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Insights into CC chemokine ligand 2 - chemokine receptor 2 molecular recognition: a step forward towards anti-chemotactic agents Katlyn Silva David, Edson R.A. Oliveira, Bruno A.C. Horta, Ana Paula Valente, and Viviane Silva de Paula Biochemistry, Just Accepted Manuscript • Publication Date (Web): 01 Jun 2017 Downloaded from http://pubs.acs.org on June 2, 2017

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Insights into CC chemokine ligand 2 - chemokine receptor 2 molecular recognition: a step forward towards anti-chemotactic agents Katlyn S. David,‡ Edson R. A. Oliveira,§ Bruno A. C. Horta,§ Ana P. Valente,k,⊥ and Viviane S. de Paula∗,‡,⊥ ‡Campus Xerém, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 25245-390, Brazil §Instituto de Qu´ımica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-909, Brazil kInstituto de Bioqu´ımica Médica, Centro Nacional de Ressonˆ ancia Magnética Nuclear Jiri Jonas, Universidade Federal do Rio de Janeiro, 21941-920, Brazil ⊥Centro de Biologia Estrutural e Bioimagem, Rio de Janeiro, 21941-920, Brazil E-mail: [email protected] Phone: +55 (21) 26791018

Running header Insights into CC chemokine ligand 2 - chemokine receptor 2 molecular recognition: a step forward towards anti-chemotactic agents

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Biochemistry

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Abstract Chemokine ligand 2 (CCL2), also known as monocyte chemo-attractant protein 1 (MCP-1), is a chemokine that recruits immune cells to inflammatory sites by interacting with the G-protein coupled receptor CCR2. The CCL2/CCR2 axis is also involved in pathological processes such as tumor growth and metastasis and hence is currently considered as an important drug target. CCL2 exists in a dynamic monomer/dimer equilibrium that is modulated by CCR2 binding. We used solution NMR spectroscopy and MD simulations to study the interactions between CCL2 and a sulfopeptide corresponding to the N-terminal sequence of CCR2 (CCR218-31 ). Peptide binding induced the dissociation of CCL2 into monomers, forming stable CCL2/CCR218-31 complexes. NMR relaxation measurements indicated that residues around the CCR218-31 binding site, which are located at the dimer interface, undergo a complex regime of motions. NMR data were used to construct a tridimensional structural model of the CCL2/CCR218-31 complex, revealing that CCR218-31 occupies a binding site juxtaposed to the dimer-interface, partially replacing monomer-monomer contacts, explaining why CCR218-31 binding weakens the dimer interface and induces dissociation. We found that the main interactions governing receptor binding are highly stable salt-bridges with conserved chemokine residues as well as hydrophobic interactions. These data provide new insights into the structure-function relationship of the CCL2-CCR2 interaction and may be helpful for the design of novel anti-chemotactic agents.

Introduction Inflammation is a defensive-orchestrated physiological response to fight infections and tissue damages. 1 However, under a wide range of circumstances, this response can be continuously activated by the immune system, and instead of conferring protection, it can lead to a deleterious scenario. The results are often characterized as persistent inflammatory diseases, with atherosclerosis as one of the most noteworthy examples. 2 Other diseases such as multiple sclerosis, asthma and cancer are also implicated with misdirected or over-exaggerated 2


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inflammatory response. 3,4 Among the multitude of molecules that mediate the inflammatory process, the CC-chemokine-ligand-2 / CC-chemokine-receptor-2 (CCL2/CCR2) axis has drawn attention due to its participation in guiding inflammatory cells to the site of inflammation. 5,6 As a consequence of this central role, CCL2/CCR2 axis is considered as a major target in numerous drug discovery studies. 7 Autoimmune-induced inflammatory diseases have been responsible to lower the quality of life of millions of people worldwide and, in this sense, efforts are still necessary to widen the options of treatment. 8 The ability of chemokines to recruit leukocytes is mediated by high-affinity interactions with G protein-coupled receptors (GPCRs) expressed in the leukocyte membranes. 6 Humans express about 40 chemokines that are broadly classified on the basis of their conserved cysteines into two major (CXC and CC) and two minor subfamilies (CX3C and C). 7 All of them present a three-dimensional structure composed by a three-stranded β-sheet followed by an α-helix, which is typically stabilized by two conserved disulfide bonds. CC and CXC chemokines form distinct quaternary structures, so that monomeric or dimeric CXC chemokines can activate CXC receptors, whereas only monomeric CC chemokines can activate CC receptors. 9–12 According to the prevalent model, the activation of chemokine receptors involves two sites of interaction. The Site-I, which is the recognition between the chemokine N-loop and the receptor N-terminal residues, and the Site-II, that is represented by interactions between the chemokine N-terminal and the receptor extracellular/transmembrane residues. 13 A key post-translational modification that is prevalent among membrane-bound proteins such as chemokine receptors is the tyrosine sulfation. Such modification on chemokine receptor N-terminal region has been described as a main factor that regulates chemokinereceptor interactions. 14 Studies using differentially sulfated N-terminal peptides from various chemokine receptors have shown that tyrosine sulfation increases the binding affinity of the receptor peptides to their cognate chemokines. 15–19 Structures of two CXC chemokines (CXCL8/interleukin-8 and CXCL12/SDF-1) 12,20 and one CC chemokine (CCL11/ eotaxin3


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1), 21 bound to N-terminal fragments of their respective receptors, have revealed details of the recognition between receptor N-terminal and chemokines N-loop regions. Additionally, it was found that the chemokine receptor CCR2 contains two sulfated tyrosine residues that modulate chemokine binding. 22

