Conformational Entropy of FK506 Binding to FKBP12 Determined by

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Conformational entropy of FK506 binding to FKBP12 determined by NMR relaxation and molecular dynamics simulations Gleb Solomentsev, Carl Diehl, and Mikael Akke Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b01256 • Publication Date (Web): 07 Feb 2018 Downloaded from http://pubs.acs.org on February 8, 2018

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Biochemistry

Conformational entropy of FK506 binding to FKBP12 determined by NMR relaxation and molecular dynamics simulations Gleb Solomentsev, Carl Diehl, and Mikael Akke* Biophysical Chemistry, Center for Molecular Protein Science, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden. ABSTRACT: FKBP12 (FK506 binding protein 12 kDa) is an important drug target. NMR order parameters, describing amplitudes of motion on the pico- to nanosecond timescale, can provide estimates of changes in conformational entropy upon ligand binding. Here we report backbone and methyl-axis order parameters of the apo and FK506-bound forms of FKBP12, based on 15N and 2H NMR relaxation. Binding of FK506 to FKBP12 results in localized changes in order parameters, notably for the backbone of residues E54 and I56 and the side chains of I56, I90, and I91, all positioned in the binding site. The order parameters increase slightly upon FK506 binding, indicating an unfavorable entropic contribution to binding of T∆S = –18 ± 2 kJ/mol at 293 K. Molecular dynamics simulations indicate a change in conformational entropy, associated with all dihedral angles, of T∆S = –26 ± 9 kJ/mol. Both these values are significant compared to the total entropy of binding determined by isothermal titration calorimetry and referenced to 1 mM reactant concentration, T∆S = –29 ± 1 kJ/mol. Our results reveal subtle differences in the response to ligand binding compared to the previously studied rapamycin–FKBP12 complex, despite the high structural homology between the two complexes and their near-identical ligand–FKBP12 interactions. These results highlight the delicate dependence of protein dynamics on drug interactions, which goes beyond the view provided by static structures, and reinforce the notion that protein conformational entropy can make important contributions to the free energy of ligand binding.

INTRODUCTION The FK506 binding protein 12 kDa (FKBP12) is a 12 kDa peptidyl-prolyl cis-trans isomerase involved in several cellular processes. It is the target of the immunosuppressive drugs FK506 and rapamycin,1, 2 which are two naturally occurring macrolides of similar structure (Figure 1), and many synthetic derivatives of these. Both FK506 (also known as tacrolimus) and rapamycin (sirolimus) bind with high affinity (sub-nanomolar dissociation constants) to FKBP12. FK506 exerts its immunosuppressant function by forming a complex with FKBP12, which then binds to and inhibits calcineurin, leading to loss of both T-lymphocyte signal transduction and interleukin-2 transcription.3 Rapamycin has similar effects on the immune system, but acts via the rapamycin–FKBP12 complex by inhibiting mTOR (mammalian or mechanistic target of rapamycin), which regulates cell growth and metabolism.4 Current understanding indicates a primary physiological role of FKBP12 in regulating signaling pathways, most notably by interacting with the ryanodine receptor calcium channel 5-7 and the transforming growth factor β type I receptor. 8, 9 More recently, multifaceted roles have emerged for FKBP12 in modulating neurodegenerative disease, which appears to be related to its cis-trans isomerase function. Inhibition of FKBP12 by FK506 has been shown to reduce α-synuclein aggregation and cell death in a Parkinson's disease model.10, 11 FKBP12 also appears to be involved in processing of the amyloid precursor protein towards the amyloidogenic pathway12 and has been shown to co-localize with neurofibrillar tangles observed in Alzheimer's disease.13 Furthermore, FKBP12 prevents aggregation of peptide segments from the tau protein.14

Several structures are available for FKBP12 in the ligandfree (apo)15-17 and ligand-bound forms.16, 18-21 The binding site is a relatively shallow hydrophobic pocket between an α-helix and a 6-stranded β-sheet and the intervening loops composed of residues 39–46, 50–56, and 82–95 (Figure 1). The pocket is delineated by a number of aromatic residues (Y26, F36, F46, W59, H87, and F99), but comparatively few methyl groups (V55, I56, and I91).19 Ligand recognition includes a number of key hydrogen bonds involving the backbone carbonyls of Q53 (via a water molecule) and E54, the backbone amide of I56, and the side chains of D37 and Y82. Comparing 14 human FKBP domains, the most conserved residues in the binding site are Y26, F46, and F99, which all interact with the most buried regions of FK506 and rapamycin, and the hydrogen bonding residues D37, I56, and Y82.22 A secondary network of hydrogen bonds between the ligand-coordinating residues and neighboring ones, several of which are located in the flanking loops, provide additional stabilization of the complex. Despite the relatively large size of the macrolide ligands, only small structural changes are observed between the various apo and ligand-bound states determined by Xray crystallography (Fig. 1), with a backbone RMSD of approximately 0.4 Å between apo and FK506-bound FKBP12. FK506 and rapamycin form highly similar complexes with FKBP12: the two drugs are virtually identical in those regions that interact with FKBP12, the drug–FKBP12 contacts involve the same atoms, and the protein structure is very similar in the two complexes with a backbone RMSD of only 0.4 Å. Similarly, all close analogues of FK506 and rapamycin bind in an equivalent

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mode, where the common ligand moieties interact with the most highly conserved residues in FKBP12.22

Figure 1. Structure of FKBP12 in the apo (red, PDB id 2PPN17), FK506-bound (cyan, 1FKJ16), and rapamycinbound (green, 1FKL16) states. (A) The FKBP12 backbone trace and the ligands are shown in stick representation with FK506 colored in dark blue and rapamycin in dark green. (B) Close-up view of the binding site with FK506 (cyan) and rapamycin (green) shown as thick sticks, the protein Cα trace as medium sticks, and side chains shown as thin sticks. The figure was produced using the PyMOL molecular graphics system (Schrödinger, LLC).

The structural similarities of the FK506–FKBP12 and rapamycin–FKBP12 complexes raise the question whether differences in the conformational dynamics of the complexes might contribute to the difference in their target specificities and consequent action on different signaling pathways. Indeed, previous work has shown that the FK506–FKBP12 and rapamycin–FKBP12 complexes exhibit very different conformational exchange processes on the micro- to millisecond timescale.23-25 It was observed that the backbone undergoes conformational exchange in apo-FKBP12, and that binding of FK506—but not rapamycin—quenches this process. In contrast, the methyl-bearing side chains show increased conformational exchange upon binding either ligand. Furthermore, the two ligands induce side-chain dynamics in different regions of the protein that are remote from the FK506/rapamycin binding site, but involved in binding additional protein targets, suggesting an allosteric role for the increased population of transient, high-energy conformational states.24, 25 A recent study on aromatic ring flips in FK506- and rapamycin-bound FKBP12 also revealed significant differences in rare large-amplitude fluctuations between the two complexes.26 Taken together, these differences in conformational

