 ± 1.3 34 ± 3 24 ± 3 105 ± 11 29 ± 4 44 ± 5 8.3 ± 1.2 70 ± 6 21 ± 3 12 ± 2 15 ± 2 23 ± 3 89 ± 10 30 ± 3 63 ± 6 13 ± 2 139 ± 14 26 ± 2 21 ± 3 14 ± 2

1.59 ± 0.07 1.75 ± 0.08 1.64 ± 0.07 1.56 ± 0.07 1.57 ± 0.07 1.61 ± 0.07 1.82 ± 0.08 1.73 ± 0.07 1.52 ± 0.07 1.65 ± 0.07 1.55 ± 0.07 1.58 ± 0.07 1.56 ± 0.07 1.53 ± 0.07 1.85 ± 0.08 1.59 ± 0.07 1.71 ± 0.07 1.58 ± 0.07 1.59 ± 0.07 1.52 ± 0.06 1.67 ± 0.07 1.62 ± 0.07 1.53 ± 0.07

7.5 ± 0.3 6.8 ± 0.4 8.0 ± 0.4 7.9 ± 0.3 7.7 ± 0.4 6.8 ± 0.3 7.3 ± 0.6 6.8 ± 0.4 8.6 ± 0.4 8.1 ± 0.3 10.2 ± 0.5 8.6 ± 0.4 9.2 ± 0.4 7.9 ± 0.4 9.1 ± 0.4 9.9 ± 0.6 9.1 ± 0.4 8.9 ± 0.5 7.8 ± 0.4 9.7 ± 0.7 7.9 ± 0.4 8.2 ± 0.4 7.9 ± 0.4

0.76 0.32 0.81 0.48 0.68 0.71 1.25 0.45 0.70 0.68 0.89 0.77 0.39 0.41 0.34 0.55 0.52 0.50 0.75 0.51 0.67 0.58 0.42

1.0 3.6 2.6 1.1 4.0 2.7 12.2 3.4 5.1 1.0 8.1 2.4 1.4 1.7 2.7 10.3 3.5 7.4 1.5 16.1 3.0 2.4 1.7

Side-chain dynamics in the free state We previously reported that 12 of 57 methyl groups in free FKBP12 undergo conformational exchange with a common correlation time of τex = 126 ± 16 μs (Fig. 3b; Table 2).36 The exchange correlation times determined for the backbone amides and side-chain methyl groups are identical within errors. Indeed, a single-exchange correlation time, τex = 121 ± 26 μs, can be fitted simultaneously to the two data sets without a statistically significant loss in the quality of the fit (p = 0.39, which does not reject the null hypothesis that the reduced model is adequate). This result provides strong evidence that the backbone and side chains probe the same conformational exchange process in the free state. In further support of this conclusion, the exchanging methyl groups are located in and around the FK506 binding site (Fig. 3b), in close proximity to those backbone amides that show exchange [compare panels (a) and (b) of Fig. 3]; note that residues 31– 48 (including strand β3) are devoid of methyl groups. The methyl groups experience chemicalshift differences [|ΔωexF(13C)|min] in the range of 0.2–0.9 ppm, which should be compared with the maximal shift change upon binding FK506, |ΔωF–B ( 13 C)|max = 2.0 ppm. Similar to the case of the backbone, there is a close correspondence between the regions that exhibit conformational exchange and those whose chemical shifts respond to FK506 binding [compare panels (b) and (d) of Fig. 3], as well as those that experience the largest structural changes upon FK506 binding (Fig. 3h). This observation reinforces the conclusion that the minor conformation sampled in the free state is similar to the ligand-bound state.

Backbone dynamics in the TSA-bound state In contrast to the free state, there is no detectable conformational exchange affecting the backbone of the FK506-bound state. This result indicates that any alternative conformational substate of the transitionstate ensemble is higher in energy by more than 5kBT (corresponding to a population of a potential highenergy state of less than 0.5%, which is approximately the detection limit in these experiments). Thus, the transition-state ensemble of FKBP12 is dominated by a single basin in the energy landscape, as probed by the backbone amides. In other words, the backbone is locked into a single conformation by tight interactions with the substrate in the transition state. Similar results have been presented previously for the amplitudes of N–H bond vector fluctuations on sub-nanosecond timescales.40,41 The present and previous results agree with the observed enthalpy– entropy compensation in the transition state across a panel of peptide substrates, which suggests that increasingly favorable interactions (enthalpy) in the transition state are compensated by an increasingly unfavorable activation entropy.42 Our work clearly shows that the number of available conformational states of the protein backbone is reduced in the transition-state ensemble compared with the free state. Side-chain dynamics in the TSA-bound state In sharp contrast to what is observed for the backbone, conformational exchange prevails for 20 methyl groups in the FK506-bound state (Fig. 3f; Table 2). The side-chain dynamics is adequately fitted to a global process with an exchange correlation time slightly shorter than that in the case of free FKBP12, τex = 71± 13 μs. The chemical-shift differences [ΔωexB (13C)|min] between the exchanging substates vary between 0.2 and 0.5 ppm, which compare well with the values for the free state (see above). The comparison of the free and TSA-bound states demonstrates that the side-chain conformational variability is retained and even increased in the latter state. Only two methyl groups, T75 and A81, show exchange in both the free and TSA-bound states, while the conformational exchange has changed from one methyl group to the other in the cases of residues I56, I76, I90, and V101. In addition, exchange contributions are seen for 14 methyl groups in the FK506-bound state that do not exhibit exchange in the free state (Table 2). In many cases, the exchanging methyl groups in the TSA-bound state are located close to residues exhibiting exchange in the free state, but their side chains point away from the active site (cf., Fig. 3b and f). Moreover, in a few cases, exchanging residues appear in more remote locations. Thus, the extended network of dynamic methyl groups in FK506-bound FKBP12 represents a shift from residues located in the immediate vicinity of the binding site in the free state to methyl groups located more distally from FK506 in the bound state. Notable examples of distal

Conformational Exchange in FKBP12

238 Table 2. Exchange parameters for methyl

13

C spins in free and FK506-bound FKBP12

Free FKBP12 Residue V4γ1 T6δ2 I7δ1 I7γ2 V23γ2 V24γ2 T27γ2 M29ɛ M49ɛ V55γ1 V55γ2 I56δ1 I56γ2 V63γ2 A64β M66ɛ T75γ2 I76δ1 I76γ2 A81β A84β I90δ1 I90γ2 I91δ1 A95β T96γ2 V98γ2 V101γ1 V101γ2 L103δ1

FK506-bound FKBP12

Φex (103 s− 2)

R1 (s− 1)

R2,0 (s− 1)

Χ2v

Rex (s− 1)

7.6 ± 0.9

2.02 ± 0.17

4.9 ± 0.4

1.29

1.0

21 ± 3

1.32 ± 0.11

5.3 ± 0.4

2.40

0.69 ± 0.06

4.5 ± 0.4

1.18

2.2

10 ± 1

0.75 ± 0.06

4.2 ± 0.4

2.09

1.3

5.3 ± 0.7

1.06 ± 0.09

4.8 ± 0.4

1.89

0.7

124 ± 15 18 ± 2

3.67 ± 0.31 1.75 ± 0.14

8.6 ± 0.8 5.2 ± 0.4

0.96 2.66

15.6 2.3

17 ± 2

0.59 ± 0.05

2.7 ± 0.2

1.39

2.1

24 ± 4 52 ± 6 31 ± 3

0.78 ± 0.07 1.07 ± 0.09 1.48 ± 0.12

2.8 ± 0.3 5.6 ± 0.5 5.5 ± 0.5

1.98 1.12 1.93

3.1 6.5 3.9

1.14 ± 0.10

4.6 ± 0.4

2.23

methyl groups that show conformational exchange only in the FK506-bound state include V23, T27, M29, M49, and A84.

Discussion The free state samples closed conformations In the absence of ligands, FKBP12 samples a minor (high-energy) substate that differs from the ground state in the conformation of the ligand binding (active site) region (Fig. 3a and b). The generally good correspondence between the regions showing chemical-shift differences between the minor and major substates in free FKBP12 and those with shift differences between the free and FK506- or peptidebound states [compare panel (a) with panels (c) and (e) and panel (b) with panel (d) of Fig. 3] strongly suggests that the minor state resembles the closed Michaelis complex. The fact that the minor substate is thermally accessible and transiently populated with an exchange rate of 8000 s− 1 is clearly of functional advantage since it implies that the conformational change needed for catalysis occurs rapidly and without great energetic cost that would otherwise need to be compensated for by binding energy between the substrate and the enzyme in its bound conformation. Similar observations have been made for a number of other proteins.9,43–48

R1 (s− 1)

R2,0 (s− 1)

Χ2v

Rex (s− 1)

17 ± 5

0.94 ± 0.05

4.3 ± 0.2

2.42

1.2

7.5 ± 3.0 19 ± 5 27 ± 7

0.65 ± 0.03 1.05 ± 0.05 0.75 ± 0.05

3.1 ± 0.2 3.9 ± 0.2 3.8 ± 0.2

1.10 2.46 3.09

0.5 1.3 2.0

28 ± 7 20 ± 5 26 ± 6 45 ± 9 29 ± 10

0.95 ± 0.05 0.36 ± 0.03 0.36 ± 0.03 1.72 ± 0.10 2.57 ± 0.14

3.8 ± 0.3 1.0 ± 0.2 0.7 ± 0.2 4.9 ± 0.3 5.9 ± 0.4

2.48 4.31 6.31 1.69 1.65

2.0 1.4 1.9 3.2 2.1

18 ± 4

0.85 ± 0.05

4.8 ± 0.3

1.16

1.3

16 ± 4 15 ± 4 18 ± 6 14 ± 5

1.10 ± 0.06 0.38 ± 0.02 1.11 ± 0.06 0.63 ± 0.03

4.5 ± 0.2 3.5 ± 0.2 4.8 ± 0.3 4.1 ± 0.4

2.13 2.26 2.24 1.54

1.2 1.1 1.3 1.0

35 ± 10 9.3 ± 3.1

2.64 ± 0.14 1.68 ± 0.08

6.1 ± 0.4 5.3 ± 0.3

2.95 2.08

2.5 0.7

19 ± 4

0.85 ± 0.04

3.4 ± 0.2

3.67

1.4

13 ± 4 13 ± 3

1.03 ± 0.06 1.15 ± 0.06

4.2 ± 0.2 3.8 ± 0.2

2.66 3.87

0.9 1.0

38 ± 8

1.49 ± 0.08

4.3 ± 0.3

3.29

2.7

2.7

18 ± 2

12 ± 2

Φex (103 s− 2)

1.6

The TSA-bound state shows differential backbone and side-chain dynamics The present results show that the responses upon binding FK506 are markedly different between the backbone and side chains. Taking the FK506-bound state to represent the transition state, the reduction in the conformational flexibility of the backbone agrees with the long-standing hypothesis that the transition state is more constrained than the Michaelis complex and the free state.3,6–8 However, it should be noted in this context that the present results correspond to a relatively minor entropic contribution to the free energy of binding that arises from the confinement to a single basin in the energy landscape, ΔS = − R∑ipiln(pi) ≤ Rln(2) = 5.8 J K − 1 mol− 1 (where pi is the population of substate i and R is the gas constant). Changes in the fluctuation amplitudes of fast timescale (picoseconds to nanoseconds) motions within each basin are expected to make larger contributions to the entropy of transition-state formation.49 Nonetheless, the present results indicate an entropic penalty against transition-state stabilization as a consequence of conformational restriction of the backbone. The residual side-chain dynamics sampled by methyl groups in the TSA-bound state suggests that the transition-state ensemble includes at least two substates with different conformations for approximately 35% of the methyl groups. Thus, as probed by the methyl groups, there is no evidence for

Conformational Exchange in FKBP12

preferential rigidification of the transition state as a consequence of transition-state stabilization. Instead, the observed redistribution of side-chain flexibility corresponds to “entropy–entropy compensation,” 50 which serves to increase the transition-state stabilization by increasing the entropy of the transition-state ensemble by a factor of ln(2), relative to the scenario where a single substate is populated (as in the case of the backbone). It would be highly interesting to complement the present data with methyl order parameters to probe fluctuations on the picosecond-to-nanosecond timescale. Conformational dynamics and interactions between FKBP12 and FK506 Binding of FK506 causes a minor change in the backbone structure of FKBP12, affecting mainly the loop regions close to the binding site (Figs. 1 and 3c). Below, we discuss in some detail the extent of correspondence between the observed conformational exchange and the structural change upon binding of FK506 with reference to both the identity of the affected residues and the magnitude of the chemical-shift differences. The chemical shift is an exquisitely sensitive probe of structural changes. The 15N chemical shift of the backbone amide depends on a number of structural factors, including hydrogen-bonding, backbone torsion (ϕ, ψ), and side-chain torsion (χ1) angles of the residue itself and of the preceding residue.51,52 Yet, it should be noted that the absence of chemical-shift changes cannot be taken as definite proof of the absence of structural change, because it is possible for the local magnetic field to be invariant to a given structural change; similarly, changes in the local magnetic field can be due to structural changes in the neighborhood of the observed residue and cannot be taken to indicate a structural change of this residue per se. The chemical-shift differences between the free and FK506-bound states (Fig. 3c and d) map closely to those regions of the structure that change upon FK506 binding (Figs. 1 and 3g and h), confirming that the chemical shifts are sensitive probes of structural changes in FKBP12. As noted above, the exchanging amide groups in the free state are located in areas that are highly affected by FK506 or peptide binding [compare panel (a) with panels (c) and (e) of Fig. 3]. Residues 26–44, 52–57, and 79–91 constitute three segments that guard the entrance to the FK506 binding pocket (Fig. 1). Conformational exchange is prominent in these areas: 42% of the available nitrogen atoms in the 26–44 stretch, 50% in 52–57, and 75% in 79–91 show significant relaxation dispersion. Residues 19– 21, 30–38, 50–57, 79–80, and 83–92 exhibit significant (0.5–2.1 Å) root-mean-square displacements (RMSDs) of their backbone N atoms upon binding of FK506 (Fig. 3g). Furthermore, the high-resolution crystal structure of free FKBP12 [Protein Data Bank (PDB) ID 2ppn; 0.92 Å] 14 resolves alternative conformations for many residues in these segments,

239 including R18, T27, M29, E31, R40, N43, L50, E60, T85, and H87, several of which also experience conformational exchange (cf., Table 1). The generally good correspondence between regions experiencing conformational exchange in the free state and those experiencing structural change upon binding FK506 is not mirrored by the residue-by-residue comparison of the corresponding chemical-shift differences. Linear regression of |ΔωexF| versus |ΔωF–B| indicates that these data are uncorrelated, regardless of whether |ΔωF–B| is evaluated based on the peptide-bound state or the FK506-bound state (rc b 0.3 for 15N and 13C; data not shown). The lack of correlation can be explained in part by the direct interactions between several residues in FKBP12 and FK506 (there is no structure available for the peptide-bound state), which are expected to have a significant influence on the chemical shifts in the FK506-bound state. We attempted to account for this fact by predicting the chemical shifts of the bound conformation (in the absence of FK506) using the programs SHIFTS53 and SHIFTX,54 but the correlation between the predicted chemical-shift differences and |ΔωexF| does not show any significant improvement. We further compared the crystal structures of free and FK506bound (PDB ID 1fkj)15 FKBP12 in terms of the structural parameters that have a major impact on the 15N chemical shift, viz. the hydrogen-bonding patterns, backbone ϕ or ψ torsion angle, or sidechain χ1 torsion angle. While 74% of the exchanging residues differ in one or several of these parameters between the two crystal structures, the data do not reveal any direct correlation between the exchanging residues and those that differ significantly in any of these parameters. We conclude that formation of the high-affinity complex is likely to involve induced-fit rearrangements of FKBP12 that go beyond the conformational changes between the ground and excited states in free FKBP12. It is also possible that the excited state includes a multitude of substates with different chemical shifts and populations. Since the relaxation dispersions are well represented by a two-state model, the interconversion between these substates is expected to be fast relative to τex. Consequently, the R1ρ data yield a value of |Δω exF | that corresponds to the difference in the population-weighted chemical shifts of ground- and excited-state ensembles, while |ΔωF–B| is heavily weighted towards the chemical shifts of the single (in the case of the backbone) bound conformation. In this model, the protein samples different substates that are capable of binding the different ligands.55 Conformational dynamics of FKBP12 and recognition of multiple targets The FKBP12 active site has been shown to bind several types of molecules. FKBP12 binds a variety of small organic compounds with partly common architectures, including TSA-based inhibitors such as FK506, ascomycin, rapamycin, and derivatives of

Conformational Exchange in FKBP12

240 these.56 In addition, FKBP12 binds a range of natural protein targets, some of which do not contain the canonical Leu–Pro motif preferred for peptidylprolyl cis–trans isomerase catalysis. For example, the binding pocket is occupied by a Leu–Leu motif in the complex with TβRI,57 and a Val-Pro motif is important for binding to the ryanodine receptor RyR1.58 Our present results on the conformational flexibility of the binding site help in explaining the capability of FKBP12 to recognize a range of targets with variable structural properties. The overall structure and backbone trace of FKBP12 do not adjust appreciably upon binding various targets, except for a few highly localized changes in the loops that surround the binding pocket (see above). The RMSDs of the Cα coordinates of FKBP12 between the FKBP12–TβRI complex and the FK506–FKBP12 and FK506–FKBP12–calcineurin complexes are less than 0.65 Å in both cases. Based on the observation that the backbone of FKBP12 is relatively rigid on the sub-nanosecond timescale, it has been proposed that the FK506–FKBP12 complex presents a rigid surface towards calcineurin (and, by inference, to other targets) that does not undergo any major rearrangement upon forming the ternary complex;41 the same conclusion has been reached for the complex with an FK506-derived neurotrophic ligand.59 By contrast, our current results paint a richer picture by demonstrating that many side chains of FKBP12 sample multiple conformations in the FK506-bound state as well. Turning next to the target recognition areas outside the FK506 binding site, we identified a conspicuous correspondence between residues that acquire conformational flexibility as a consequence of FK506 binding and those that have been implicated in target binding. It is likely that target recognition involves selection of preexisting sidechain conformers and that the apparent promiscuity of FKBP12 is made possible by the multiple conformations presented to target proteins. Below, we summarize the present results in light of the available structures and mutagenic analyses of various FKBP12–target complexes. In the FKBP12–FK506–calcineurin complex (PDB ID 1tco),60 the regions contacting calcineurin comprise residues 32–38, 40–47, 53, 54, and 87–90 (Fig. 4). Residues 37–46 and 87–90 undergo subtle conformational rearrangements upon binding calcineurin, including a change in the side-chain rotamer of I90. The structural change of these regions has been noticed previously as a potential prerequisite for surface complementarity and target recognition.60 Mutagenesis studies have highlighted residues K34, R42, and I90 as particularly important for calcineurin recognition.61,62 How do our results relate to these observations? There is no methyl-bearing residue to probe the dynamics in the protruding loop where R42 is situated (none between residues 31 and 48), but T21 and V23 are close to K47, while T27 and M29 are close to the region 34–39. Of these probes, V23, T27, and M29 sample multiple conformations in the FK506-bound state but not in the free state. The same

Fig. 4. Regions of FKBP12 interacting with target proteins. The surface representation of the FK506-bound state of FKBP12 is color-coded to highlight residues that interact with RyR1 (red), calcineurin (yellow), mTOR/ FRAP (blue), or both of the latter two proteins (green). The approximate locations of key residues are indicated.

is true for V55 (next to residue 54). I90 undergoes exchange in both states. Apparently, the regions that are critical for calcineurin recognition emerge as hot spots of conformational variability upon binding to FK506. The FKBP12–rapamycin complex inhibits the protein kinase mTOR/FRAP in a manner that involves few, but significant, points of contact between FKBP12 and mTOR/FRAP.63,64 In the five structures of the FKBP12–rapamycin–mTOR/FRAP complex available in the PDB†65 (PDB IDs 1nsg, 1fap, 2fap, 3fap, and 4fap),63,66 the contacts between FKBP12 and mTOR/FRAP cover the regions spanning residues 44–47 and 85–90 in FKBP12 (Fig. 4). The binding surface thus overlaps in part with that for calcineurin; additionally, in this case, I90 changes its conformation between the binary and ternary complexes.63 While the ligands (FK506 versus rapamycin) differ between these two cases, the fact that partly the same residues are involved in forming the intermolecular contacts suggests that the conformational exchange observed for the FK506–FKBP12 complex might serve a role in adapting the recognition surface to fit either of the two targets. The interaction between FKBP12 and the ryanodine receptors can be inhibited by FK506, indicating that FK506 and a receptor segment compete for the same binding site in FKBP12. Residues Q3, R18, and M49 have been shown by mutagenesis to be particularly important for the specificity of the interaction between FKBP12 and RyR1,67 but none of these residues is located in the FK506 binding pocket. Q3 is located on the surface on the back side of the protein in the views shown in Figs. 1, 3, and 4. R18 and M49 are distal from the FK506 binding site and situated close together (Fig. 4), with their † www.pdb.org

Conformational Exchange in FKBP12

methylene groups forming a hydrophobic surface patch that has been implicated in the binding of RyR1.67 It seems likely that residues Q3, R18, and M49 are involved in interactions that govern the selective binding of RyR1 over RyR2. However, a low-resolution (16 Å) cryo-electron microscopy model of the FKBP12–RyR1 complex suggests that Q3, E31, and D32 of FKBP12 face RyR1, while R18 and M49 point away from the modeled interface.68 At this low resolution, the model leaves unexplained both the role of R18/M49 and the dissociation of the FKBP12–RyR1 complex by FK506. As stated above, M49 shows exchange only in the FK506-bound state. Q3 is next to V4 and close in space to T75, both of which show exchange in the FK506–FKBP12 complex. V4 samples alternative conformations only in the FK506-bound state, while T75 is flexible also in the free state. These observations provide a third example of how the conformational exchange induced by FK506 involves regions on the surface that are important for target recognition. To summarize, binding of FK506 results in a redistribution of side-chain dynamics to incorporate methyl groups that are remote from the ligand binding pocket. Several of these methyl groups are located on the surface of FKBP12, in regions known to be involved in forming the interfaces of ternary complexes. We speculate that the residual conformational side-chain heterogeneity detected in this work might be an important factor in the pluripotency of FKBP12. Concluding remarks Comparisons of the free and FK506-bound FKBP12 reveal dramatic differences between the backbone and side chains in their dynamic responses to ligand binding. Experiments were performed for both backbone amide nitrogen atoms and side-chain methyl carbon atoms, thereby ensuring sampling of the conformational dynamics throughout the protein, including the active-site pocket interacting with FK506 and other ligands, as well as the surface regions that interact with natural target proteins. The relaxation dispersions reveal extensive flexibility in the free state of FKBP12, while the conformational exchange in the FK506bound state is restricted to the side chains. These results have a bearing both on the concept of transition-state stabilization and the promiscuous recognition of protein targets by FKBP12.

Materials and Methods Sample preparation Recombinant FKBP12 was expressed in Escherichia coli BL21⁎ grown with 15NH4Cl and optional 13C-labeled glucose as the exclusive nitrogen and carbon sources, respectively. Samples used in 13C relaxation experiments were obtained by preparing 50%/50% (v/v) H2O/D2O growth media to obtain 13CHD2 methyl moieties. The

241 proteins were purified as previously described.36 The NMR samples were made with 25 mM potassium phosphate buffer in 90%/10% (v/v) H2O/D2O at pH 7.0 with trace amounts of 2,2-dimethyl-2-silapentane-5-sulfonic acid and NaN3. The 15N relaxation experiments were performed on samples labeled exclusively with 15N FKBP12, using 0.6 mM FKBP12 to study the free state and 1 mM FKBP12 + 1.7 mM FK506 for the TSA-bound state. A sample of 1.0 mM 15N FKBP12 with the addition of approximately 2.5 mM Nsuccinyl-Ala-Leu-Pro-Phe-p-nitroanilide was used for the peptide binding studies. Samples uniformly enriched with 13 C and 15N and randomly partially (50%) enriched with 2H were used in the 13C methyl experiments—1.5 mM FKBP12 for the free state and 2 mM FKBP12 + 2.5 mM FK506 for the TSA-bound state. Experiments for resonance assignments utilized 1.8 mM uniformly 13C + 15N-labeled FKBP12 together with the 13C/15N/50% 2H-labeled FKBP12/ FK506 sample. NMR experiments All experiments were run on Varian INOVA spectrometers at a temperature of 20.0 ± 0.1 °C. Resonance assignments and 15N relaxation experiments were performed at a static magnetic field strength of B0 = 14.1 T, while 13 C relaxation experiments were acquired at B0 = 11.7 T. Previous amide backbone assignments69 were verified using three-dimensional 1H–15N TOCSY (total correlation spectrometry)–HSQC (heteronuclear singlequantum coherence).70,71 The amide backbone assignments were transferred from the free state to the TSAbound state based on a three-dimensional HNCA spectrum.72 Side-chain assignments were obtained using three-dimensional HCCH–TOCSY spectra.73 Each 15N relaxation experiment was acquired with 128 × 1024 complex points (F1 × F2) and spectral widths of 2000 and 9210 Hz, respectively. The 15N relaxation experiments on free FKBP12 were acquired using 16 R1ρ data points and 2 points per decay. Relaxation delays of 0–480 ms at a spin-lock field strength of ω1/2π = 1032 Hz were used, with offsets ranging from −400 to 50,000 Hz. To accurately quantify the dispersion step, we acquired 1–3 additional near-resonance data points with offsets less than ±67 Hz and a weak spin-lock field of ω1/2π = 168 Hz using the pulse sequence described by Massi et al.74 The 15N relaxation experiments on the FKBP12–FK506 complex were acquired with 15 spin-lock offsets using 3–10 points per relaxation decay. Relaxation delays were 0–180 ms at ω1/2π = 1032 Hz, with offsets ranging from −800 to 30,000 Hz. Each data set consisted of 64 × 512 complex points (F1× F2), with spectral widths of 2500 and 8000 Hz, respectively. The 13C relaxation experiments on the FKBP12–FK506 complex were run with 36 R1ρ data points and 15–20 points per decay. Relaxation delays of 0–400 ms and spin-lock field strengths ranging between ω1/2π = 816 and 3208 Hz were used, with offsets ranging from −15,000 to 250 Hz. All data were processed using NMRPipe.75 Intensities were measured from 3 × 5 (F1 × F2) data points centered on the peak maximum. Monoexponential decay curves were fitted to the intensity-versus-relaxation delay data sets. The R1ρ rates were fitted using simplex routines in Matlab (The Mathworks, Inc.) to the equations for fast exchange, given by:
ð1Þ R1U = R1 cos2 u + R2;0 + Rex sin2 u; where R1 is the longitudinal relaxation rate, R2,0 is the transverse relaxation rate in the absence of exchange, Rex is the exchange contribution, and θ is the tilt angle used

Conformational Exchange in FKBP12

242 in the dispersion experiment. In the case of two-state exchange, Rex is given by: Rex =

DN2 pA pB kex DN2 pA ð1  pA Þkex Aex kex = = 2 ; ð2Þ 2 2 2 2 kex + Neff kex + Neff kex + N2eff

where pA and pB are the relative populations of the two states, Δω and kex are the chemical-shift difference and rate of exchange between these, respectively, and ωeff = ω1/cosθ is the effective spin-lock field. The fast-exchange limit should hold in the present application because kex ≈ 8000 s− 1 (determined by simultaneous fitting to 15N and 13C relaxation-dispersion data) is significantly greater than the chemical-shift differences expected between the conformationally exchanging open and closed states, as gauged from the maximum chemicalshift differences between the free and bound states, |ΔωF–B (15N)|b 2.3 ppm and |ΔωF–B(13C)| b 2.0 ppm (see Results). Thus, kex/ΔωF–B(15N) is N9 and kex/ΔωF–B(13C) is N 5, which should be compared with the cutoff value of kex/Δω= 4, above which Eq. (2) adequately describes the exchange contribution to R1ρ.76 As an alternative and unbiased indication, we consider the average standard deviation in the chemical shift of the relevant spins as obtained from BioMagResBank‡ to obtain an estimate of the average expected chemical-shift difference between exchanging conformations. For backbone 15N spins in non-proline residues, this value is 4.0 ppm, whereas for methyl 13C spins, it is 1.6 ppm, corresponding to kex/Δω values of 5 and 6, respectively. Finally, as a consistency check, the minimum chemical-shift differences obtained from the fitted Φex values correspond to kex/ΔωexF(15N) ≤ 10 and kex/ΔωexF (13C) ≤ 11. The F-statistic was used to discriminate between the twoparameter (R1 and R2) and four-parameter (R1, R2, Φex, and kex) dispersion fits, with p b 0.01 considered significant.77 The dispersion step, Rex, needs to exceed ∼0.5 s− 1 to be quantified reliably in the above analysis, which limits the detected chemical-shift differences to |Δωex| N 20 Hz. Error estimates for the 15N data points were obtained from the background noise in the spectra. Error estimates for the 13C R1ρ relaxation rates were obtained from jackknife simulations. Calibrations of the applied spin-lock field strength were performed as described previously,78 resulting in errors of less than 6%. Global fits of the dispersion data were made using in-house software, with errors estimated using jackknife procedures. Structural comparisons of free and FK506-bound FKBP12 were based on the crystal structures of these species (PDB IDs 2ppn and 1fkj, respectively). The coordinate RMSDs of the backbone N and methyl C atoms were calculated by superimposing all heavy atoms of the structures. Hydrogen bonds were identified based on the criteria that the donor–acceptor (N–O) distance is less than 2.4 Å and that the angle between the N–H and N– O vectors is less than 35°. All figures representing protein structures were prepared using MOLMOL.79

Acknowledgements This work was supported by the Swedish Research Council, the Knut and Alice Wallenberg ‡ http://www.bmrb.wisc.edu

Foundation, and the Göran Gustavsson Foundation for Research in Medicine and the Natural Sciences. We thank Michael Rosen (University of Texas Southwestern, Dallas, TX) and Timothy Logan (National High Field Magnet Laboratory, Tallahassee, FL) for their generous gifts of FKBP12 clones.

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