The solution structure of human CCL2 has been determined by NMR spectroscopy 23 and showed that CCL2 dimerizes via an intermolecular N-terminal antiparallel β-sheet recognition. These interactions also involve the N-loop (residues that range from I5 to T16) and other three aminoacids, E50, I51 and C52. CCL2 monomer and dimer are in slow exchange equilibrium and can be observed as separate sets of 1 H-15 N HSQC resonances. Tan and colleagues found that an N-terminal CCR2 peptide sulfated at Y26 and Y28 predominantly binds to monomeric CCL2 and this interaction shifts the monomer-dimer equilibrium to favour the monomeric form. 19 It was also shown that an obligate monomeric mutant CCL2 (P8A) binds more tightly to CCR2-derived sulfopeptides than obligate dimeric mutant CCL2 (T10C). 19 Although the above experiments strongly suggest that sulfation of Y26 and Y28 in CCR2 can modulate the monomer-dimer equilibrium of CCL2, the structural basis of the molecular recognition between CCL2 and CCR2 remain unknown.

In this work, we have studied the structure and dynamics of CCL2-CCR2 sulfopeptide complex using solution NMR spectroscopy and molecular dynamics (MD) simulations. We provided molecular evidence of how hydrophobic and electrostatic interactions jointly contribute to the stabilization of the chemokine-receptor complex. The NMR data and MD simulations revealed that sulfated N-terminal of CCR2 triggers CCL2 dimer dissociation with altered dynamics. A more detailed description of the CCL2-CCR2 binding mechanism is proposed here, which may be useful to assist further pharmacological research on novel modulators of chemotaxis. 4


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Methods Cloning, expression and purification of CCL2 The CCL2 WT (corresponding to residues 1-76) was cloned into the expression vector pET32a into the 5’ NcoI and 3’ BamHI restriction sites (Genscript). The expression in pET-32a vector provides a fusion partner Thioredoxin A (TRX) in the N-terminal of the protein. The fusion construct comprises a 163 residues N-terminal expression/purification tag for a total length of 239 amino acids and molecular weight of 26 kDa. 13

C/15 N and

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N-labelled NMR samples were produced in Escherichia coli Rosetta-gami

B (DE3) harboring the expression construct. The bacterial culture was grown in M9 minimal medium supplemented with [13 C]-glucose (3 g/L) and/or 15 NH4 Cl (1 g/L), as the only sources for carbon and nitrogen, respectively. The M9 medium was also supplemented with yeast nitrogen base without aminoacids 1.7 g/L (SIGMA) and MEM Vitamin Solution (SIGMA). Ampicillin (50 µg/mL) and Chloramphenicol (34 µg/mL) were incorporated to the selective medium. The cells were cultured at 30 o C under agitation (200 rpm) until optical density (OD600 ) reached 0.7. Subsequently, protein expression was induced with 0.4 mM isopropyl-βD-thiogalactopyranoside (IPTG) for 18 h under agitation at 20 o C. The cells were harvested by centrifugation, resuspended in lysis buffer [50 mM Tris-HCl (pH 7.6), 500 mM NaCl, 10 mM Imidazole, 10 µg/mL lysozyme and 1mM phenylmethanesulfonyl fluoride (PMSF)] and disrupted by sonication. The cellular debris were separated by centrifugation at 6000g for 30 min at 4 o C. Soluble protein in the supernatant of cell lysate was purified by a Ni2+ affinity chromatography step using a His-Trap HP 5 mL column (GE Healthcare) previously equilibrated in 50 mM Tris–HCl buffer (pH 7.6), 500 mM NaCl, 10 mM imidazole. TRXHis6 -CCL2 was eluted in 50 mM Tris–HCl buffer (pH 7.6), 500 mM NaCl, 200 mM imidazole and subsequently dialyzed against 50 Tris-HCl buffer (pH 7.6), 200 mM NaCl. The removal of the N-terminal TRX-His6 tag was achieved using 5U of enterokinase (Invitrogen). The efficiency of enterokinase cleavage was monitored by SDS-PAGE. CCL2 was further purified 5


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to homogeneity by reverse phase chromatography using a C8 column PRP-3 (Hamilton 7.0 mm x 305 mm) with a linear gradient of acetonitrile (10 to 90%) containing 0.1% TFA. The resulting sample was freeze-dried under vacuum. SDS-PAGE revealed protein purity of >98%.

NMR Experiments 15

N labeled CCL2 samples were prepared in 20 mM Tris-HCl buffer, 24 0.02% NaN3 , 90/10%

(vol/vol) H2 O/D2 O, pH 7.0. NMR experiments were performed on a Bruker DRX 600 equipped with a 1 H,15 N,13 C TXI-cryoprobe or Bruker Avance III 800-MHz instruments at 25 o C. CCL2 1 H-15 N assignments were transferred from previously published chemical shift table. 23 The synthetic sulfopeptide (purity >95%) corresponding to the N-terminal of chemokine receptor CCR2 was purchased from American Peptide. The peptide has the following sequence: CCR2(sulfated)-

18 EEVTTFFDs YDs YGAP31 .

A stock solution of the CCR2 sul-

fopeptide (1.5 mM) prepared in the same buffer was added to the protein sample and a series of 1 H-15 N HSQC spectra were collected until essentially no changes in the chemical shifts were observed. The final protein/peptide molar ratio for the CCL2 was 1:3. The pH at each step of the titration was kept constant. Chemical shift perturbations were calculated using CcpNmr analysis. 25

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N backbone dynamics

Relaxation experiments were recorded at 0.18 mM and 1 mM

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N-labeled CCL2 at field of

600 MHz. The NMR relaxation data at 1 mM supports the formation of a trimer or even a tetramer, considering that CCL2 forms a higher order structure (i.e., oligomer) at high concentrations. For this reason, all experiments were performed at 0.18 mM (Fig. S1).

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N

R1 and R2 relaxation rates were measured from spectra with different relaxation delays: 50, 100 (duplicate), 200, 250, 500, 750, and 1000 ms for R1 and 16, 48, 80 (duplicate), 6


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112, 144, 176, 208 ms for R2 . The errors in the peak intensities were calculated from the duplicate experiments. The 1 H–15 N heteronuclear NOEs were determined from the ratio of peak intensities with and without the saturation of the amide protons. Errors in the heteronuclear NOE values were calculated from the peak intensities and noise levels in the reference and saturated spectra. Relaxation data curve-fitting was performed using CcpNmr analysis.

Model free analysis In order to characterize the ps-ns time-scale backbone dynamics and explore the potential occurrence of conformational exchange (Rex ) on the µs-ms time-scale, we have analyzed the

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N relaxation data recorded as described above and using TENSOR2. 26 Relaxation

parameters were fitted according to the Lipari-Szabo model-free formalism. 27 In order to extract the intramolecular dynamics, S2 (squared order parameter of the NH bond vector) and Rex (exchange broadening) were derived from the 15 N-R1 , R2 and 1 H-15 N NOE datasets (for both the free- and bound-CCL2) using the common models of the spectral density function. The diffusion tensors for spherical and axially symmetric diffusion models were estimated on the basis of

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N-R2 /R1 values for rigid amides and which had NOE ratios

> 0.65. The TENSOR2 program was used to define a motional model for CCL2 free and CCL2-CCR218-31 complex with an axially symmetric diffusion model in the presence of an anisotropic tensor.

Computational details Molecular docking using HADDOCK Molecular docking of the CCR218-31 peptide to CCL2 monomer (PDB id: 1DON) 23 was carried out using HADDOCK 2.1 28–30 so as to generate an interaction model for this protein complex. The calculations were performed using NMR chemical shift mapping as ambiguous 7


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interaction restraints to define the residues potentially involved in binding. The CCL2 residues considered as “Active” were A4, I5, A7, F15, K19, I20, V22, R24, L25, A26, T45, I46, V47, A48, K49, E50 and I51. Active residues for CCR218-31 included E18, E19, T21, T22, F23, F24, D25, sY26, D27, sY28 and G29, and were defined from 2D TOCSY experiments for the free peptide and in the presence of

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N CCL2. Routinely, HADDOCK uses a three-

stage search procedure: rigid-body docking (it0-step), flexible interface docking (it1-step) and a step considering a water layer around the complex (water-step). The present protocol used 5000 steps for it0-step, of which the top 200 models were selected for it1-step. During this stage, the N-terminal region of CCL2 and the entire CCR218-31 were allowed to be fully flexible during the calculations. These 200 models were further refined by the water-step and ranked according to the HADDOCK score, which considers the van der Waals, electrostatic and restraint energies, as well as an empirical desolvation penalty. 31 The calculated models were finally clustered using a 7.5 ˚ A interface RMSD cutoff.

Simulated systems

The systems considered in this work were the CCL2 dimer extracted from the previously determined NMR structure deposited under PDB code 1DON 23 and the best model of CCL2CCR218-31 obtained from molecular docking using the HADDOCK platform. Parametrization of the non-standard residue sY (TYS - sulfotyrosine) within the AMBER ff99SB-ILDN force field 32 was based on the Y (TYR - tyrosine) residue. The difference between Y and sY is the replacement of the hydroxyl group by a sulfate group. The partial atomic charges for the atoms in the sY residue were derived by using the standard RESP procedure 33 and a full description of the sY residue is shown in Table S1. Missing atoms were added using the Swiss PDB Viewer (SPDBV) program. 34 8


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Simulation protocol

The MD simulations were performed using the GROMACS simulation package 35,36 version 5.1.2 together with the AMBER ff99SB-ILDN force field 32 and the TIP3P water model. 37 The hydrogen atoms were added according to a neutral pH condition. The simulations were carried out under periodic boundary conditions based on rectangular boxes. Newton’s equations of motion were integrated using the leapfrog scheme 38 with a time step of 2 fs. All bonds involving hydrogen atoms were constrained using the LINCS procedure. 39 A cutoff distance of 10 ˚ A was used for the truncation of the Lennard-Jones interaction and the shortrange electrostatic interactions. The smooth particle mesh Ewald (SPME) method 40 was used with a 32 × 32 × 32 grid and a fourth-order interpolation scheme to compute the longrange electrostatic interactions. The pairlists were generated and updated according to the Verlet scheme. 41 The center of mass motion was removed every 100 ps. Systems were energy minimized with 5000 steps of the Steepest-descent algorithm and subsequently equilibrated. Equilibrations were performed using positional restraints on the protein atoms and lasted 300 ps. The simulations were run under the NPT ensemble (constant number of particles N , pressure P , and temperature T ) with a reference pressure P of 1 bar and reference temperature T of 298.15 K. The temperature was maintained by a velocity-rescaling algorithm 42 with the protein and solvent degrees of freedom separately coupled to a temperature bath at temperature T , with a coupling constant of 0.1 ps. Aside from the equilibration that was performed using a Berendsen thermostat 43 with compressibility of 4.5 e−5 bar−1 and relaxation time of 2 ps, the pressure was maintained close to the reference pressure P by isotropic coupling using the Parrinello-Rahman algorithm. 44 The production runs had a duration of 500 ns and were performed in triplicates. Each simulation used different seeds for initial velocities assignment. The equilibration steps were not considered for analysis and were not represented in the time series. 9


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Trajectory analysis Root mean square deviations (RMSD) were calculated for all Cα atoms of chain A after leasts square fitting to the central (excluding termini) Cα atoms of the CCL2 monomer (residues 6 to 70). RMSF data were calculated using all Cα coordinates. Hydrogen bond analysis was performed using a combination of standard GROMACS tools and personal scripts. The occupancies of hydrogen bonds were calculated by dividing the occurrence of a given bond by the total number of frames corresponding to the last 100 ns of the MD trajectory. For solvent-accessible surface area analysis (SASA), the software NACCESS version 2.1.1 45,46 was employed. The results were averaged on a per residue basis considering the last 100 ns.

Results NMR mapping of CCL2 residues altered by CCR2 sulfopeptide binding Previous studies have shown that the N-terminal region of CCR2 is critical for binding and recognition of CCL2. 11,19 Here, we investigated the CCR2-sulfopeptide-binding region of CCL2 by using a peptide sequence (18 EEVTTFFDs YDs YGAP31 ) spanning residues 18-31 corresponding to the extracellular N-terminal domain of CCR2 (CCR218-31 ). Note that this peptide presents two sulfated tyrosine residues: one at position 26 (sY26) and another at position 28 (sY28). We employed the classical structure-activity relationship (SAR) using NMR spectroscopy 47 to characterize the regions perturbed by peptide binding. The 1 H, 15

N HSQC spectrum of free CCL2 previously assigned 23 was used to monitor chemical shift

changes of amide resonances upon binding to CCR218-31 (Fig. 1a and Fig. S2). By measuring the chemical shifts perturbations (CSP) of all backbone amide cross-peaks caused by the presence of the peptide, we were able to identify large changes of CSP that correlated with CCR218-31 binding. Considering the CSP data (Fig. 1b), the most perturbed 10


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Figure 1: Interaction of CCL2 with CCR2 sulfopeptide by NMR chemical shift perturbation (CSP) analysis. (A) A section of the 1 H,15 N HSQC spectra of the free CCL2 (magenta) at 0.18 mM and in the presence of CCR2 sulfopeptide at 1:3 molar ratio (green). (B) CSP map of CCL2-CCR2 interactions. The statistical significance of the change seen for an individual residue can be judged by the dotted lines. The sequence-specific secondary structural elements from the NMR structure of CCL2 (PDB entry 1DON) are shown on the top of the CSP map.

amides are those that belong to residues A4, I5, A7 (N-terminal), F15, K19, I20 (N-loop), V22, R24 (310 -turn), L25, A26 (β1 strand), T45-I51 (β2-β3 loop and β3 strand). These data are in agreement with a previously published study describing similar regions in an obligate monomeric mutant CCL2 (P8A) for the interaction with CCR218-31 . 19 Three basic aminoacids (K19, R24 and K49) and twelve hydrophobic aminoacids (A4, I5, A7, F15, I20, V22, L25, A26, I46, V47, A48 and I51) had backbone (15 N and 1 H) resonances significantly 11


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affected by peptide binding (Fig. 1b), indicating that both electrostatic and hydrophobic interactions contribute to CCR218-31 binding. It is also important to note that the values of the dissociation constant Kd of wild type CCL2-CCR2 complex were previously reported and range from 13.9 (± 1.2) µM (for monomer CCL2) to 37.8 (± 4.3) µM (for dimer CCL2). 19

Binding to CCR2 sulfopeptide promotes CCL2 dissociation Chemokine dimer dissociation has been proposed to be a required step for activation of the chemokine receptors. 11,17,48 The interactions between CCL2 and CCR2 N-terminal region have been investigated by NMR spectroscopy and it was suggested that CCL2 dimers dissociate upon binding to CCR218-31 . 19 However, apart from the current knowledge, the dynamical behavior of this complex formation is still a matter of debate. Aiming to better characterize the CCL2-CCR2 recognition, we used

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N spin-relaxation NMR methods to

examine the ps-ns and µs-ms timescale conformational dynamics of the free CCL2 and the CCR218-31 -bound complex. Fig. 2 shows the 15 N-R1 and R2 relaxation rates, and 1 H-15 N heteronuclear Nuclear Overhauser Enhancement (15 N,1 H-NOE) for free CCL2 (red circles) and CCL2-CCR218-31 complex at a 1:3 molar ratio (black circles) measured at 600 MHz. The median R1 , R2 and

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N,1 H-

NOE at 600 MHz for free CCL2 are 1.11 (± 0.18) s-1 , 14.83 (± 3.6) s-1 and 0.69 (± 0.35), respectively. Interestingly, we observed a general decrease in R2 in the backbone of the bound state of CCL2 compared to its free form. The median R1 , R2 and 15 N,1 H-NOE at 600 MHz for CCL2-CCR218-31 complex are 1.18 (± 0.21) s-1 , 11.50 (± 4.8) s-1 and 0.73 (± 0.37), respectively. Notably, both R2 and R2 /R1 showed a region exhibiting extensive µs-ms conformational exchange, in the bound state of CCL2, encompassing part of the CCR218-31 binding site residues from β0, N-loop, β1/β2 loop (30s loop), β2 and β3 strands. The R2 relaxation rate for the CCL2-CCR218-31 complex at a 1:1 molar ratio is 14.84 (± 4.7) s-1 . This value is almost the same as the R2 value for CCL2 free in solution 14.83 (± 3.6) s-1 , indicating that equimolar concentration of the peptide is not sufficient to promote CCL2 dissociation 12


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(Fig. S3). For this reason we used 1:3 ratio of CCL2-CCR218-31 complex in order to shift the equilibrium towards monomers, obtaining a R2 value of 11.50 (± 4.8) s-1 .

Figure 2: Backbone dynamic changes upon CCR2 sulfopeptide binding to CCL2. 15 N relaxation parameters measured at 600 MHz; (A) R1 , (B) R2 , (C) R2 /R1 and (D) 1 H-15 N heteronuclear NOE for the backbone amide groups obtained in the free CCL2 (red circles) and in the complex with CCR2 sulfopeptide (1:3 equivalent molar ratio, black circles). To further characterize fast internal motions and conformational exchange in the backbone of CCL2 and to evaluate the existence of rotational diffusion anisotropy, the relaxation parameters were analyzed using the Lipari-Szabo model-free formalism. 27 A correlation time for protein tumbling (τ c ) of 11.3 ns at 25 o C was estimated from the R2 /R1 ratio excluding those residues exhibiting below-average 15 N,1 H-NOEs values ( 2.5 Hz are shown. (B) Rex mapped on the structure of CCL2 in the bound state (structure derived from HADDOCK model). Low values indicate rigidity, whereas higher values indicate conformational exchange. Color coding by Rex (in Hz) is as follows: dark blue, Rex ∼ 0; light blue, 2 4; magenta, 4 8; red, 8 12; orange, 12 16; yellow, 16. (C) CCL2 dimer with the representation of residues presenting large Rex values (between 10 and 22 s-1 ) that are directly involved with (red) or near (yellow) the CCR218-31 binding site. These residues are represented for the chain A (in blue), while the backbone of chain B (in orange) is also depicted to facilitate the visualization of the dimer interface.

changes in the order parameters upon CCR218-31 binding (Fig. 4b). The decrease in S2 is associated with an increase in conformational entropy. 56 The binding of CCR218-31 causes an increase in the amplitude of amide-bond fluctuations on the ps–ns timescale. More specifically, these gains of flexibility are mainly located in the N-loop, β1/β2 loop and β3strand, therefore there is a correlation between order parameter changes and direct ligand 16


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Figure 4: Effect of CCR2 sulfopeptide binding on CCL2 order parameters. (A) Generalized order parameters (S2 ) for each residue in the free CCL2 (red) and the CCL2CCR218-31 complex (black). (B) Differences in order parameters (∆S2 ) between CCL2CCR218-31 complex and free CCL2 for each residue. ∆S2 is given as S2 (after binding) - S2 (before binding), so negative ∆S2 values denote enhanced flexibility of the protein backbone upon binding. (C) Root mean square fluctuations (RMSF) of complexed or non-complexed systems averaged considering the last 100 ns of three 500ns-MD runs. (D) Surface representation of CCL2 monomer where low ∆RMSF values (red) correspond to regions that are more rigid in the bound form (CCL2-CCR218-31 ), whereas high ∆RMSF values (blue) correspond to regions that are more flexible in the bound state.

contacts. Typically, the formation of a complex is usually accompanied by a reduction in conformational entropy and an increase in S2 . Similar decreases in S2 upon complex formation have been reported before and proposed to contribute significantly to the stability of the complex by decreasing its Gibbs free energy due to an increase in conformational 17


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entropy. 57–59 The dynamic nature of the CCL2-CCR218-31 complex is complemented by the molecular dynamics (MD) simulations. Two systems were considered, the free CCL2 dimer and the CCL2-CCR218-31 complex. The root-mean-square fluctuations (RMSF), calculated over the last 100 ns of 500 ns MD simulation trajectories, show that almost all CCL2 residues became more flexible upon CCR218-31 binding (Fig. 4c). This is especially more evident for the Nterminal region, where the ∆RMSF values (RMSFbound − RMSFfree ) were much more positive when compared to the rest of the protein (Fig. 4d).

Structural model of CCL2-CCR2 sulfopeptide complex The CCL2 structure reveals that the basic and hydrophobic residues identified by NMR chemical shift mapping form a contiguous surface within a monomer. From the relaxation parameters we have concluded that upon CCR218-31 binding, the monomer form of CCL2 is prevalent within its dimer-monomer equilibrium. To gain insights into the structure of the complex, we carried out molecular docking calculations using the HADDOCK program. 29 For that we used CSP data (from both CCL2 and CCR218-31 ) as ambiguous interaction restraints (AIRs) to drive the docking process. The CCL2 monomer and CCR218-31 were used as starting structures for the docking process. For CCL2, the residues highlighted in Fig. 1 were defined as active in HADDOCK (A4, I5, A7, F15, K19, I20, V22, R24, L25, A26, T45, I46, V47, A48, K49, E50 and I51). The active residues of CCR218-31 were those that changed when comparing the 2D total correlated spectroscopy (TOCSY) spectrum of free and bound states (E18, E19, T21, T22, F23, F24, D25, sY26, D27, sY28 and G29), assuming that these residues participate in the binding process (Fig. S4). We performed different HADDOCK runs to ensure that the input constraints did not favor specific structural models and that a significant number of possible binding geometries in the monomer were considered. In our analyses, the NMR data were consistent with only one model, in which the N-terminal region of CCL2 monomer and the CCR218-31 were considered fully flexible during the calculations. 18


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The lowest energy structure obtained from the NMR-restrained docking of the CCL2CCR218-31 complex is displayed in Fig. 5. Specific interactions formed between CCL2 and

Figure 5: Molecular docking of the CCL2-CCR218-31 complex. (A) Ribbon structure of CCL2 bound to CCR218-31 . The chemokine is shown as gold ribbon and the CCR218-31 is shown as a red ribbon with side chains sticks for sY26 and sY28. Residues showing substantial CSPs upon CCR2 titration are colored in green. The conserved R24 and K49 residues of CCL2 are shown as green sticks. Hydrogen bonds and salt bridges formed with the sulfate moieties (obtained by HADDOCK output) are shown as yellow dashed lines. (B) CCL2 surface and CCR218-31 ribbon representations with sY residues shown as red sticks (C) Electrostatic potential was generated with the program Pymol (Delano Scientific LLC). The surface polarity is scaled with colors, using blue for the positively charged surface and red for the negatively charged surface, while a white color indicates nonpolar patches. (D) Structural detail exhibiting the residues K49, sY26, R24 and sY28 interacting in a chain of alternating charges.

CCR218-31 involve six intermolecular hydrogen bonds between residues T10, F15, T16, R18, K35 and T45 of CCL2 and residues E19, D25, sY26, D27, E18 and sY26 of CCR218-31 , 19


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respectively. In addition, two salt bridges between residues R24 and K49 of CCL2 and residues sY26 and sY28 of CCR218-31 were identified. Note that, the functionally important CCR2 residues, sY26 and sY28, were found to form contacts with the CCL2 structure in this model. The model predicts that these residues, together with residues D25 and D27, lie on a positively charged surface of CCL2 as shown by the electrostatic potential depicted in Fig. 5c. Interestingly, the antiparallel β-sheets, which stabilize the dimer interface, undergo a big change in conformation upon CCR218-31 binding, leading to a complete loss of these structural elements. This observation explains the increase in the N-terminal flexibility. MD simulations were also used to assess the time evolution of the interactions between CCL2 and CCR218-31 . Overall, both systems remained stable during the course of the simulation, although more pronounced conformational changes were observed considering the N-terminal region of the CCR218-31 peptide (see RMSD plots in Fig. S5a). In our model, the peptide occupies a slit (of approximately 18 nm) perpendicular to the β-sheet and next but not superposing the dimer interface (Fig. S5b). The interactions between the C-terminal region of CCR218-31 and the positively charged surface of CCL2 were highly stable and showed a very good agreement with those predicted by the docking model. However, the N-terminal of CCR218-31 underwent significant deviations from the docking model. It was interesting to observe that the agreement with the primary experimental data (CSP in Fig. 1b) improved after the simulation, indicating that the simulation was able to further optimize the structural model. In order to identify complementary polar interactions between CCL2 and CCR218-31 , the intermolecular hydrogen bonds were calculated over the last 100 ns of the trajectories (Table 1). The most notable residue pairs involved in stable hydrogen bonds between the C-terminal region of CCR218-31 and CCL2 are: sY26-T45, sY26-R24, sY28-R24, sY28-S21, A30-K19, T22-Y13, T22-N14, D25-N14 and sY26-K49. Most of these interactions participate in a chain of alternating charges, which neutralizes the binding pocket of the C-terminal region (Fig. 5d). These interactions are stable throughout the entire simulation. In special, the residue R24 that was initially involved in a salt-bridge with sY28 suffered 20


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Table 1 Hydrogen bonds between CCL2 and CCR2 peptide averaged over the last 100 ns of the MD simulation. The columns are displayed as follows: Residue denoted by its number, one-letter code and the name of the atom directly involved in the hydrogen bond (within brackets); Atom Number as in the simulation file and Occupancy. The results are sorted by decreasing value of occupancy and only those with occupancy above 20% are shown. For sake of clarity, residues belonging to the CCR2 peptide are highlighted in bold. Residue(Atom name) Donor Acceptor 45T(HG1) 26sY(O2S) 24R(H11) 26sY(O1S) 24R(HE) 28sY(O3S) 21S(H) 28sY(O3S) 24R(H21) 28sY(O1S) 20V(H) 9V(O) 19L(H) 30A(O) 13Y(H) 22T(OG1) 14N(H) 22T(OG1) 22T(HG1) 1Q(OE1) 49K(HZ1) 26sY(O3S) 29R(H21) 19E(O) 18R(H11) 29G(O) 21T(H) 50E(OE1) 21S(HG) 28sY(O3S) 29R(H11) 19E(O) 21T(HG1) 50E(OE1) 26sY(H) 16T(OG1) 1Q(H1) 22T(O) 14N(D21) 25D(OD2) 22T(H) 1Q(OE1) 14N(D21) 25D(OD1) 21S(H) 28sY(O2S) 18R(HE) 29G(O)

Atom Number Donor Acceptor 724 1371 372 1370 369 1408 312 1408 375 1406 1260 128 271 1431 164 1299 185 1299 1300 14 789 1372 460 1258 263 1421 1276 805 319 1408 457 1258 1286 805 1356 228 2 1302 194 1352 1290 14 194 1351 312 1407 260 1421

Occupancy [%] 98.8 94.8 93.0 90.5 86.8 81.8 77.1 76.3 67.3 47.1 44.9 43.9 35.4 34.9 34.2 33.7 32.7 31.9 28.2 25.7 24.4 23.4 23.4 21.9

a conformational change within the first 40 ns of the trajectory and started to occupy the space between sY26 and sY28, forming two salt-bridges that remained stable for the rest of the simulation (Fig. S6). To understand how the binding between CCL2 and CCR218-31 affects the exposure of residues to solvent we proceeded with a solvent-accessible surface area analysis (SASA). 21


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Considering the last 100 ns of the CCL2 and CCL2-CCR218-31 trajectories, we observed different patterns of exposure that were in line with our previous findings. Fig. 6 shows CCL2 residues that become less (highlighted in blue) or more (highlighted in green) solvent exposed upon peptide binding. Among the residues that are less exposed to solvent, mainly

Figure 6: Analysis of the solvent-accessible surface area (SASA). The SASA were computed on a per-residue basis for both CCL2 dimer (free) and CCL2-CCR218-31 complex (bound) considering the last 100 ns of the simulations. (A) Difference of SASA between the two systems, ∆SASA, calculated as SASAfree − SASAbound . Residues showing ∆SASA greater than 20 ˚ A2 (highlighted in blue) were considered as relevantly less exposed to the solvent in the bound state compared to the free state, whereas residues with ∆SASA lower than -20 ˚ A2 (highlighted in green) were considered to be more exposed. The above limits are represented as dashed lines. Structural representations of (B) complex and (C) CCL2 dimer, showing the relevant residues extracted from the ∆SASA plot using a surface representation and colored accordingly. we observed R18, R24 and K49. As indicated before, these residues participate directly in 22


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interactions between CCL2 and CCR218-31 , and compose the borders of the shallow cleft present in CCL2 where the CCR218-31 lays on (Fig. 6a and b). On the other hand, residues that mainly compose the N-terminal CCL2 region, such as D3, I5, N6, P8 and V9 became more exposed to solvent since they belong to CCL2 the monomer-monomer interface region (Fig. 6a and c).

Discussion The current knowledge concerning chemokine systems is very limited considering their biological importance and their high level of complexity. This is extremely unfortunate, since it is known that several chemokine axes play important roles in many diseases. A better description of the mechanisms by which chemokines exert their biological function is highly desirable and should walk hand-in-hand with the pharmacological study of new drugs. In this sense, we investigated the CCL2/CCR2 axis due to its relevance in atherosclerosis, cancer, multiple sclerosis, among others. By studying the interaction between CCL2 and a sulfopeptide corresponding to the N-terminal region of human CCR2, we could envision novel characteristics regarding the process of CCL2-CCR2 binding under a physiological environment. Data obtained from NMR and molecular modeling analyses supported previous findings and provided structural and dynamical details that may be useful in pharmacological attempts to modulate chemotaxis. According to the commonly-accepted two-site model of interaction, 13,60,61 the chemokine receptor makes use of its N-terminal region to interact with chemokines. This is considered to be the first step of the binding process that probably occurs between many combinations of chemokine-chemokine receptors. In a later step, the N-terminal region of the chemokine is assumed to interact with the extracellular loops and transmembrane segments of the receptor. This mechanistic view can be considered as a general and simplistic picture of a more complex process of which the details may vary according to the many peculiarities of 23


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different chemokine systems. In the specific case studied here, a more complete mechanistic proposal must take into account the accumulated experimental data concerning CCL2 and CCR2 binding. Most of these recent evidences are due to the substantial contribution of Tan and colleagues 19 and, according to their work: (i) there is a prevalence of the dimeric form of CCL2 in solution, although monomer and dimer coexist in equilibrium; (ii) the addition of a CCR2 N-terminus peptide to this solution, shifts the equilibrium towards the monomeric form of CCL2 (as also observed herein), suggesting that binding of CCR2 to CCL2 dimer weakens the dimer interface; and (iii) the obligate CCL2 monomer P8A induces a similar biological response compared to the WT CCL2, whereas the obligate dimer T10C is significantly less effective. Altogether, this collection of evidences suggests that the CCL2 monomer is key to produce the biological response and that the monomer-dimer equilibrium is probably part of a complex control mechanism in chemokine receptor activation. The structural and dynamical analysis provided by the present work supports the above mechanistic description and reveals important interaction sites that could modulate receptor activation. The NMR chemical shift perturbations measured in the present work are in line with previous studies, 17,19,21,55 supporting that the N-terminal region of CCR2 receptor mainly interacts with the N-terminal, N-loop and β2-β3 structural elements of CCL2. Comparison of the dynamical behavior between the free CCL2 and CCR218-31 -bound CCL2 reveals that CCR218-31 binding enhances fast motions (Fig. 3 and 4), suggesting that the binding process is accompanied by an increase in conformational entropy. In addition, the observed increase in Rex values upon binding indicates an ensemble of conformations that interconvert on the µs–ms timescale. The free CCL2 is dynamically inert on the µs–ms timescale and upon CCR218-31 binding, the slow dynamics is enhanced with a redistribution of motion throughout the backbone. These results support the idea that the recognition of CCR218-31 may have a significant entropic contribution and may be governed by conformational selection rather than by an induced-fit mechanism. 62 On the basis of NMR chemical shift perturbations, a structural model for the interaction 24


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of CCL2 and CCR218-31 was derived using the HADDOCK program. This model was further refined by molecular dynamics simulations. According to this model, the CCR218-31 segment occupies an extended cleft on the surface of CCL2 between the N-terminal region and the β-sheet region of the chemokine. This binding pocket does not overlap, but is instead juxtaposed to the dimer interface. This is in agreement with the assumption that the N-terminal region of CCR2 is able to bind to the CCL2 dimer and may weaken the dimer interface. In fact, we observed that CCR218-31 binding perturbs several residues that make monomermonomer contacts (most residues belong to the N-loop; see Figs. 3, 4 and 6). The most relevant residues of CCL2 that appear to contribute to the binding of CCR218-31 are the conserved residues R24 and K49 (sequence alignment shown in Fig. S7). Our structural model reveals specific interactions between the CCR218-31 sulfated tyrosines sY26 and sY28 and residues R24 and K49. The interaction mode of this 4 residues form a chain of alternating charges that neutralize the binding pocket (Fig. 5) and, due to this highly complementary electrostatic interactions, this binding mode may be extremely relevant for receptor selectivity. In special, it is important to highlight the role of sY26. Experimental results have shown that sulfation of Y26 plays a major role in the binding affinity. 19 This is readily explained by our molecular model since this residue lies exactly in between R24 and K49, forming very stable interactions. Another relevant feature extracted from the molecular dynamics simulations and NMR experiments is the increase in flexibility of CCL2 N-terminal upon binding. This is justified since most of the CCR218-31 bound form of CCL2 are present in the monomeric form and the N-terminal, that was previously making monomer-monomer contacts at the dimer interface, becomes more solvent exposed and flexible, thus ready to interact with the extracellular regions and transmembrane segments of the receptor. Notably, the orientation of CCL2 shown in Fig. 5 is consistent with the model of Zheng and colleagues, 63 which suggested the CCL2 N-terminal region insertion into the hydrophobic transmembrane segments of CCR2 receptor. 25


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To date, only one structure of a CC chemokine in complex with a receptor peptide has been reported. 21 The authors described the structure of CCL11 in complex with a CCR3 sulfopeptide and, similarly to the present work, the binding site is formed by the N-loop and β2-β3 regions of CCL11. However, it is important to note that the orientation of CCR3 peptide on the CCL11 surface differs substantially from that observed in the CCL2CCR218-31 complex. More specifically, the CCR2 sulfopeptide lies perpendicular to the β3 strand of CCL2 (Fig. 5) in contrast to the CCR3 that lies approximately antiparallel to the β3 strand of CCL11. 21 Our structural model is also in agreement with the representation proposed for the full CCR2-CCL2 complex. 63 The latter illustrates an extensive binding interface and corroborates with the perpendicular orientation of CCR218-31 in relation to the β3 strand of CCL2. Additionally, this orientation is in agreement with the proposed mechanism for CCL2 dissociation (by weakening the dimer interface) and is probably a required orientation to expose the N-terminal region of the bound chemokine to perform the additional interactions with the receptor extracellular loops and transmembrane region. Another interesting aspect concerning the binding of CCL2 to CCR2 is the possibility of a cross-talk in which the first binding stage (site I) would involve one receptor and the second stage (site II) would involve another molecule of the receptor. 64 In fact, such a possibility is supported by the results of Monteclaro and Charo. 61 Considering this aspect and all the other above evidences related to the mechanistic possibilities, we have prepared a pictorial representation trying to embrace the relevant features for the CCL2-CCR2 binding process (Fig. 7). Considering drug-development applications, chemokines and their receptors have become an attractive segment for modulation due to their role in inflammatory-based diseases and other conditions, such as HIV infection and cancer. 7 The increasing number of clinical trials that are based on restricting specific chemoattractant signals highlights the tendency of pharmaceutical industries to invest in this field. Successful examples of marketed therapeutic agents include the receptor antagonists “Maraviroc” 65 that targets the CCR5 and “Plerix26


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Figure 7: Model for the putative two-site mechanism for the interaction between CCL2 and CCR2 receptor. (A and B) The first step of receptor recognition, the N-loop (site I) of CCL2 probes the N-terminal region of CCR2, weakening the CCL2 dimer interface. (C) Upon CCR2 binding and CCL2 dissociation, CCL2 exhibits an increase in thermal motions in ps-ns and µs–ms timescale. (D) the N-terminal region of CCL2 (site II) becomes more solvent exposed and flexible, thus ready to interact with the extracellular regions and transmembrane segments of the receptor, triggering its activation. (E) Putative cross-talk in which the first binding stage (site I) would involve one receptor and the second stage (site II) would involve another molecule of the receptor. 64

afor” 66 that targets the CXCR4, which are used against HIV and cancer, respectively. Apart from the great challenges in understanding the human chemotaxis system, these achievements reflect the feasibility of developing new pharmaceutical options in this field. A matter that draws attention in such drug-developing area is the many different kinds of approaches used to regulate chemotaxis. 67–71 Basically, the main efforts are directed to the screening of small molecules capable of binding to chemokine receptors. 72 However, instead of solely relying on screening routines, a better strategy could also involve structural and biological insights. In this sense, data obtained from our CCL2-CCR218-31 model may provide new expectations in drug discovery. The highly-stable manner by which the CCR218-31 -sulfotyrosines interact with their basic-counterpart residues (R24 and K49) suggests a drugable pocket of 27


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modulation in CCL2. Molecules with the right structural arrangement and with suitable complementary physico-chemical properties able to mimic sY26 and sY28 interactions could serve as potential CCL2 inhibitors. Since this binding pocket presents conserved residues, molecules binding to this region could potentially bind to other chemokines. This lack of specificity could be a desirable feature for a new drug if one considers the elevated level of redundancy within the chemokine signaling. According to Solari et al., 7 antagonizing a specific chemokine or chemokine receptor would not be enough to regulate signaling, since other chemokines can perform the same function. Under this scenario, it would be an attractive point to consider the application of peptidomimetics as a novel anti-chemotactic approach targeting a conserved binding region.

Supporting information Fig. S1 - Backbone

15

N relaxation parameters for CCL2; Fig. S2 - NMR analysis of the

interaction between CCL2 and CCR218-31 ; Fig. S3 - Backbone

15

N relaxation parameters of

CCL2-CCR218-31 complex; Fig. S4 - NMR analysis of the interaction between CCR218-31 and CCL2; