Tyrosine Sulfation Restricts the Conformational Ensemble of a Flexible

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Tyrosine sulfation restricts the conformational ensemble of a flexible peptide, strengthening the binding affinity to an antibody Kazuhiro Miyanabe, Takefumi Yamashita, Yoshito Abe, Hiroki Akiba, Yuichiro Takamatsu, Makoto Nakakido, Takao Hamakubo, Tadashi Ueda, Jose Caaveiro, and Kouhei Tsumoto Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00592 • Publication Date (Web): 23 Jun 2018 Downloaded from http://pubs.acs.org on June 27, 2018

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Biochemistry Miyanabe et al.

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Tyrosine sulfation restricts the conformational ensemble of a flexible peptide, strengthening the binding affinity to an antibody. Kazuhiro Miyanabe,1 Takefumi Yamashita,2 Yoshito Abe,3 Hiroki Akiba,4,5 Yuichiro Takamatsu,6 Makoto Nakakido,4 Takao Hamakubo,6 Tadashi Ueda,3 Jose M. M. Caaveiro,7,* and Kouhei Tsumoto1,4,5,8,*

1

Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo,

Bunkyo-ku, Tokyo 113-8656, Japan, 2Laboratory for Systems Biology and Medicine, RCAST, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan, 3Laboratory of Protein Structure, Function, and Design, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan, 4Department of Bioengineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan, 5Laboratory of Pharmacokinetic Optimization, Center for Drug Design Research, National Institutes of Biomedical Innovation, Health and Nutrition, 7-6-8 Saito-Asagi, Ibaraki City, Osaka, 567-0085 Japan, 6Quantitative Biology and Medicine, Research Center for Advanced Science and Technology (RCAST), The University of Tokyo, Meguro-ku, Tokyo 153-8904, Japan, 7Laboratory of Global Healthcare, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan, 8Laboratory of Medical Proteomics, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

Running Title: Recognition of a sulfated peptide by an antibody

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Keywords Tyrosine sulfation, post-translational modification, protein-protein interactions, MD simulations, thermodynamics, protein flexibility.

ABSTRACT Protein tyrosine sulfation (PTS) is a post-translational modification regulating numerous biological events. PTS generally occurs at flexible regions of proteins, enhancing intermolecular interactions between proteins. Because of the high flexibility associated to the regions where PTS is generally encountered, an atomic-level understanding has been difficult to achieve by X-ray crystallography or NMR techniques. In this study, we focused on the conformational behavior of a flexible sulfated peptide, and its interaction with an antibody. Molecular dynamics (MD) simulations and thermodynamic analysis indicated that PTS reduced the main-chain fluctuations upon the appearance of sulfate-mediated intramolecular H-bonds. Collectively, our data suggested that one of the mechanisms by which PTS may enhance protein-protein interactions consist in the limitation of conformational dynamics in the unbound state, thus reducing the entropy loss upon binding and boosting the affinity for its partner.

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INTRODUCTION Protein tyrosine sulfation (PTS) is an important post-translational modification of proteins, which mediates various biological processes such as signal transduction, inflammatory responses, and virus infection, among others.1,2 Investigating the mechanism of how PTS influences these events at the molecular level would contribute to increase our precise understanding of this important protein modification. PTS generally appears in the flexible regions of proteins, enhancing their interaction with cognate receptors.3,4 Previous studies reported that the recognition of motifs bearing PTS is driven by direct polar interactions with the partner protein via the sulfate group.4-9 Because sulfated regions are flexible, changes in the dynamic behavior and/or conformation upon sulfation are additional mechanisms to strengthen protein-protein interactions. Indeed similar explanations have been proposed for other post-translational modifications.10-13 However, the effect of PTS on the conformation of the sulfated regions has not been examined with the same level of intensity, since the sulfated regions occur in highly flexible segments of proteins, making their analysis by traditional structural techniques such as X-ray crystallography and NMR more challenging.14,15 As a result, such as mechanism (conformational change) has not been considered except in one example of an unusually stable peptide.16 A more intense scrutiny into this problem is necessary to improve our mechanistic understanding, and to elucidate how PTS may affect protein–protein interactions in general. In this study, molecular dynamics (MD) simulations, NMR, calorimetric analysis, and X-ray crystallography revealed that sulfation of a flexible peptide increased the number of intramolecular hydrogen bonds in the unbound form, reducing the fluctuations of the main chain region and strengthening the binding affinity to an antibody.

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MATERIALS AND METHODS Preparation of the antibody The preparation of antibody was previously described.17 Briefly, scFv 4B08 bearing a pelB leader sequence and a hexahistidine tag was inserted in the expression vector pRA2.18 The scFv was expressed in Escherichia coli BL21 (DE3) as inclusion bodies. The inclusion bodies were sequentially washed with solutions containing 1% Triton X-100, acetone, and deionized H2O, and following that, they were solubilized in a buffer containing 6 M guanidinium chloride, 20 mM TRIS (pH 8.0), 500 mM NaCl, and 5 mM imidazole. The scFv was purified from the supernatant of the solubilized inclusion bodies by nickel affinity chromatography. The solubilized scFv was refolded by stepwise dialysis.19 The final step of purification was size exclusion chromatography to remove unwanted aggregates.

Peptides Synthesized peptides were purchased from Medical & Biological Labolatories Co., Ltd. (Nagoya, Japan) or Scrum Inc. (Tokyo, Japan) at a purity > 95%. The sequences of the peptides were: pep1, DINYYTSEP; Sulfo-pep1, DIN(Tys)(Tys)TSEP.

NMR NMR spectra of sulfated and non-sulfated peptides were recorded on a Bruker AVANCE600 spectrometer (Bruker Biospin Corporation, Billerica, MA) at 20 °C, except where indicated. Peptides at 2 mg/ml were dissolved in a water/deuterium solution (90:10) and the pH adjusted to 5.0. The assignments of the signals were carried out by two-dimensional DQF-COSY, total correlation spectroscopy (TOCSY), and ROESY. All chemical shifts were referenced to tetramethylsilane. For the measurement of the temperature coefficients,20 the chemical shifts of amide protons of the 1D 1H-NMR spectra at 15, 20, 25, 30 and 35 °C were recorded. When significant overlap occurred, the 2D spectra was used instead. We also noticed minor peaks in 4


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the 1D-spectra. These peaks were assigned to Ser7 and Glu8 and likely corresponded to a low-populated conformation caused by cis-trans isomerization of the neighboring residue Pro9. JNH-CH coupling constants were determined using the 1D spectra at 20 °C, except Tyr4 and Tyr5 of the nonsulfated peptide for which the spectra at 35 °C were used instead because of signal overlap at lower temperature.21 Chemical shift index of alpha protons were calculated according to a previous report.22

Isothermal titration calorimetry (ITC) The interaction between antibody and peptide was monitored by ITC in a MicroCal Auto-iTC200 or an iTC200 instrument (Malvern). The purified antibody was dialyzed overnight in PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4・12H2O, 1.76 mM KH2PO4, pH 7.4). Peptides were diluted in the buffer used for the dialysis of the antibody. The concentration of scFv, pep1, and Sulfo-pep1 were determined by absorbance. The antibody at a concentration of 9–11 μM was placed in the cell, and the peptides at 100–120 μM in the syringe. The measurements were performed with a reference power of 5 μcal/s, and a stirring rate of 750 rpm. The experiment consisted of a single injection of 0.5 μL and 18 injections of 2 μL injection each with an interval of 180 seconds between injections. The thermodynamic parameters were calculated with Origin 7.0 (OriginLab) using a single-site binding model.

Crystallization, data collection and refinement. Crystallization of Sulfo-pep–4B08 complex was described previously.17 Briefly, scFv was dialyzed overnight in MES buffer (10 mM MES, 30 mM NaCl, pH 5.9). Sulfo-pep1 was resuspended in dialysis buffer at 200 µM and mixed with the protein (100 µM). And equal volume of reservoir buffer (0.1 M sodium acetate, 1.4 M ammonium sulfate, pH 4.8) was mixed with 2 µL of the peptide-antibody complex and incubated at 20 °C by the hanging-drop

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vapor diffusion method. Crystals were harvested, briefly transferred to the same solution supplemented with 20% glycerol, and stored in liquid N2 until data collection. Diffraction data were collected at beamline BL5A of the Photon Factory (Tsukuba, Japan) under cryogenic conditions (100 K). Data were indexed, integrated with MOSFLM,23 and scaled with SCALA or AIMLESS.24 The structure of the complex between Sulfo-pep1 and scFv 4B08 was determined by the method of molecular replacement as described previously25 with PHASER.26 Refinement was performed with COOT27 and REFMAC5.28 Structural validation was performed with COOT and PROCHECK.29 Data collection and refinement statistics are summarized in Table 2.

MD simulations The operational conditions of the MD simulations were previously described.17 Briefly, we employed the NAMD 2.10 package30 with the CHARMM22 force field,31 and the CMAP backbone energy correction.32 The force field of sulfated tyrosine was generated from the force field of tyrosine and methyl sulfate by the CGenFF program.33 The extended peptide (φ / ϕ angle = 180°) was solvated with TIP3P water under periodic boundary conditions.34 Sodium and chloride ions were included to neutralize the protein charge, after which additional ions were added to a salt concentration of 0.14 M. The time step was set to 2 fs throughout the simulations. A cutoff distance of 10 Å for Coulomb and van der Waals interactions was selected. Coulomb interactions were evaluated through the particle mesh Ewald method. First, the energy minimization was performed for each system with the conjugate gradient method (5000 steps). Then five Langevin dynamics simulations were conducted for 50 ps to heat the systems gradually from 0 to 298 K. After 200 ps equilibration under the NPT constant condition, the production run was performed for each system in the NPT ensembles at 298 K for 200 ns. Coordinates were saved every 10 ps.

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RESULTS AND DISCUSSION Conformational ensemble of the peptide in solution As a model system we employed a peptide from the N-terminal region of the human CC chemokine receptor type 5 (CCR5), a region naturally modified by PTS. The peptide of sequence DINYYTSEP (termed pep1), and its sulfated counterpart (termed Sulfo-pep1) of sequence DIN(Tys)(Tys)TSEP (Tys refers to sulfated tyrosine) were obtained by chemical synthesis. Since short peptides are highly flexible, their conformational ensemble in solution is often studied by MD simulations.35,36 In a previous study, we carried out MD simulations of pep1 in the unbound state.17 Herein we have performed equivalent MD simulations for the sulfated peptide Sulfo-pep1, comprising five independent trajectories lasting 200 ns each (Supporting movies S1 and S2 ). The conformation of sulfated and non-sulfated peptide was monitored every 100 ps, resulting in 10,000 independent structures. The analysis of their trajectories revealed two interesting features resulting from PTS, (i) the emergence of a new conformation in the ensemble in solution, and (ii) an overall reduction of the flexibility of the main chain (Figure 1). First, the effect of sulfation on the conformational ensemble of the peptide was estimated by the average dihedral angles of the main chain using circular analysis (Figure 1a, Table S1).37 As a result, the average value of three dihedral angles revealed three statistically meaningful changes: Ile2ψ from 75 ± 28° to 8.5 ± 29°; Asn3ψ from 81 ± 11° to 17 ± 11°; and Ser7ψ from 13 ± 7.1° to 69 ± 23°). In structural terms, the sulfate group of Tys5 transiently formed a few intramolecular H-bonds with main chain atoms of Asp1, Ile2 and Asn3, and with the side chain atom of Asn3 (Figure 1c). Also, the sulfate group of Tys4 formed a transient and low populated intramolecular H-bond with Ser7. An intramolecular H-bond was considered to appear when the distance between donor and acceptor was below a cutoff value of 3.5 Å, and the angle between donor, hydrogen atom, and an acceptor was less than 150° (Figure 1d).38 In the MD simulations of Sulfo-pep1 these intermolecular H-bonds appeared in 8-15 % of the frames. 7


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Figure 1. Structural and dynamic properties of the peptide. (a) Average and (b) variance of the dihedral angles of each of the residues of pep1 and Sulfo-pep1. The dihedral angles were determined with UCSF Chimera. The mathematical definition of the average and the variance are described in Table S1. (c) Illustrative conformation of Sulfo-pep1 in which the side chain of Tys5 forms several hydrogen bonds with main chain atoms of the peptide. (d) The fraction of structures displaying intramolecular H-bonds between the side-chain oxygen of Tys5 (O1S, O2S or O3S) and the main chain nitrogen of Asp1, Ile2, Asn3, or the side chain of Asn3 (Asn3-sc) is indicated. Intramolecular H-bonds were considered to occur when the distance between donor and acceptor was less than 3.5 Å, and the angle between the atoms participating was less than 150°. The distance and angles were determined with UCSF Chimera.

These intramolecular H-bonds suggested that the N-terminal and central region of Sulfo-pep1 is transiently stabilized (reduced flexibility) in the unbound form. Therefore, we subsequently examined the effect of PTS on the fluctuation of main-chain atoms by monitoring the variance of each dihedral angle during the MD simulations trajectories (Figure 1b, Table S1). The variance of three main-chain dihedral angles decreased in the simulations of Sulfo-pep1 with respect to the nonsulfated peptide. The value of the variance of Ile2ψ decreased from 0.56 ± 0.15 to 0.29 ± 0.15; that of Asn3φ from 0.48 ± 0.17 to 0.12 ± 0.04; and that of Tyr4ψ from 0.66 ± 0.07 to 0.42 ± 0.12. Tys4 is sandwiched between residues Asn3 and Tys5 both forming intramolecular H-bonds, and itself engages in a low populated intramolecular H-bond with Ser7, explaining its lower mobility. No other residue with statistically significant differences was found.

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Further insights were obtained by NMR. The amide proton-Cα proton coupling constant obtained from the two-dimensional double quantum filtered correlation spectroscopy (DQF-COSY) experiment (Figure S1) and the chemical shift change of the proton-Cα with respect to random coil values (Figure S2) are both consistent with an extended conformation (random coil) of both the sulfated and the non-sulfated peptides. No sign of definite conformation was found in the rotating frame nuclear Overhauser effect spectroscopy (ROESY) experiment (data not shown). In contrast, the temperature dependence 1-D 1H-NMR spectra of pep1 in solution showed a significant difference with respect to that of Sulfo-pep1 (Figure 2). The NH-signal of Ile2 in pep1 was broad and lacked detail at 15 and 20 °C, and at temperatures greater than 20 °C was barely visible. In contrast, the signal of Ile2 in Sulfo-pep1 was clearly recognizable at all temperatures. These differences indicate that the amide proton of Ile2 in the nonsulfated peptide is located in a more flexible environment by multiple conformations and/or rapid water exchange. In addition, the temperature coefficients suggested that H-bond formation is more likely in the sulfated that in the non-sulfated peptide (Figure S3).20

Figure 2. Temperature-dependence of the 1D 1H-NMR spectra of peptides. 1-D NMR spectra of (a) pep1 and (b) Sulfo-pep1 at five different temperatures (15, 20, 25, 30 and 35 °C). The spectra are shown in increasing order of temperature. The arrow points at the region where the signal corresponding to Ile2 appeared. Peptide concentration was 2 mg/ml.

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Collectively, the MD simulations showed that sulfate-mediated intramolecular H-bonds restrict the fluctuations of the main chain of the modified peptide compared with the nonsulfated peptide. NMR, a technique responsive in a different time-scale to that of MD simulations also suggested that sulfation reduced the flexibility of the peptide.

Energetic contribution to binding (ITC) To determine if PTS also influenced the interaction of the peptide with a model binding partner, we employed a single-chain variable fragment (scFv) construct of a previously reported monoclonal antibody (4B08).17 Isothermal titration calorimetry (ITC)39,40 was employed to determine the binding affinity and thermodynamic parameters of the interaction of 4B08 with pep1, or Sulfo-pep1, at five different temperatures ranging from 15 °C to 35 °C (Table 1, Figure 3a).

Figure 3. Effect of Tyr sulfation on the binding of peptide to antibody. (a) Binding of pep1 (left) and Sulfo-pep1 (right) to 4B08 at 30 °C as determined by ITC. The upper panels correspond to the titration kinetics, whereas the lower panels represent the integrated binding isotherms. Molar ratio refers to the relative concentration of peptide-to-protein in the cell. The binding enthalpy (ΔH) and the dissociation constant (KD) were obtained by non-linear regression of the integrated data to a one-site binding model with the program ORIGIN. (b) Relationship between temperature and change of binding enthalpy for pep1 (orange) and Sulfo-pep1 (green). A clear correlation is observed for pep1 (R2 = 0.98) as well as for Sulfo-pep1 (R2 = 0.99). The change of heat capacity for the binding of each peptide to the antibody was determined from the slope of the linear regression.

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Table 1. Thermodynamic parameters of binding of pep1 and Sulfo-pep1 to scFv.a peptide

pep1

Sulfo-pep1

a

Temp. (°C) 15 20 25 30 35 15 20 25 30 35

ΔG n (kcal mol-1) 0.97 ± 0.03 −9.2 ± 0.2 0.97 ± 0.04 −9.0 ± 0.1 0.91 ± 0.04 −8.8 ± 0.1 0.92 ± 0.01 −8.7 ± 0.1 0.90 ± 0.03 −8.6 ± 0.1 1.08 ± 0.02 −9.2 ± 0.1 1.04 ± 0.02 −9.2 ± 0.1 1.04 ± 0.10 −9.1 ± 0.1 1.01 ± 0.01 −8.9 ± 0.1 0.99 ± 0.01 −8.9 ± 0.2

ΔH (kcal mol-1) −13.3 ± 0.2 −14.8 ± 0.4 −16.8 ± 0.1 −17.3 ± 0.1 −18.9 ± 0.8 −12.2 ± 0.3 −13.5 ± 0.1 −14.4 ± 0.5 −15.1 ± 0.3 −16.1 ± 0.2

−TΔS (kcal mol-1) 4.1 ± 0.6 5.8 ± 0.5 8.0 ± 0.2 8.6 ± 0.2 10.3 ± 0.9 2.9 ± 0.4 4.2 ± 0.2 5.3 ± 0.6 6.1 ± 0.4 7.3 ± 0.4

KD ΔCp (nM) (cal mol-1K-1) 110 ± 37 190 ± 19 370 ± 5.6 −280 510 ± 39 850 ± 33 98 ± 12 140 ± 19 220 ± 48 −190 310 ± 52 510 ± 140

Each experiment was performed three times. The error corresponds to the standard deviation.

It was observed that the binding affinity of the sulfated peptide to the antibody to the antibody was modestly but consistently strengthened with respect to the nonsulfated peptide at all temperatures except at 15 ºC. The relative affinity KDnonsulfated / KDsulfated determined at 15, 20 25, 30, and 35 °C was 1.19 ± 0.52, 1.40 ± 0.33, 1.77 ± 0.41, 1.71 ± 0.41, and 1.82 ± 0.56, respectively. The interaction of pep1 with 4B08 was characterized by a large and favorable enthalpy change (ΔH = −16.8 ± 0.1 kcal mol-1 at 25 °C; hereafter this temperature is applied to all thermodynamic descriptions), and opposed by a less unfavorable entropy change (−TΔS = 8.0 ± 0.2 kcal mol-1). Interestingly, the increase of affinity in the sulfated peptide was characterized by a smaller entropy loss (−TΔS = 5.3 ± 0.4 kcal mol-1) and not by a gain in enthalpy, something that could have been expected if additional interactions between the sulfate group and the antibody were present. In general, the negative contribution of the entropic term to peptide–protein interactions is a combination of the loss of conformational dynamics, and the loss of translational and rotational degrees of freedom.41 Consistent with that argument, in the sulfated peptide the restriction of the atomic fluctuations of the main chain in the unbound state resulted in a decrease of the unfavorable change of entropy. The value of the enthalpy change is generally influenced by the rearrangement of inter-/intramolecular interactions in the unbound state with respect to the bound state. The formation of transient intramolecular H-bonds and van der Waals interactions 11


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in Sulfo-pep1 prior to the binding to the antibody would explain the smaller contribution of the enthalpy change of the sulfated peptide to the complex with 4B08 with respect to the nonsulfated peptide.39 The sulfation also increased (less negative) the value of the heat capacity change (ΔCp) from -280 cal mol-1 K-1 in pep1 to -190 cal mol-1 K-1 in Sulfo-pep1 (Figure 3b). Since lower values of heat capacity change are a function of the dehydration from the apolar interaction surface, the values obtained for pep1 and Sulfo-pep1 may reflect more limited conformational re-arrangements in the sulfated peptide.42,43

Crystal structure of Sulfo-pep1 in complex with 4B08 To obtain a detailed atomic picture of the interaction between the sulfated peptide and the antibody, we determined the crystal structure of the complex at 1.59 Å resolution (Table 2). This structure was compared with the previously determined structure of the complex between pep1 and 4B08 antibody at 1.35 Å resolution (PDB entry code 5YD3)17. The two structures were very similar to each other (RMSD4B08 = 0.58 ± 0.06 Å, RMSDpeptide = 0.10 ± 0.04 Å). The binding surface of Sulfo-pep1 (1,046 Å2) was slightly greater than that of pep1 (995 ± 31 Å2) (Table S2), although that difference was centered on Ile2 belonging to the relatively disordered N-terminal region (high B-factors) with no apparent influence for binding and not contacting the sulfated Tys residues (Figure S4). The residue Asp1 of the peptide was not observed in the electron density (disordered). Similarly, the intermolecular and intramolecular H-bonds, and the single salt bridge in the complex of pep1 with 4B08 were conserved in the complex of Sulfo-pep1 with 4B08, and their distances essentially identical to each other (Figure 4a, Figure S5, and Table S3, S4). Importantly, the side chain of the Tys residues did not engage in interactions with the receptor. Although the complexes were crystallized in different space-groups, the temperature factors (B-factors) of the peptides in the complex were comparable to each other (Figure S4), suggesting minimal differences in their dynamic behavior in the bound form. Collectively, the 12


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crystal structures did not revealed any significant feature that could explain the different binding properties between the peptides, suggesting that in this particular case PTS exerted its influence primarily in the unbound state.

Table 2. Data collection and refinement statistics. Statistical values given in parenthesis refer to the highest resolution bin. Data Collection Space Group Unit cell a, b, c (Å) α, β, γ (°) Resolution (Å) Wavelength Observations Unique reflections Rmerge. (%) CC1/2 I / σ (I) Multiplicity Completeness (%)

4B08 + sulfated pep1 P 21 21 21 45.25, 56.61, 97.92 90.0, 90.0, 90.0 26.5 – 1.59 (1.625 – 1.59) 1.000 310,377 (13,334) 32,983 (1,456) 8.0 (83.1) 0.999 (0.802) 18.4 (2.2) 9.4 (9.2) 95.6 (85.6)

Refinement Statistics Resolution (Å) Rwork / Rfree (%)a No. atoms (protein) No. atoms (peptide) No. atoms (solvent) No. atoms (other) B-factor (protein) (Å2) B-factor (peptide) (Å2) B-factor (solvent) (Å2) B-factor (other) (Å2) Ramachandran Plot Preferred Regions (%) Allowed Regions (%) Outliers (%) RMSD Bond (Å) RMSD Angle (°) PDB entry code a

26.5 – 1.59 15.7 / 19.4 1,986 78 266 11 19.4 28.9 40.0 30.0 91.7 7.8 0.5 0.013 1.60 5YY4

Rfree was calculated as Rwork taking 3% of data not included in the refinement.

Effect of PTS in the binding energy Our data indicated that the surge of affinity upon PTS resulted in a decrease of the unfavorable entropy change (-TΔΔS = 2.7 kcal mol-1). Of the three possible causes

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(dehydration, conformational restriction, and formation of salt-bridges),39 our data support the hypothesis that the reduction of entropy loss is the consequence of the restriction of fluctuations of the peptide in the unbound state. Moreover, the close analysis of the value of enthalpy change and the heat capacity change also support the idea that PTS affects the conformational dynamics of pep1 in solution, as explained below.

Figure 4. X-ray crystal structure of pep1 and Sulfo-pep1 in complex with antibody. X-ray crystal structure of (a) pep1 (PDB entry code 5YD3) and (b) Sulfo-pep1 (this work) bound to 4B08. The antibody is shown in gray. The peptide pep1 (orange), Sulfo-pep1 (green), and key residues of the paratope (gray) are depicted with sticks. Intermolecular hydrogen bonds between the peptide and the protein are shown with black dotted lines. (c) Fraction of intramolecular hydrogen bonds within pep1 or Sulfo-pep1. The definition of intramolecular hydrogen bonds was the same as in Figure 1. (d) BSA of the peptide in the unbound state was calculated from the MD simulations. BSA was defined as the difference between the maximum value of solvent accessible surface area (SASAmax) and the actual value of SASA at each simulation time-point. The value of SASAmax corresponding to pep1 and Sulfo-pep1 were 1,663 Å2 and 1,770 Å2, respectively. The SASA values were calculated with VMD version 1.9.2. The normalized distribution plots were calculated with R (version 3.2.3, https://www.R-project.org/) using the kernel density estimation.

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In principle, the decline of the favorable enthalpy change (ΔΔH = 2.4 kcal mol-1) could not be explained from the set of polar interactions observed in the crystal structures, since they were essentially identical to each other. To explain this decline it is necessary to take into account that the average number of intramolecular H-bonds formed within Sulfo-pep1 in solution was 2.15 ± 0.33, whereas that within pep1 was only 1.25 ± 0.09 (Figure 4c). The extra intramolecular H-bonds in the unbound state of Sulfo-pep1 correspond mainly to Tys5 (0.56 ± 0.22 bonds). Since this residue change its conformation when bout to the antibody, these H-bonds must be broken and rearranged upon complexation (for example with H-bonds with water molecules), resulting in an approximately null contribution to the change of enthalpy. Considering that an intra H-bond between a sulfate group and an amide generates approximately -2.9 kcal mol-1,44 the greater number of intramolecular H-bonds of the sulfated peptide (∆H-bonds = ~0.90) was consistent with the magnitude of the decrease of favorable enthalpy change with respect to the nonsulfated peptide (2.0 ± 0.7 kcal mol-1). The greater (less negative) value of the heat capacity change of the interaction of Sulfo-pep1 with respect to pep1 (ΔΔCp = 90 cal mol-1 K-1) cannot be explained from the value of the peptide-antibody binding interface in the crystal structure, since the values were very similar to each other (see above). We also calculated the buried surface area (BSA) of each peptide in the unbound form (Figure 4d). No notable differences were found: the BSA of Sulfo-pep1 was 277 ± 15 Å2, whereas that of pep1 was 251 ± 6 Å2. The gain of heat capacity in the sulfated peptide could thus be explained by the described differences in the conformational freedom and intramolecular non-covalent bonds between the peptides.45,46

Restriction of fluctuations in solution enhances interaction The formation of direct polar interaction between the sulfate group and the partner protein explain several cases of sulfation-mediated protein-protein interactions.4,5,8,9 However, this 15


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mechanism cannot be applied in our case, since the sulfate groups did not engage in direct polar interaction with the antibody 4B08. In our model system we propose that the restriction of the main chain fluctuation in the unbound state strengthens the affinity (Figure 5). MD simulations and NMR suggested that the sulfation of pep1 restricts the conformation of pep1 in solution. In particular, MD simulations revealed a populated conformation in the ensemble characterized by the appearance of additional intramolecular H-bonds involving the sulfate group of Tys5, and to a lesser extent Tys4. These conformations were not present in the crystal structure of the complex with 4B08, resulting in conformational changes that limited the potential of sulfation to boost the affinity for this particular model system.47,48 Considering that the recognition of disordered segment of proteins bearing PTS by cognate receptors may not require such conformational changes from the unbound to the bound state47, a greater effect in the affinity may be observed in other recognition phenomena mediated by PTS.

Figure 5. Effect of sulfation in the dynamics of a peptide in solution and its recognition by an antibody. The figure shows the entropy profile of the peptide along the reaction coordinate. In the unbound form, pep1 appears as an ensemble comprising a greater number of conformations that in the sulfated peptide because of the formation of additional intramolecular H-bonds in the latter. The restriction imposed by the sulfated group in the unbound state thus decreases the entropy loss when recognized by the antibody, resulting in higher affinity for the receptor.

16


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Conclusion Protein tyrosine sulfation (PTS) is a major post-translational modification appearing generally on the disordered region of proteins, enhancing protein–protein interactions. However, the mechanism explaining how sulfation affects the conformational ensemble of the flexible region, and how it might affect the interaction between proteins, has not been clarified to date. Using a model peptide and its specific antibody, our research revealed a novel mechanism by which PTS may strengthen the affinity of protein-protein interactions. We propose that PTS reduces the fluctuation of the disordered region in the unbound form, decreasing the entropic penalty of the interaction and boosting the binding affinity.

ASSOCIATED CONTENT Supporting information The supporting information section includes five figures, four tables, and two movies. AUTHOR INFORMATION Corresponding Authors Jose M.M. Caaveiro, E-mail: [email protected] Kouhei Tsumoto, E-mail: [email protected] Author Contributions KM designed research, performed experiments (protein purification, ITC, MD simulations, and crystallization), analyzed and discussed the results, and wrote the manuscript. TY designed research, analyzed and discussed the results, and contributed new computational tools/models. YA designed research, performed NMR experiments, and analyzed that data. HA analyzed and discussed the results. YT contributed new computational tools. MN analyzed and discussed the results. TH contributed new reagents/materials. TU designed research. J.M.M.C. designed research, performed experiments (crystal manipulation, crystallographic data collection and 17


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processing), analyzed and discussed the results, and wrote the manuscript. KT designed the overall study, designed research, analyzed and discussed the results, and made critical revisions to the manuscript. Funding Sources This work was supported by the Funding program for world-leading Innovative R&D on Science and Technology (FIRST) from JSPS, and by JSPS Grants-in-Aid for Scientific Research 25249115 (K.T.), 15K06962 (J.M.M.C.), 15H05752, 15KT0103 (T.Y.) and SIP project SM4I (T.Y.). Funding sources had no role in study design, in collection, analysis and interpretation of the data, in the writing of the report, or in the decision to submit the article for publication. Notes The coordinates and structure factors for the structure of scFv antibody 4B08 in complex with sulfated pep1 (PDB entry code 5YY4) have been deposited in the PDB. Conflict of interest statement The authors declare that they have no conflicts of interest. Acknowledgements We thank the staff of the Photon Factory (Tsukuba, Japan) for excellent technical support. Access to beamlines BL5A was granted by the Photon Factory Advisory Committee (project 2016G199). This research used computational resource of Tsubame through the HPCI System Research Project (hp160077, hp170130). Abbreviations PTS, protein tyrosine sulfation; MD, molecular dynamics; DQF-COSY, double quantum filtered correlation spectroscopy; ROESY, rotating frame nuclear Overhauser effect spectroscopy; scFv, single-chain variable fragment; ITC, isothermal titration calorimetry; TOCSY, total correlation spectroscopy.

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REFERENCES (1) Stone, M. J., Chuang, S., Hou, X., Shoham, M., and Zhu, J. Z. (2009) Tyrosine sulfation: An increasingly recognised post-translational modification of secreted proteins, Nat. Biotechnol. 25, 299-317. (2) Yang, Y. S., Wang, C. C., Chen, B. H., Hou, Y. H., Hung, K. S., and Mao, Y. C. (2015) Tyrosine sulfation as a protein post-translational modification, Molecules 20, 2138-2164. (3) Kehoe, J. W., and Bertozzi, C. R. (2000) Tyrosine sulfation: A modulator of extracellular protein-protein interactions, Chem. Biol. 7, R57-R61. (4)

Ludeman, J. P., and Stone, M. J. (2014) The structural role of receptor tyrosine sulfation in chemokine recognition, Br. J. Pharmacol. 171, 1167-1179.

(5) Albrizio, S., Carotenuto, A., Fattorusso, C., Moroder, L., Picone, D., Temussi, P. A., and D'Ursi, A. (2002) Environmental mimic of receptor interaction: Conformational analysis of CCK-15 in solution, J. Med. Chem. 45, 762-769. (6)

Liu, C. C., Brustad, E., Liu, W. S., and Schultz, P. G. (2007) Crystal structure of a biosynthetic sulfo-hirudin complexed to thrombin, J. Am. Chem. Soc. 129, 10648-10649.

(7) Qin, L., Kufareva, I., Holden, L. G., Wang, C., Zheng, Y., Zhao, C. X., Fenalti, G., Wu, H. X., Han, G. W., Cherezov, V., Abagyan, R., Stevens, R. C., and Handel, T. M. (2015) Crystal structure of the chemokine receptor CXCR4 in complex with a viral chemokine, Science 347, 1117-1122. (8) Somers, W. S., Tang, J., Shaw, G. D., and Camphausen, R. T. (2000) Insights into the molecular basis of leukocyte tethering and rolling revealed by structures of P- and E-selectin bound to SLe(X) and PSGL-1, Cell 103, 467-479. (9) Thompson, R. E., Liu, X. Y., Ripoll-Rozada, J., Alonso-Garcia, N., Parker, B. L., Pereira, P. J. B., and Payne, R. J. (2017) Tyrosine sulfation modulates activity of tick-derived 19


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

Miyanabe et al.

Page 20 of 30 bi-2018-001433

thrombin inhibitors, Nat. Chem. 9, 909-917. (10) Kiyoshi, M., Tsumoto, K., Ishii-Watabe, A., and Caaveiro, J. M. M. (2017) Glycosylation of IgG-Fc: a molecular perspective, Int. Immunol. 29, 311-317. (11) Tompa, P. (2016) The principle of conformational signaling, Chem. Soc. Rev. 45, 4252-4284. (12) Wilson, I. A., and Stanfield, R. L. (1994) Antibody-antigen interactions - New structures and new conformational-changes, Curr. Opin. Struct. Biol. 4, 857-867. (13) Xin, F., and Radivojac, P. (2012) Post-translational modifications induce significant yet not extreme changes to protein structure, Bioinformatics 28, 2905-2913. (14) Huang, C. C., Lam, S. N., Acharya, P., Tang, M., Xiang, S. H., Hussan, S. S. U., Stanfield, R. L., Robinson, J., Sodroski, J., Wilson, I. A., Wyatt, R., Bewley, C. A., and Kwong, P. D. (2007) Structures of the CCR5 N terminus and of a tyrosine-sulfated antibody with HIV-1 gp120 and CD4, Science 317, 1930-1934. (15) Szyperski, T., Guntert, P., Stone, S. R., and Wuthrich, K. (1992) Nuclear-magnetic-resonance solution structure of hirudin(1-51) and comparison with corresponding 3-dimensional structures determined using the complete 65-residue hirudin polypeptide-chain, J. Mol. Biol. 228, 1193-1205. (16) Fournie-Zaluski, M. C., Belleney, J., Lux, B., Durieux, C., Gerard, D., Gacel, G., Maigret, B., and Roques, B. P. (1986) Conformational-analysis of cholecystokinin CCK26-33 and related fragments by H-1-NMR spectroscopy, fluorescence-transfer measurements, and calculations, Biochemistry 25, 3778-3787. (17) Miyanabe, K., Akiba, H., Kuroda, D., Nakakido, M., Kusano-Arai, O., Iwanari, H., Hamakubo, T., Caaveiro, J.M.M., Tsumoto, K. (2018) Intramolecular H-bonds govern the recognition of a flexible peptide by an antibody, J. Biochem. 164, 1-12. (18) Makabe, K., Asano, R., Ito, T., Tsumoto, K., Kudo, T., and Kumagai, I. (2005) Tumor-directed lymphocyte-activating cytokines: refolding-based preparation of 20


Page 21 of 30 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


bi-2018-001433

recombinant human interleukin-12 and an antibody variable domain-fused protein by additive-introduced stepwise dialysis, Biochem. Biophys. Res. Commun. 328, 98-105. (19) Tsumoto, K., Shinoki, K., Kondo, H., Uchikawa, M., Juji, T., and Kumagai, I. (1998) Highly efficient recovery of functional single-chain Fv fragments from inclusion bodies overexpressed in Escherichia coli by controlled introduction of oxidizing reagent application to a human single-chain Fv fragment, J. Immunol. Methods 219, 119-129. (20) Cierpicki, T., and Otlewski, J. (2001) Amide proton temperature coefficients as hydrogen bond indicators in proteins, J. Biomol. NMR 21, 249-261. (21) Pardi, A., Billeter, M., and Wuthrich, K. (1984) Calibration of the angular-dependence of the amide proton-C-alpha proton coupling-constants, 3jhn-alpha, in a globular protein, J. Mol. Biol. 180, 741-751. (22) Wishart, D. S., Sykes, B. D., and Richards, F. M. (1992) The chemical-shift index - a fast and simple method for the assignment of protein secondary structure through NMR-Spectroscopy, Biochemistry 31, 1647-1651. (23)

Leslie, A. G. W. (2006) The integration of macromolecular diffraction data, Acta Crystallogr. D Struct. Biol. 62, 48-57.

(24) Evans, P. (2006) Scaling and assessment of data quality, Acta Crystallogr. D Struct. Biol. 62, 72-82. (25) Kudo, S., Caaveiro, J. M. M., Nagatoishi, S., Miyafusa, T., Matsuura, T., Sudou, Y., and Tsumoto, K. (2017) Disruption of cell adhesion by an antibody targeting the cell-adhesive intermediate (X-dimer) of human P-cadherin, Sci. Rep. 7, 39518. (26) McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C., and Read, R. J. (2007) Phaser crystallographic software, J. Appl. Crystallogr. 40, 658-674. (27) Emsley, P., Lohkamp, B., Scott, W. G., and Cowtan, K. (2010) Features and development of Coot, Acta Crystallogr. D Struct. Biol. 66, 486-501. (28) Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Refinement of 21


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

Miyanabe et al.

Page 22 of 30 bi-2018-001433

macromolecular structures by the maximum-likelihood method, Acta Crystallogr. D Struct. Biol. 53, 240-255. (29)

Laskowski, R. A., Macarthur, M. W., Moss, D. S., and Thornton, J. M. (1993) Procheck - a program to check the stereochemical quality of protein structures, J. Appl. Crystallogr. 26, 283-291.

(30) Phillips, J. C., Braun, R., Wang, W., Gumbart, J., Tajkhorshid, E., Villa, E., Chipot, C., Skeel, R. D., Kale, L., and Schulten, K. (2005) Scalable molecular dynamics with NAMD, J. Comput. Chem. 26, 1781-1802. (31) MacKerell, A. D., Bashford, D., Bellott, M., Dunbrack, R. L., Evanseck, J. D., Field, M. J., Fischer, S., Gao, J., Guo, H., Ha, S., Joseph-McCarthy, D., Kuchnir, L., Kuczera, K., Lau, F. T. K., Mattos, C., Michnick, S., Ngo, T., Nguyen, D. T., Prodhom, B., Reiher, W. E., Roux, B., Schlenkrich, M., Smith, J. C., Stote, R., Straub, J., Watanabe, M., Wiorkiewicz-Kuczera, J., Yin, D., and Karplus, M. (1998) All-atom empirical potential for molecular modeling and dynamics studies of proteins, J. Phys. Chem. B 102, 3586-3616. (32) MacKerell, A. D., Feig, M., and Brooks, C. L. (2004) Improved treatment of the protein backbone in empirical force fields, J. Am. Chem. Soc. 126, 698-699. (33) Vanommeslaeghe, K., Hatcher, E., Acharya, C., Kundu, S., Zhong, S., Shim, J., Darian, E., Guvench, O., Lopes, P., Vorobyov, I., and MacKerell, A. D. (2010) CHARMM general force field: A force field for drug-like molecules compatible with the CHARMM all-atom additive biological force fields, J. Comput. Chem. 31, 671-690. (34) Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W., and Klein, M. L. (1983) Comparison of simple potential functions for simulating liquid water, J. Chem. Physics. 79, 926-935. (35) Daura, X., Jaun, B., Seebach, D., van Gunsteren, W. F., and Mark, A. E. (1998) Reversible peptide folding in solution by molecular dynamics simulation, J. Mol. Biol. 22


Page 23 of 30 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


bi-2018-001433

280, 925-932. (36) Ho, B. K., and Dill, K. A. (2006) Folding very short peptides using molecular dynamics, PLoS Comput. Biol. 2, 228-237. (37) Yamashita, T. (2016) On the accurate molecular dynamics analysis of biological molecules, AIP Conf. Proc. 1790, 20026. (38) McDonald, I. K., and Thornton, J. M. (1994) Satisfying hydrogen-bonding potential in proteins, J. Mol. Biol. 238, 777-793. (39) Falconer, R. J. (2016) Applications of isothermal titration calorimetry - the research and technical developments from 2011 to 2015, J. Mol. Recognit. 29, 504-515. (40) Freire, E., Mayorga, O. L., and Straume, M. (1990) Isothermal titration calorimetry, Anal. Chem. 62, A950-A959. (41)

London, N., Movshovitz-Attias, D., and Schueler-Furman, O. (2010) The structural basis of peptide-protein binding strategies, Structure 18, 188-199.

(42)

Loladze, V. V., Ermolenko, D. N., and Makhatadze, G. I. (2001) Heat capacity changes upon burial of polar and nonpolar groups in proteins, Protein Sci. 10, 1343-1352.

(43) Yokota, A., Tsumoto, K., Shiroishi, M., Nakanishi, T., Kondo, H., and Kumagai, I. (2010) Contribution of asparagine residues to the stabilization of a proteinaceous antigen-antibody complex, HyHEL-10-hen egg white lysozyme, J. Biol. Chem. 285, 7686-7696. (44) Rapp, C., Klerman, H., Levine, E., and McClendon, C. L. (2013) Hydrogen bond strengths in phosphorylated and sulfated amino acid residues, PLoS One 8, e57804. (45) Gomez, J., Hilser, V. J., Xie, D., and Freire, E. (1995) The heat-capacity of proteins, Proteins 22, 404-412. (46) Sturtevant, J. M. (1977) Heat-capacity and entropy changes in processes involving proteins, Proc. Natl. Acad. Sci. USA 74, 2236-2240. (47) Krieger, J. M., Fusco, G., Lewitzky, M., Simister, P. C., Marchant, J., Camilloni, C., 23


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Feller, S. M., and De Simone, A. (2014) Conformational recognition of an intrinsically disordered protein, Biophys. J. 106, 1771-1779. (48) Vogt, A. D., and Di Cera, E. (2013) Conformational selection is a dominant mechanism of ligand binding, Biochemistry 52, 5723-5729.

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Tys5 Tyrosine sulfation Page 25 of 30 Biochemistry

O2S

O1S

1 2 3 4 5 6 7

Transient stabilization by intramolecular H-bonds

O3S Asn3-sc

Thr6

Asp1 Ile2

Glu8

Asn3

ACS Paragon Plus Environment Tys4

Biochemistry

pep1 Sulfo-pep1

180

b

pep1

0.8

Sulfo-pep1

135 0.6

90

Variance

45 0 -45 -90

0.4 0.2

-135 -180

0

Ile2

c

Asn3

Tyr4/Tys4 Tyr5/Tys5

Tys5

Ser7

Glu8

Ile2

Tyr4/Tys4 Tyr5/Tys5

Thr6

Ser7

Glu8

0.25 0.20

O3S

Asn3-sc Asp1 Ile2 Asn3

Glu8

Asn3

d

O2S O1S

Thr6

Thr6

Fraction

a Average angle (°)

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

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0.15 0.10

0.05

Tys4

0

Asp1


Ile2

Asn3

Asn3-sc

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b

pep1

E8

N3

Y5 Y4

S7

T6

Sulfo-pep1

S7 N3 Tys5 Tys4 E8

I2

35 °C

4 E8

N3

N3

Y5

S7

T6

S7

Y4

N3

E8

T6

T6

1

I2

I2

20 °C N3

E8

Y5 S7

Y4

0

T6

I2

8.6

I2

2

Y4

S7

T6

35 °C

I2

Tys5 Tys4

T6

30 °C

S7 N3 Tys5 Tys4 E8

8.2

8.0

7.8

2

T6

25 °C

1

S7 N3 Tys5 Tys4 E8

T6

20 °C N3 S7 E8 Tys5 Tys4

0

T6

15 °C

15 °C

8.4

3

S7

3

25 °C Y5

N3 E8

I2

30 °C Y5

E8

Y4

[rel]

a [rel]

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

Biochemistry

7.6

7.4

8.6

8.4

[ppm]

8.2

8.0

[ppm]


7.8

7.6

7.4

Biochemistry

a 10

20

30

40

50

0

60

0

10

20

30

40

50

60

-10

0

-0.2

-0.1

-0.3

-0.3

-0.4 0

-0.4 0

-4.0 -8.0 -12.0

-12

-0.2

ΔH (kcal mol-1)

μcal sec -1

-0.1

kcal mol-1 of injectant

μcal sec -1

b

Time (min)

Time (min) 0

kcal mol-1 of injectant

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

Page 28 of 30

-4.0 -8.0

-14

-16

-18

-12.0

-16.0

-20

-16.0

0

0.5

1.0

1.5

Molar ratio

2.0

0

0.5

1.0

1.5

Molar ratio

2.0


15

20

Temperature (°C) 25

30

35

Page 29 of 30 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

Biochemistry


Biochemistry

Conformational Ensemble Fluctuation

Sulfo-pep1

pep1

pep1

Sulfo-pep1

Average S of Sulfo-pep1

Small ﬂuctuation

nb

ou

pl ex

nd

Single chain Fv

om

Reduce average ﬂuctuation (ΔΔS

Tyrosine Sulfation Restricts the Conformational Ensemble of a Flexible

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