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exchange suggests a functional role of dynamics in target recognition and allostery. In addition to the sampling of distinct, high-energy states mentioned above, conformational fluctuations within the ground state also influence protein function, because they contribute to conformational entropy, which has emerged as an important factor in ligand binding and allostery.27-33 NMR order parameters34-36 describe the probability distribution of bond vector orientations, which is related to conformational entropy.28, 37, 38 Changes in order parameters upon ligand binding thus provide an estimate, subject to a number of limitations,33, 39, 40 of conformational entropy contributions to the free energy of binding. In the case of globular, wellfolded proteins it is expected that conformational entropy originates primarily from the ensemble of states sampled on sub-nanosecond timescales.33, 40, 41 Several previous NMR studies have been carried out to characterize the dynamics of apo and ligand-bound FKBP12 on the pico- to nanosecond timescale.25, 42-45 Seminal studies carried out by Moore and co-workers during the early 90's focused on changes in backbone order parameters of FKBP12 upon binding of FK506.42, 43 In their initial study of free FKBP12,42 these authors reported difficulties in observing residues in the loop regions flanking the active site cleft (residues 34–45 and residues 78–95), indicating that significant exchange dynamics was present in these regions. Follow-up work indicated reduced amplitudes of motion in the 80’s loop (residues 78–95) upon binding of the FK506 inhibitor, but otherwise few notable differences between the apo and FK506-bound states.43 FKBP12 is an actively pursued drug target22, 46 and commonly used as a model system in computational studies of ligand binding and drug design.47-53 Computational approaches employing molecular dynamics simulations often utilize experimental order parameters as a means to validate the resulting trajectories.54-58 Thus, it is essential for this purpose that the field has access to an accurate set of order parameters for FKBP12 in its apo and ligand-bound states. Developments over the last two decades have resulted in improved characterization of protein dynamics by NMR relaxation methods,59-61 enabling us to provide an updated set of order parameters for FKBP12. Here we extend previous results on fast timescale conformational fluctuations of FKBP12 and address the role of conformational entropy in ligand binding. We characterized the fast timescale (ps–ns) backbone and side chain dynamics of apo and FK506-bound FKBP12 in terms of model-free order parameters.35, 36 The results for apo FKBP12 are in excellent agreement with those reported recently by Sapienza et al.,25 making a strong case for the accuracy of the order parameters. Together with our present results on FK506-FKBP12, these data serve as an update of the original 15N backbone order parameters published by the Vertex group in 1993–4.42, 43 In addition, we report for the first time 2H methyl-axis side chain order parameters for both apo and FK506bound FKBP12. We have also carried out 40 ns long molecular dynamics (MD) simulations for each of the apo and FK506-bound states that augment the experimental data by offering mechanistic insights into differences in


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Biochemistry

order parameters and estimates of the total conformational entropy of ligand binding to FKBP12. Finally, we present a detailed analysis of differences in order parameters between the FK506- and rapamycin-bound states, revealing subtle variations between these two states in their conformational fluctuations, which apparently result from differences in drug interactions with FKBP12 that cannot be resolved from the static structures alone. MATERIALS AND METHODS NMR Sample Preparation. FKBP12 expression and purification was performed as reported previously.23, 24 15N and 15N/13C/2H labeled samples were prepared in 25 mM phosphate buffer, pH 7.0, 90%/10% (v/v) H2O/D2O. The resulting apo-FKBP12 sample concentrations were 1.1 and 1.5 mM for the 15N and 15N/13C/2H samples respectively. The FK506 ligand was dissolved in a stock solution of ethanol. FK506 bound samples were prepared by titrating the ligand into apo-FKBP12 samples while monitoring the FKBP12 chemical shifts in the 15N heteronuclear single-quantum coherence (HSQC) spectra. At the end of the titration the total amount of added ethanol was 2% (v/v). All NMR samples included small amounts of NaN3, 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) and D2O. Backbone 15N Relaxation Experiments. R1, R2, and {1H}–15N nuclear Overhauser effect (NOE) experiments62, 63 targeting the 15N spins of the backbone were performed on apo and FK506-bound FKBP12 at static magnetic field strengths of 11.7 and 14.1 T and a temperature of 293 K. Typically 8–12 data points were acquired in an interleaved manner with relaxation relays in the range of 0–1 s for R1 or 0–0.192 s for R2, using a recycle delay between experiments of 2.0 s. R2 was measured using a 1.2 ms delay between refocusing pulses in the CPMG train. {1H}–15N heteronuclear NOEs were measured in an interleaved manner using a 1H saturation time of 7 s and a recycle delay of 10 s between acquisition and the first 15N pulse in both the NOE and the control experiments. For the saturation of the proton magnetization, 180° pulses were used.64 Spectral widths were 8013 and 1835 Hz in the 1H and 15N dimensions, respectively, sampled over 1024 and 128 points. The carrier frequencies of 1H and 15N were placed on the water frequency and in the center of the backbone amide region, respectively. Side-Chain Methyl 2H Relaxation Experiments. We measured the following 2H relaxation rates: R(Dz), longitudinal magnetization; R(3Dz2–2), quadrupolar order; R(D+), in-phase transverse magnetization; and R(D+Dz+DzD+), antiphase transverse magnetization.65 Experiments were performed on 2H–13C-labeled methyl groups of apo and FK506-bound FKBP12 at magnetic field strengths of 11.7 and 14.1 T and a temperature of 293 K. The spectral widths were 8012 Hz and 3000 Hz in the 1H and 13C dimensions, respectively, covering 1024 and 84 points. The B1 field strength of the 2H channel was 1.05 kHz. Relaxation data were acquired using interleaved delays in the range 0–40 ms for R(Dz) and R(3Dz2–2) and 0–20 ms for R(D+) and R(D+Dz+DzD+) and sampled by 8–12 data points. A recycle delay of 2.0 s was used in all experiments.

Data Processing and Model-free Optimization. NMR data were processed using NMRPipe.66 Errors in the relaxation decays were estimated based on the noise in the spectrum. Peak intensities were measured as the summed signal in windows of 5×3 (1H×15N/13C) points centered on the peak. The signal-to-noise ratio (S/N) was estimated by calculating the standard deviation of 200 samples of integrated 5×3 windows in empty regions of each spectrum. Mono-exponential functions were fitted to the R1 and R2 decays using the LevenbergMarquardt minimization routine, as implemented in Cprograms developed in-house. Errors in the fitted parameters were estimated from 1000 synthetic data sets created using Monte Carlo simulations. Each NOE was calculated as the ratio of the peak intensitites in the saturated and unsaturated experiments. The S/N was estimated as described above, and the errors in the NOE were determined by error propagation. Initial estimates of the overall correlation time, τc, was based on the trimmed mean value of R2/R1; the trimmed mean and standard deviation were calculated for each data set of R1, R2, and NOE, where residues outside of two standard deviations from the mean were excluded in a single pass. Relaxation data were interpreted using the model-free (MF) formalism35, 36 with an N–H bond length of 1.02 Å and a CSA of 172 ppm, as implemented in relax version 1.3 or 4.0.2.67, 68 Initial estimates for backbone order parameters were based on the local τm model and these values were used as seeds for fitting of higher order diffusion tensors. Four models were used to fit backbone MF parameters: S2 (model 1); S2 and τe (model 2); S2 and Rex (model 3); and S2, τe, and Rex (model 4). Model-free parameters for the side-chain methyl groups were optimized against the experimental relaxation rates using in-house routines implemented in MATLAB. S2 and τf were fitted assuming an isotropic diffusion tensor.69 Enforcing an isotropic diffusion model resulted in systematic errors of < 3% in the back-calculated 2H relaxation rates; this value was much less than the typical variation between states. The global correlation time was determined by 15N relaxation, as described above. The weighted mean value of the order parameter (for a given state) was calculated as: 〈〉 = / / (1/ )

where xi is S2 for residue i, σi is the standard error of S2 for residue i, and the sum runs over all residues.70 The weighted pairwise RMSD in order parameters between two states (e.g., between apo and FK506-bound) was calculated as:

= / / (1/ )

where xi is the difference ∆S2 between states for residue i, σi is the standard error in ∆S2 for residue i, and the sum runs over all residues. The conformational entropy of the backbone and the methyl-bearing side chains was estimated from the order parameters using the dictionary approach presented by Li and Brüschweiler.41


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Molecular Dynamics Simulations and Trajectory Analyses. Molecular dynamics simulations were carried out using GROMACS v 4.0.5,71, 72 using the Amber99SB force field.73 Force field parameters for the FK506 ligand were based on the General Amber Force Field (Gaff).74 Long-range electrostatics were treated with particle mesh Ewald summation (PME).75-77 Hydrogen atoms were constrained using the SHAKE algorithm.78 Starting structures for the simulations of apo and FK506-bound FKBP12 were taken from PDB entries 2PPN17 and 1FKF,19 respectively. The structures were solvated and slowly heated in 20 ps increments to a temperature of 294 K, with starting velocities generated from a MaxwellBoltzmann distribution at 60 K and with a harmonic positional restraint force constant of 5000 kJ/mol/nm applied to the heavy atoms. With each incremental trajectory segment (of 20 ps) the restraint force constant was halved progressively to finally reach 300 kJ/mol/nm. For subsequent simulations, the positional restraints were not applied. An initial trajectory with a total length of 10 ns was carried out for both FK506bound and apo FKBP12. Using restart coordinates taken at 2.5, 5.0, 7.5 and 10.0 ns, we initiated 4 simulations, each 10 ns long, resulting in a total of 40 ns of simulation time for each of the two states. Backbone and side-chain order parameters were evaluated using the iRED method,79 using a 1 ns window as described by Genheden et. al.80 Conformational entropy was estimated from MD simulations based on dihedral angle sampling histograms as described previously.81, 82 A conformational entropy term was calculated for each replicated simulation and averaged across the four simulations for the two states (apo and FK506-bound FKBP12). All backbone and side-chain dihedral angles were included in the calculations. The entropy S for each simulation was calculated as = − ln( )

where the R is the universal gas constant, pi is the probability of dihedral angle state i. The conversion of the difference in entropy between the apo and FK506-bound states with respect to the bin width was assessed by calculating S for each state with bin widths varying from 2° to 10°. Convergence was reached with a 5° bin width, which was used in the calculations reported below. RESULTS AND DISCUSSION We carried out 15N and 2H relaxation experiments on apo and FK506-bound FKBP12 at two static magnetic field strengths, B0 = 11.7 and 14.1 T, and a temperature of 293 K. The results enable us to evaluate the change in subnanosecond intramolecular dynamics of the backbone and methyl-bearing side chains upon binding of FK506 by FKBP12, and to compare with the results obtained previously for rapamycin-bound FKBP12.25 We also carried out MD simulations of apo and FK506-bound FKBP12, which we validated against the experimental order parameters and further analyzed to extract the conformational entropy of the protein. 15N relaxation data (R1, R2, and NOE) could be measured for 91 and 96 backbone amides in apo and FK506-bound FKBP12, respectively. The larger number of probes avail-

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able in FK506-FKBP12 reflects the lower degree of overlap and less exchange broadening of the NMR spectrum of this state, which can be attributed to the stabilizing effect of the inhibitor in regions of the protein involved in ligand binding.42, 43 The average relative errors in the R1, R2, and NOE are: 0.4%, 0.4–0.8%, and 2–5%, respectively, for apo FKBP12 and 0.5–0.5%, 0.4–0.8%, and 2% for FK506-FKBP12 (where the ranges indicate the variation between data sets acquired at different B0 field strengths). 2H relaxation rates could be measured for 41 and 40 methyl groups in apo and FK506-bound FKBP12, respectively. The average relative errors in the relaxation rates R(Dz), R(3Dz2–2), R(D+), and R(D+Dz+DzD+) are: 2–3%, 7–9%, 3–5%, and 6–7%, respectively, for apo FKBP12 and 5–6%, 6–12%, 3–4%, and 6–8% for FK506-FKBP12. Model-Free Analysis. We analyzed the backbone 15N relaxation data sets using the model-free formalism.35, 36 The apo and FK506-bound states were both best fit using a prolate diffusion tensor with a global correlation time (τc) of 7.9 and 8.4 ns, respectively, and anisotropy (D /D⊥) of 1.27 and 1.19, respectively. A recent study by Sapienza and coworkers reported τc values of 6.8 and 7.1 ns for FBKP12 in the free and rapamycin-bound states, respectively, at 298 K.25 The correlation times measured here are in excellent agreement with those determined previously,25 taking into account the 5 K difference in temperature between the two studies and the fact that rotational correlation times of macromolecules in dilute aqueous solutions vary with temperature roughly as 0.2 ns/K due to the nearly linear temperature dependence of the factor η(T)/T, which appears in the Stokes-Einstein equation, with η representing the solvent viscosity. The similar correlation times of apo FKBP12 and the ligandbound complexes are expected, because the apo and ligand-bound states have nearly identical structures. At the same time, the consistently higher values of τc observed for both ligand-bound states compared to apo are explained by the protrusion into the solvent of the peripheral moieties of the ligands (Fig. 1). We note that the present τc values differ significantly from older results by Cheng et al., which yielded τc = 9.2 and 9.3 ns for the apo and FK506-bound states at 303 K.42, 43 The higher values of τc determined previously likely arise from the higher protein concentration (8 mM compared to 1.5 mM), which might lead to intermolecular interactions causing a reduction in rotational diffusion. The full set of optimized backbone MF parameters is listed in Supplementary Information Tables S1 and S2. The majority of residues were fitted with either MF models 3 or 4, i.e., including an Rex term, but in half of the cases the resulting value is small (< 1 s–1). Such small Rex terms commonly arise as a consequence of minor differences between the solution and crystal structures in the orientations of the N–H bond vector in the principal-axis frame of the diffusion tensor. FK506-FKBP12 has only 6 residues with Rex > 2 s–1, with the largest values observed for Q53, E54 and V55. This exchange was not observed in previous 15N R1ρ experiments,24, 25 indicating that the exchange rate is relatively slow such that it affects the relaxation decay sampled by the present R2 CPMG experiment, but is quenched by the higher refocusing fields


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Biochemistry

used in the R1ρ experiment. The MF fits for apo-FKBP12 yield 15 residues with Rex > 2 s–1. These exchange contributions agree well with those determined by R1ρ experiments,24 thus indicating that the MF fits accurately portray the slow timescale dynamics in apo FKBP12 (Supplementary Fig. S1). Reproducibility of Order Parameters for Apo FKBP12. A detailed comparison of our present results on apo FKBP12 with order parameters published by Sapienza et al.25 shows excellent agreement between the two data sets (Fig. 2A, B). It should be noted that the present data were acquired at a temperature of 293 K, while the work of Sapienza et. al. was carried out at 298 K. Therefore our data yield slightly higher order parameters, with the average difference being = 0.01 for the backbone and = 0.03 for the methyl axes (ignoring residues at the termini). The modest temperature dependence of S2 is expected for a well-ordered protein.83 The weighted pairwise RMSD between the two sets of backbone order parameters is 0.02 and the maximum absolute difference is |∆S2|max = 0.09. Only 9 residues have |∆S2| > 0.03. The methyl-axis order parameters have a weighted pairwise RMSD of 0.04 and |∆S2|max = 0.2. Two independent studies, carried out in two different labs, using slightly different experimental methodology, have thus arrived at very similar order parameters. We conclude that the excellent agreement vouches for a high level of accuracy in the determined order parameters for apo FKBP12, and we expect that the same holds true also for the ligand-bound states. In analyzing differences in order parameters between any two states (e.g., apo and ligand-bound, or FK506and rapamycin-bound), one should keep in mind that the level of reproducibility of the apo data sets a limit on how small values of ∆S2 one can interpret with confidence. Thus, the RMSD between apo data sets indicates that, on average, values of ∆S2 < 0.02 (backbone) and 0.04 (methyls) are not significant. Below, we carry out comparisons of order parameters for the apo and FK506bound states, as well as the rapamycin-bound state. In so doing, we report all ∆S2 ≥ 0.02 (backbone) or 0.04 (methyls) that are statistically significant given the estimated uncertainties of the two residue-specific S2 values that go into the pairwise comparison.

S2 from this work, while black dots indicate S2 determined by Sapienza et al.25 (A) 15N backbone amide S2. (B) 2H methyl-axis S2.

Backbone Order Parameters: Changes Upon FK506 Binding. The backbone order parameters of apo and FK506-bound FKBP12 are compared in Supplementary Fig. S2. The individual S2 values range between 0.72–0.95 in apo-FKBP12 and 0.77–0.96 in FK506-FKBP12, the weighted mean values are 0.86 ± 0.03 in both cases, and the weighted pairwise RMSD is 0.02, indicating that, on average, ligand binding does not induce a significant change in backbone order parameters. However, residue-by-residue pairwise comparisons show that individual residues are indeed affected by FK506 binding (Fig. 3A). Residues showing significant changes in backbone S2 are mapped onto the structure in Fig. 3C. Several of these residues, but not all, also show chemical shift changes upon ligand binding; conversely, many residues that show shift changes do not exhibit changes in order parameters (Fig. S3). As would be expected, residues that become rigidified upon FK506 binding are found primarily in the active site. Specifically, the segment 47–56 includes several residues with ∆S2 ≥ 0.02. We note that the 80's loop does not stand out in this comparison, in contrast to previous results42, 43 but similar to recent results for rapamycin binding.25 Several of the observed changes in S2 can be understood in terms of the structure of the FK506–FKBP12 complex. The largest increases in S2 occur for residues Q53, E54, and I56 (∆S2 = 0.04–0.16). These changes are explained by the interactions with FK506. As outlined above, the carbonyl of E54 hydrogen bonds to a hydroxyl of FK506,19 and the amide of I56 forms a hydrogen bond to the lactone carbonyl of the α-keto amide region of the pipecolic ring that is conserved in all determined structures of FKBP12 in complex with FK506-related compounds.51. Surprisingly, these changes were not observed in the studies by Cheng et al.42, 43 We also observe a stiffening of F48, which seems to propagate via hydrogen bonds across the β-sheet to residue C22 (∆S2 = 0.04; hydrogen bonded to F48) and further to K105 (∆S2 = 0.05; hydrogen bonded to V23). Reduced backbone order parameters in the bound state are observed for residues G58 and V61 on the external side of the helix located next to the segment 50–56, which shows increases in S2 (Fig. 3A, C).

Figure 2. Comparison of order parameters for apo FKBP12 determined in two independent studies. Solid bars indicate


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1. The figure was produced using the PyMOL molecular graphics system (Schrödinger, LLC).

Figure 3. Difference in order parameters between the apo and FK506-bound forms of FKBP12, ∆S2 = S2(FK506) – S2(apo). Residues shown in blue (∆S2 > 0) and red (∆S2 < 0) become more rigid and flexible, respectively, upon binding of FK506. (A) ∆S2 from 15N backbone data. (B) ∆S2 from 2H methyl data. (C) ∆S2 for backbone amides mapped onto the FK506-FKBP12 structure (PDB id 1FKJ16) with cyan and pink indicating 0.02 ≤ |∆S2| < 0.04 and dark blue and red indicating |∆S2| ≥ 0.04. (D) ∆S2 for methyl axes mapped onto the FKBP12 structure. Methyl-bearing side chains with |∆S2| > 0.05 are shown in space-filling representation with cyan and pink indicating methyl groups with 0.05 ≤ |∆S2| < 0.15, and blue and red indicating ∆S2 ≥ 0.15. For reference, the backbone is color coded as in panel C. FK506 is shown in green. The view is similar, but not identical, to that in Fig.

Methyl-Axis Order Parameters: Changes Upon FK506 Binding. As a complement to the backbone 15N relaxation study, we also performed 2H relaxation measurements on methyl-bearing side chains. We interpreted the resulting relaxation rates in terms of methyl-axis order parameters, following established methods,65, 69 with τc fixed at the value determined from the 15N relaxation data. The methyl-axis MF parameters are listed in Tables S3 and S4. The S2 values range between 0.31–0.96 in apoFKBP12 and 0.28–0.98 in FK506-FKBP12 (Fig. S2). Thus, the variation in S2 among residues is considerably greater for the side chains than for the backbone, as usually found.84 Values of S2 ≥ 0.90 are observed primarily for the short side chains of alanine and threonine, but also for I56γ 2 in the bound state, where it packs against FK506, and for M66ε (in both states), which packs tightly into the interior of the hydrophobic core. By contrast, the other two methionines, M29 and M49, are located on the surface of the protein and are highly flexible in both states, 0.30 ≤ S2 ≤ 0.33. Upon binding FK506, the change in order parameter is correspondingly greater for the side chains than for the backbone, as might be expected from the greater variation in S2 values for the former. There is a modest trend of loss of flexibility upon ligand binding (Fig. 3B, D), with = 0.04 and a weighted pairwise RMSD of 0.06. Order parameters could be measured in both states for 33 methyl groups. I91δ1 shows the largest increase in S2 (∆S2 = 0.52) and packs directly against FK506. The neighboring methyl group I90δ1 also shows an increase with ∆S2 = 0.16. A sizeable increase in S2 is observed also for I56 γ 2, ∆S2 = 0.16 , which also packs against FK506. Both I56 and I91 are highly conserved across the FKBP family in humans,22 suggesting that these tight interactions with the ligand might be representative for FKBP12 complexes in general. In addition, ∆S2 > 0.05 is observed for residues V23, L50, V63, T75, V98, and L103. L50 and I56 are located in a segment of residues showing increased backbone order parameters upon FK506 binding (cf. Fig. 3C and D). Further, the side chains of V63 and L103 are located close to those of the 50's segment, while the side chains of T75 and V98 pack against one another. One group of residues showing ∆S2 < –0.05 form a single interacting cluster involving V24, M66, L74, and V101, while A64 and L106 are both located close to this group, but not in direct contact (Fig. 3D). Thus, there is a tendency for clusters or segments of neighboring residues to respond to ligand binding in a similar fashion, suggesting that damping of conformational fluctuations propagates through the protein structure via direct interactions. We note that the cluster of side chains with ∆S2 < 0 is bracketed by residues showing increased S2 values upon FK506 binding, indicating that the reduced fluctuations of the surrounding side chains free up conformational space for the neighboring residues. Order Parameters from Molecular Dynamics Simulations. We carried out a series of MD simula-


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tions, resulting in a total of 40 nanoseconds of simulation data on both apo and FK506-bound FKBP12. We calculated backbone and side-chain order parameters from the MD trajectories of both states and compared the results to the experimental data (Fig. S4). In comparing the NMR- and MD-derived order parameters for the apo and FK506-bound FKBP12, one of the most noteworthy aspects is the general similarity in the trends of both the backbone and methyl-axis order parameters. Backbone order parameters are reproduced with similar accuracy for the apo and FK506-bound states with an RMSD of 0.05. These values are on par with previous benchmarking of MD simulations against experimental NMR data,81, 85 but slightly worse than the RMSD between independent NMR data sets for apo FKBP12 (with an RMSD of 0.02, as reported above) and other proteins.83, 85 The RMSD between experimental data sets offers a limit on the level of agreement that can reasonably be expected when assessing the performance of MD simulations in reproducing order parameters. Methyl side-chain order parameters show a lower degree of agreement, presumably owing to the much greater range of S2 values for these moieties (see above), yielding RMSDs of 0.18 and 0.19 for the apo and FK506-bound state, respectively. Again, these RMSD values are comparable with previous results,86 but higher than the RMSD of 0.05 between the two experimental data sets. While the MD simulations do not precisely reproduce the experimental order parameters for individual residues, they are clearly capable of capturing the trends along the protein sequence with fair agreement (Fig. S4). Notably, the MD simulations reproduce the lower order parameters of the 80's loop in both the apo and FK506-bound states, although the precision in S2 is lower in general for flexible residues as a consequence of limited sampling. Thus, the MD simulations and NMR data both indicate that the sub-nanosecond timescale motions of the 80's loop do not become highly restricted upon ligand binding. Differences in Order Parameters Between FK506 and Rapamycin Bound FKBP12. Given the excellent reproducibility of the order parameters for apo FKBP12, it is of interest to carry out a direct comparison of the responses to binding either FK506 or rapamycin. In comparing the two ligand-bound states, we find a high level of agreement for individual residues with weighted pairwise RMSDs of 0.02 and 0.04 for the backbone and methyls, respectively (Fig. S5). Partly as a consequence of the 5 K difference in temperature between the two data sets, FK506-FKBP12 has slightly higher order parameters on average, with = 0.01 for both the backbone and methyl axes, similar to the results from the two data sets for apo FKBP12. Elevated differences in S2 between the two ligand-bound states could arise for residues whose order parameters are more temperature dependent than others, but given the generally good agreement between the two apo data sets, we expect that such effects are minor. Statistically significant differences in S2 between individual residues in the FK506- and rapamycin-bound states are highlighted in Fig. 4. These differences primarily involve residues that are located close to the bound ligand. In several cases, the affected residues also experi-

ence changes in S2 with respect to the apo state (cf. Fig. 3). The backbone amide of I56 shows higher S2 in rapamycin-bound FKBP12 than in the FK506-bound state (Fig. 4A, C). As noted above, the amide of I56 hydrogen bonds to a carbonyl of the ligand in both cases. Thus, the higher S2 likely indicates a stronger interaction with the ligand in the case of rapamycin-bound FKBP12, in agreement with theoretical predictions.87

Figure 4. Difference in order parameters between the FK506-FKBP12 and rapamycin-FKBP12 complexes, ∆S2 = S2(FK506) – S2(rapamycin). Residues shown in blue (∆S2 > 0) and red (∆S2 < 0) are more rigid and flexible, respectively, in FK506-FKBP12 than in rapamycin-FKBP12. (A) ∆S2


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from 15N backbone data. (B) ∆S2 from 2H methyl data. (C) ∆S2 for backbone amides mapped onto the FK506-FKBP12 structure (PDB id 1FKJ16) with cyan and pink indicating 0.02 ≤ |∆S2| < 0.04 and dark blue and red indicating |∆S2| ≥ 0.04. (D) ∆S2 for methyl axes mapped onto the FKBP12 structure. Methyl-bearing side chains with |∆S2| ≥ 0.05 are shown in space-filling representation with cyan and pink indicating methyl groups with 0.05 ≤ |∆S2| < 0.15, and dark blue and red indicating |∆S2| ≥ 0.15. For reference, the backbone is color coded as in panel C. FK506 is shown in green. The view is identical to that in Fig. 3. The figure was produced using the PyMOL molecular graphics system (Schrödinger, LLC).

Differences are also observed for the segment 33–41, where S2 is higher in the FK506-bound state for G33, K34, and D41, whereas the opposite holds for F36, S38, and S39. The latter three residues are located in the outermost strand that forms part of the active site lip, while the former three are located in loop regions bracketing this strand. In addition, K47 and F48, as well as S77 and D79, next to the 80's loop, have higher S2 in the FK506-bound state. Inspection of the two complexes does not reveal any obvious differences in the interactions between FKBP12 and FK506 or rapamycin that can explain these differences. Only 7 methyl-axis order parameters are significantly different between the FK506 and rapamycin-bound states (Fig. 4B, D). S2 is higher in the FK506-bound state for V2γ1, I7δ1, L30δ2 (partially hidden behind I91 in the view of Fig. 4D), and I91δ1. The side chains of L30 and I91 pack against one another. In contrast, S2 is higher in the rapamycin-bound state for I7γ2, T27γ2, and I91γ2. Again, these differences in order parameters are not readily explained by the structures of the two complexes. Conformational Entropy of Ligand Binding. An estimate of the change in conformational entropy associated with ligand binding can be gauged from the order parameters of the free and bound states.28, 37, 38, 41 We interpreted the backbone and methyl-axis order parameters determined by NMR in terms of conformational entropies associated with the backbone and side-chain dihedral angle fluctuations, using the dictionary approach,41 and calculated the entropic contributions from these degrees of freedom to the free energy of ligand binding, ∆Sbb,NMR and ∆Ssc,NMR. For comparison, we also calculated the corresponding entropies of rapamycin binding based on published data.25 The resulting changes in conformational entropy upon binding FK506 or rapamycin are identical within errors for both the backbone and methyl-bearing side chains (Table 1). Furthermore, we estimated the conformational entropy from the MD-generated ensembles of apo and FK506bound FKBP12, using one-dimensional dihedral angle probability distributions evaluated as histograms with 5° bins. This approach neglects correlations among dihedral angles, but has proven highly useful as a first-order approximation,88 particularly when comparing two different states of the same protein.89 Table 1 lists the resulting conformational entropies determined by NMR and MD. To enable comparison with the NMR results, the MD data are broken down into separate contributions from the backbone (φ, ψ, ω) dihedral angles,

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∆Sbb,MD, and the methyl-bearing side chains (χ angles), ∆Ssc,MD, and we include only those residues for which we have NMR relaxation data. In addition, the MDgenerated ensemble yields the total conformational entropy, ∆Stot,MD, of the protein calculated by including all dihedral angles. The MD-derived conformational entropies compare well with those determined by NMR, as expected from the comparison of order parameters described above. Comparing the contributions to T∆∆S from those backbone amides and side chain methyls for which experimental data are available, we find that these are identical within errors (Table 1). The change in conformational entropy upon ligand binding is far from uniform across the protein structure, and instead appears to be localized to certain areas, as also reflected by the experimental order parameters (Figs. 3–4). Table 1. Overall Thermodynamics and Conformational Entropy of Ligand Binding to FKBP12 Protein complex

FK506FKBP12e

RAPFKBP12f

∆Go (kJ/mol)a

–52 ± 1

–53 ± 1

∆Ho (kJ/mol) b

–65 ± 1

–81 ± 1

–T∆So (kJ/mol)

13 ± 1

28 ± 1

–T∆S1mM (kJ/mol)

29 ± 1

44 ± 1

–T∆Sbb,NMR (kJ/mol)

7.1 ± 0.4

8.3 ± 1.1g

–T∆Ssc,NMR (kJ/mol)

10.6 ± 1.3

9.7 ± 0.6g

–T∆Sbb,MD (kJ/mol) c

7±8

–

–T∆Ssc,MD (kJ/mol) c

4±9

–

–T∆Stot,MD (kJ/mol) d

26 ± 9

–

a Data from Bierer et al.,4 and Connelly et al.90; b Data from Connelly et al.91; c Including only residues for which NMR data are also available. d Including all dihedral angles. Error estimates of entropies calculated from MD trajectories are based on the standard deviation calculated over 4 individual trajectories; e T = 293 K; f T = 298 K; g Data from Sapienza et al.25

Table 1 offers a comparison with the the standard-state entropy of binding (T∆So), estimated from ITC data90-92 and affinity measurements.4, 90, 93 Table 1 also shows the entropy of binding referenced to the protein and ligand concentrations used in the NMR experiments, obtained as T∆S1mM = T∆So + RTln(cL), where cL is the total concentration of ligand (which in the present case is equal to the total protein concentration).31 Notably, T ∆Stot,MD is identical to T∆S1mM, within errors. This remarkable agreement is surely fortuitous, since T∆S1mM (or T∆So) includes not only contributions from protein conformational entropy, but also from desolvation, as well as conformational entropy of the ligand, and translationalrotational degrees of freedom of both ligand and protein.


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Nonetheless, the results clearly indicate that protein conformational entropy contributes significantly to the free energy of binding. Concluding Remarks. Our results highlight delicate changes in protein conformational dynamics caused by ligand binding. Binding of either FK506 or rapamycin has distinct effects on the backbone and methyl-axis order parameters of FKBP12 that can be interpreted in a structural framework. Changes in order parameters upon ligand binding are observed for networks of residues that are connected via backbone hydrogen bonds or sidechain packing interactions. Similarly, different responses to binding of FK506 or rapamycin appear to involve dynamical modes that propagate through such interactions. The net effect is a redistribution of accessible conformational states upon ligand binding that contributes significantly to the overall entropy of binding.

ASSOCIATED CONTENT Supporting Information. Figures showing comparisons of: Rex terms from model-free fits and R1ρ relaxation dispersion experiments; experimental S2 (backbone and side chain) for apo and FK506-FKBP12; residues showing chemical shift changes upon ligand binding; S2 (backbone and side chain, apo and FK506-FKBP12) from NMR and MD; experimental S2 (backbone and side chain) for FK506FKBP12 and rapamycin-FKBP12. Tables of backbone and side-chain model-free parameters for apo and FK506FKBP12. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * [email protected].

Funding Sources This work was supported by the Swedish Research Council (2014-5815), the Göran Gustafsson Foundation for Research in Natural Sciences and Medicine, and the Knut and Alice Wallenberg Foundation (KAW 2013.0022).

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We thank Kristofer Modig for helpful discussions.

ABBREVIATIONS DSS, 4,4-dimethyl-4-silapentane-1-sulfonic acid; FKBP12, FK506 Binding Protein 12 kDa; HSQC, heteronuclear single-quantum coherence; iRED, isotropic reorientational eigenmode dynamics; ITC, isothermal titration calorimetry; MD, molecular dynamics; PME, particle mesh Ewald; RMSD, root-mean-square deviation.

REFERENCES (1) Vézina, C., Kudelski, A., and Sehgal, S. N. (1975) Rapamycin (AY-22,989), a new antifungal antibiotic, J. Antibiot. 28, 721–726. (2) Kino, T., Hatanaka, H., Hashimoto, M., Nishiyama, M., Goto, T., Okuhara, M., Kohsaka, M., Aoki, H., and Imanaka, H. (1987) FK-506, a novel immunosuppressant isolated from a Streptomyces.1. Fermentation, isolation, and physicochemical and biological characteristics, J. Antibiot. 40, 1249-1255.

(3) Liu, J., Farmer, J. D., Lane, W. S., Friedman, J., Weissman, I., and Schreiber, S. L. (1991) Calcineurin is a common target of cyclophilin-cyclosporine-A and FKBP-FK506 complexes, Cell 66, 807-815. (4) Bierer, B. E., Mattila, P. S., Standaert, R. F., Herzenberg, L. A., Burakoff, S. J., Crabtree, G., and Schreiber, S. L. (1990) Two Distinct Signal Transmission Pathways in T Lymphocytes Are Inhibited by Complexes Formed between an Immunophilin and Either FK506 or Rapamycin, Proc. Natl. Acad. Sci. U.S.A. 87, 9231-9235. (5) Brillantes, A. M. B., Ondrias, K., Scott, A., Kobrinsky, E., Ondriasova, E., Moschella, M. C., Jayaraman, T., Landers, M., Ehrlich, B. E., and Marks, A. R. (1994) Stabilization of calciumrelease channel (ryanodine receptor) function by FK506binding protein, Cell 77, 513-523. (6) Bultynck, G., De Smedt, P., Rossi, D., Callewaert, G., Missiaen, L., Sorrentino, V., De Smedt, H., and Parys, J. B. (2001) Characterization and mapping of the 12 kDa FK506bindign protein (FKBP12)-binding site on different isoforms of the ryanodine receptor and of the inositol 1,4,5-triphosphate receptor, Biochem. J. 354, 413-422. (7) Gaburjakova, M., Gaburjakova, J., Reiken, S., Huang, F., Marx, S. O., Rosemblit, N., and Marks, A. R. (2001) FKBP12 binding modulates ryanodine receptor channel gating, J. Biol. Chem. 276, 16931–16935. (8) Wang, T. W., Li, B. Y., Danielson, P. D., Shah, P. C., Rockwell, S., Lechleider, R. J., Martin, J., Manganaro, T., and Donahoe, P. K. (1996) The immunophilin FKBP12 functions as a common inhibitor of the TGF beta family type I receptors, Cell 86, 435-444. (9) Chen, Y. G., Liu, F., and Massague, J. (1997) Mechanism of TGF beta receptor inhibition by FKBP12, EMBO J. 16, 38663876. (10) Deleersnijder, A., Van Rompuy, A. S., Desender, L., Pottel, H., Buee, L., Debyser, Z., Baekelandt, V., and Gerard, M. (2011) Comparative Analysis of Different Peptidyl-Prolyl Isomerases Reveals FK506-binding Protein 12 as the Most Potent Enhancer of alpha-Synuclein Aggregation, J. Biol. Chem. 286, 26687-26701. (11) Gerard, M., Deleersnijder, A., Daniels, V., Schreurs, S., Munck, S., Reumers, V., Pottel, H., Engelborghs, Y., Van den Haute, C., Taymans, J. M., Debyser, Z., and Baekelandt, V. (2010) Inhibition of FK506 Binding Proteins Reduces alphaSynuclein Aggregation and Parkinson's Disease-Like Pathology, J. Neurosci. 30, 2454-2463. (12) Liu, F. L., Liu, T. Y., and Kung, F. L. (2014) FKBP12 regulates the localization and processing of amyloid precursor protein in human cell lines, J. Biosciences 39, 85-95. (13) Sugata, H., Matsuo, K., Nakagawa, T., Takahashi, M., Mukai, H., Ono, Y., Maeda, K., Akiyama, H., and Kawamata, T. (2009) A peptidyl-prolyl isomerase, FKBP12, accumulates in Alzheimer neurofibrillary tangles, Neurosci. Lett. 459, 96-99. (14) Ikura, T., and Ito, N. (2013) Peptidyl-prolyl isomerase activity of FK506 binding protein 12 prevents tau peptide from aggregating, Protein Eng. Des. Sel. 26, 539-546. (15) Moore, J. M., Peattie, D. A., Fitzgibbon, M. J., and Thomson, J. A. (1991) Solution structure of the major binding protein for the immunosuppressant FK506, Nature 351, 248250. (16) Wilson, K. P., Yamashita, M. M., Sintchak, M. D., Rotstein, S. H., Murcko, M. A., Boger, J., Thomson, J. A., Fitzgibbon, M. J., Black, J. R., and Navia, M. A. (1995) Comparative X-ray structures of the major binding-protein for the immunosuppressant FK506 (Tacrolimus) in unliganded form and in complex with FK506 and rapamycin, Acta Crystallogr. D 51, 511-521. (17) Szep, S., Park, S., Boder, E. T., Van Duyne, G. D., and Saven, J. G. (2009) Structural coupling between FKBP12 and buried water, Proteins: Struct., Funct., Bioinf. 74, 603-611. (18) Michnick, S. W., Rosen, M. K., Wandless, T. J., Karplus, M., and Schreiber, S. L. (1991) Solution structure of FKBP, a


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

rotamase enzyme and receptor for FK506 and rapamycin, Science 252, 836-839. (19) van Duyne, G. D., Standaert, R. F., Karplus, P. A., Schreiber, S. L., and Clardy, J. (1991) Atomic-Structure of Fkbp-Fk506, an Immunophilin-Immunosuppressant Complex, Science 252, 839-842. (20) Holt, D. A., Luengo, J. I., Yamashita, D. S., Oh, H. J., Konialian, A. L., Yen, H. K., Rozamus, L. W., Brandt, M., and Bossard, M. J. (1993) Design, synthesis, and kinetic evaluation of high-affinity FKBP ligands and the X-ray crystal structures of their complexes with FKBP12, J. Am. Chem. Soc. 115, 99259938. (21) Burkhard, P., Taylor, P., and Walkinshaw, M. D. (2000) X-ray structures of small ligand-FKBP complexes provide an estimate for hydrophobic interaction energies1, J. Mol. Biol. 295, 953-962. (22) Blackburn, E. A., and Walkinshaw, M. D. (2011) Targeting FKBP isoforms with small-molecule ligands, Curr. Opin. Pharmacol. 11, 365-371. (23) Brath, U., Akke, M., Yang, D., Kay, L. E., and Mulder, F. A. A. (2006) Functional dynamics of human FKBP12 revealed by methyl 13C rotating frame relaxation dispersion NMR spectroscopy, J. Am. Chem. Soc. 128, 5718-5727. (24) Brath, U., and Akke, M. (2009) Differential response 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. 387, 233-244. (25) Sapienza, P. J., Mauldin, R. V., and Lee, A. L. (2011) Multi-Timescale Dynamics Study of FKBP12 Along the Rapamycin-mTOR Binding Coordinate, J. Mol. Biol. 405, 378394. (26) Yang, C.-J., Takeda, M., Terauchi, T., Jee, J., and Kainosho, M. (2015) Differential large-amplitude breathing motions in the interface of FKBP12-drug complexes, Biochemistry 54, 6983-6995. (27) Cooper, A., and Dryden, D. T. F. (1984) Allostery without conformational change. A plausible model, Eur. Biophys. J. 11, 103-109. (28) Akke, M., Brüschweiler, R., and Palmer, A. G. (1993) NMR order parameters and free energy: An analytical approach and its application to cooperative Ca2+ binding by calbindin D9k, J. Am. Chem. Soc. 115, 9832-9833. (29) Bracken, C., Carr, P. A., Cavanagh, J., and Palmer, A. G. (1999) Temperature dependence of intramolecular dynamics of the basic leucine zipper of GCN4: implications for the entropy of association with DNA, J. Mol. Biol. 285, 2133-2146. (30) Lee, A. L., Kinnear, S. A., and Wand, A. J. (2000) Redistribution and loss of side chain entropy upon formation of a calmodulin-peptide complex, Nature Struct. Biol. 7, 72-77. (31) Gilson, M. K., and Zhou, H. X. (2007) Calculation of protein-ligand binding affinities, Annu. Rev. Bioph. Biomol. Struct. 36, 21-42. (32) Tzeng, S. R., and Kalodimos, C. G. (2012) Protein activity regulation by conformational entropy, Nature 488, 236240. (33) Akke, M. (2012) Conformational dynamics and thermodynamics of protein–ligand binding studied by NMR relaxation, Biochem. Soc. Trans. 40, 419-423. (34) Wennerström, H., Lindman, B., Söderman, O., Drakenberg, T., and Rosenholm, J. B. (1979) 13C magnetic relaxation in micellar solutions. Influence of aggregate motion on T1, J. Am. Chem. Soc. 101, 6860-6864. (35) Halle, B., and Wennerström, H. (1981) Interpretation of magnetic resonance data from water nuclei in heterogeneous systems, J. Chem. Phys. 75, 1928-1943. (36) Lipari, G., and Szabo, A. (1982) Model-free approach to the interpretation of nuclear magnetic resonance relaxation in macromolecules. I. Theory and range of validity, J. Am. Chem. Soc. 104, 4546-4559.

Page 10 of 13

(37) Yang, D., and Kay, L. E. (1996) Contributions to conformational entropy arising from bond vector fluctuations measured from NMR-derived order parameters: Application to protein folding, J. Mol. Biol. 263, 369-382. (38) Li, Z., Raychaudhuri, S., and Wand, A. J. (1996) Insights into the local residual entropy of proteins provided by NMR relaxation, Protein Sci. 5, 2647-2650. (39) Dhulesia, A., Bodenhausen, G., and Abergel, D. (2008) Predicting conformational entropy of bond vectors in proteins by networks of coupled rotators, J. Chem. Phys. 129, 095107095105. (40) Akke, M. (2014) Order parameters and conformational entropies derived from combined NMR and molecular dynamics approach, eMagRes 3, 267-274. (41) Li, D. W., and Brüschweiler, R. (2009) A Dictionary for Protein Side-Chain Entropies from NMR Order Parameters, J. Am. Chem. Soc. 131, 7226-7227. (42) Cheng, J.-W., Lepre, C. A., Chambers, S. P., Fulghum, J. R., Thomson, J. A., and Moore, J. M. (1993) 15N NMR relaxation studies of the FK506 binding protein: Backbone dynamics of the uncomplexed receptor, Biochemistry 32, 90009010. (43) Cheng, J. W., Lepre, C. A., and Moore, J. M. (1994) 15N NMR relaxation studies of the FK506 binding protein: Dynamic effects of ligand binding and implications for calcineurin recognition, Biochemistry 33, 4093-4100. (44) Lepre, C. A., Cheng, J.-W., and Moore, J. M. (1993) Dynamics of a receptor-bound ligand by heteronuclear NMR: FK506 bound to FKBP-12, J. Am. Chem. Soc. 115, 4929-4930. (45) Sich, C., Improta, S., Cowley, D. J., Guenet, C., Merly, J.P., Teufel, M., and Saudek, V. (2000) Solution structure of a neurotrophic ligand bound to FKBP12 and its effect on protein dynamics, Eur. J. Biochem. 267, 5342-5354. (46) Liu, F., Wang, Y. Q., Meng, L., Gu, M., and Tan, R. Y. (2013) FK506-binding protein 12 ligands: a patent review, Expert. Opin. Ther. Pat. 23, 1435-1449. (47) Jayachandran, G., Shirts, M. R., Park, S., and Pande, V. S. (2006) Parallelized-over-parts computation of absolute binding free energy with docking and molecular dynamics, J. Chem. Phys. 125, 084901-084912. (48) Deng, Y. Q., and Roux, B. (2009) Computations of Standard Binding Free Energies with Molecular Dynamics Simulations, J. Phys. Chem. B 113, 2234-2246. (49) Olivieri, L., and Gardebien, F. (2011) Molecular Dynamics Simulations of a Binding Intermediate between FKBP12 and a High-Affinity Ligand, J. Chem. Theory Comput. 7, 725-741. (50) Olivieri, L., and Gardebien, F. (2014) Structure-Affinity Properties of a High-Affinity Ligand of FKBP12 Studied by Molecular Simulations of a Binding Intermediate, Plos One 9, e114610. (51) Bizzarri, M., Marsili, S., and Procacci, P. (2011) Intraligand Hydrophobic Interactions Rationalize Drug Affinities for Peptidyl-Prolyl Cis-Trans Isomerase Protein, J. Phys. Chem. B 115, 6193-6201. (52) General, I. J., Dragomirova, R., and Meirovitch, H. (2011) Calculation of the Absolute Free Energy of Binding and Related Entropies with the HSMD-TI Method: The FKBP12-L8 Complex, J. Chem. Theory Comput. 7, 4196-4207. (53) General, I. J., and Meirovitch, H. (2013) Absolute Free Energy of Binding and Entropy of the FKBP12-FK506 Complex: Effects of the Force Field, J. Chem. Theory Comput. 9, 46094619. (54) Case, D. A. (2002) Molecular dynamics and NMR spin relaxation in proteins, Acc. Chem. Res. 35, 325-331. (55) Beck, D. A. C., Jonsson, A. L., Schaeffer, R. D., Scott, K. A., Day, R., Toofanny, R. D., Alonso, D. O. V., and Daggett, V. (2008) Dynameomics: mass annotation of protein dynamics and unfolding in water by high-throughput atomistic molecular dynamics simulations, Protein Eng. Des. Sel. 21, 353-368. (56) Maragakis, P., Lindorff-Larsen, K., Eastwood, M. P., Dror, R. O., Klepeis, J. L., Arkin, I. T., Jensen, M. Ø., Xu, H.,


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Trbovic, N., Friesner, R. A., Palmer, A. G., and Shaw, D. E. (2008) Microsecond molecular dynamics simulation shows effect of slow loop dynamics on backbone amide order parameters of proteins, J. Phys. Chem. B 112, 6155-6158. (57) Trbovic, N., Kim, B., Friesner, R. A., and Palmer, A. G. (2008) Structural analysis of protein dynamics by MD simulations and NMR spin-relaxation, Proteins: Struct., Funct., Bioinf. 71, 684-694. (58) Lange, O. F., van der Spoel, D., and de Groot, B. L. (2010) Scrutinizing Molecular Mechanics Force Fields on the Submicrosecond Timescale with NMR Data, Biophys. J. 99, 647-655. (59) Palmer, A. G., Kroenke, C. D., and Loria, J. P. (2001) Nuclear magnetic resonance methods for quantifying microsecond-to-millisecond motions in biological macromolecules, Methods Enzymol. 339, 204-238. (60) Palmer, A. G. (2004) NMR characterization of the dynamics of biomacromolecules, Chem. Rev. 104, 3623-3640. (61) Palmer, A. G., and Massi, F. (2006) Characterization of the dynamics of biomacromolecules using rotating-frame spin relaxation NMR spectroscopy, Chem. Rev. 106, 1700-1719. (62) Kördel, J., Skelton, N. J., Akke, M., Palmer, A. G., and Chazin, W. J. (1992) Backbone dynamics of calcium-loaded calbindin D9k studied by two-dimensional proton-detected NMR spectroscopy, Biochemistry 31, 4856-4866. (63) Farrow, N. A., Muhandiram, R., Singer, A. U., Pascal, S. M., Kay, C. M., Gish, G., Shoelson, S. E., Pawson, T., FormanKay, J. D., and Kay, L. E. (1994) Backbone dynamics of a free and a phosphopeptide-complexed src homology 2 domain studied by 15N NMR relaxation, Biochemistry 33, 5984-6003. (64) Ferrage, F., Cowburn, D., and Ghose, R. (2009) Accurate Sampling of High-Frequency Motions in Proteins by Steady-State N-15-{H-1} Nuclear Overhauser Effect Measurements in the Presence of Cross-Correlated Relaxation, J. Am. Chem. Soc. 131, 6048-+. (65) Millet, O., Muhandiram, D. R., Skrynnikov, N. R., and Kay, L. E. (2002) Deuterium spin probes of side-chain dynamics in proteins. 1. Measurement of five relaxation rates per deuteron in 13C-labeled and fractionally 2H-enriched proteins in solution, J. Am. Chem. Soc. 124, 6439-6448. (66) Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J., and Bax, A. (1995) NMRPipe: A multidimensional spectral processing system based on UNIX pipes, J. Biomol. NMR 6, 277-293. (67) d'Auvergne, E. J., and Gooley, P. R. (2008) Optimisation of NMR dynamic models I. Minimisation algorithms and their performance within the model-free and Brownian rotational diffusion spaces, J. Biomol. NMR 40, 107-119. (68) d'Auvergne, E. J., and Gooley, P. R. (2008) Optimisation of NMR dynamic models II. A new methodology for the dual optimisation of the model-free parameters and the Brownian rotational diffusion tensor, J. Biomol. NMR 40, 121133. (69) Skrynnikov, N. R., Millet, O., and Kay, L. E. (2002) Deuterium spin probes of side-chain dynamics in proteins. 2. Spectral density mapping and identification of nanosecond time-scale side-chain motions, J. Am. Chem. Soc. 124, 64496460. (70) Bevington, P. R., and Robinson, D. K. (1992) Data reduction and error analysis for the physical sciences, WCB/McGraw-Hill, New York. (71) Berendsen, H. J. C., Van der Spoel, D., and Van Drunen, R. (1995) Gromacs - a Message-Passing Parallel MolecularDynamics Implementation, Comput. Phys. Commun. 91, 43-56. (72) Hess, B., Kutzner, C., van der Spoel, D., and Lindahl, E. (2008) GROMACS 4: Algorithms for highly efficient, loadbalanced, and scalable molecular simulation, J. Chem. Theory Comput. 4, 435-447. (73) Hornak, V., Abel, R., Okur, A., Strockbine, B., Roitberg, A., and Simmerling, C. (2006) Comparison of multiple Amber

force fields and development of improved protein backbone parameters, Proteins: Struct., Funct., Bioinf. 65, 712-725. (74) Wang, J. M., Wolf, R. M., Caldwell, J. W., Kollman, P. A., and Case, D. A. (2004) Development and testing of a general amber force field, J. Comp. Chem. 25, 1157-1174. (75) Darden, T., York, D., and Pedersen, L. (1993) Particle Mesh Ewald - an N.Log(N) Method for Ewald Sums in Large Systems, J. Chem. Phys. 98, 10089-10092. (76) Essmann, U., Perera, L., Berkowitz, M. L., Darden, T., Lee, H., and Pedersen, L. G. (1995) A Smooth Particle Mesh Ewald Method, J. Chem. Phys. 103, 8577-8593. (77) Darden, T. (1999) Ionic charging free energies using Ewald summation, IMA Vol. Math. Appl. 108, 149-161. (78) Ryckaert, J.-P., Ciccotti, G., and Berendsen, H. J. C. (1977) Numerical integration of the cartesian equations of motion of a system with constraints: Molecular dynamics of nalkanes, J. Comput. Phys. 23, 327-341. (79) Prompers, J. J., and Brüschweiler, R. (2002) General framework for studying the dynamics of folded and nonfolded proteins by NMR relaxation spectroscopy and MD simulation, J. Am. Chem. Soc. 124, 4522-4534. (80) Genheden, S., Diehl, C., Akke, M., and Ryde, U. (2010) Starting-Condition Dependence of Order Parameters Derived from Molecular Dynamics Simulations, J. Chem. Theory Comput. 6, 2176-2190. (81) Diehl, C., Genheden, S., Modig, K., Ryde, U., and Akke, M. (2009) Conformational entropy changes upon lactose binding to the carbohydrate recognition domain of galectin-3, J. Biomol. NMR 45, 157-169. (82) Solomentsev, G. Y., English, N. J., and Mooney, D. A. (2012) Effects of External Electromagnetic Fields on the Conformational Sampling of a Short Alanine Peptide, J. Comp. Chem. 33, 917-923. (83) Mandel, A. M., Akke, M., and Palmer, A. G. (1996) Dynamics of ribonuclease H: Temperature dependence of motion on multiple time scales, Biochemistry 35, 16009-16023. (84) Igumenova, T. I., Frederick, K. K., and Wand, A. J. (2006) Characterization of the fast dynamics of protein amino acid side chains using NMR relaxation in solution, Chem. Rev. 106, 1672-1699. (85) Showalter, S. A., and Bruschweiler, R. (2007) Validation of molecular dynamics simulations of biomolecules using NMR spin relaxation as benchmarks: Application to the AMBER99SB force field, J. Chem. Theory Comput. 3, 961-975. (86) Showalter, S. A., Johnson, E., Rance, M., and Brüschweiler, R. (2007) Toward quantitative interpretation of methyl side-chain dynamics from NMR by molecular dynamics simulations, J. Am. Chem. Soc. 129, 14146-14147. (87) Murata, K., Fedorov, D. G., Nakanishi, I., and Kitaura, K. (2009) Cluster Hydration Model for Binding Energy Calculations of Protein-Ligand Complexes, J. Phys. Chem. B 113, 809-817. (88) Prompers, J. J., and Brüschweiler, R. (2000) Thermodynamic interpretation of NMR relaxation parameters in proteins in the presence of motional correlations, J. Phys. Chem. B 104, 11416-11424. (89) Li, D. W., Showalter, S. A., and Brüschweiler, R. (2010) Entropy localization in proteins, J. Phys. Chem. B 114, 1603616044. (90) Connelly, P. R., Aldape, R. A., Bruzzese, F. J., Chambers, S. P., Fitzgibbon, M. J., Fleming, M. A., Itoh, S., Livingston, D. J., Navia, M. A., Thomson, J. A., and Wilson, K. P. (1994) Enthalpy of Hydrogen-Bond Formation in a Protein-Ligand Binding Reaction, Proc. Natl. Acad. Sci. U.S.A. 91, 1964-1968. (91) Connelly, P. R., Thomson, J. A., Fitzgibbon, M. J., and Bruzzese, F. J. (1993) Probing Hydration Contributions to the Thermodynamics of Ligand-Binding by Proteins - Enthalpy and Heat-Capacity Changes of Tacrolimus and Rapamycin Binding to Fk506 Binding-Protein in D2O and H2O, Biochemistry 32, 5583-5590. (92) Connelly, P. R., and Thomson, J. A. (1992) HeatCapacity Changes and Hydrophobic Interactions in the Binding


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of Fk506 and Rapamycin to the Fk506 Binding-Protein, Proc. Natl. Acad. Sci. U.S.A. 89, 4781-4785. (93) Graziani, F., Aldegheri, L., and Terstappen, G. C. (1999) High throughput scintillation proximity assay for the identification of FKBP-12 ligands, J. Biomol. Screen. 4, 3-7.


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Biochemistry


Conformational Entropy of FK506 Binding to FKBP12 Determined by

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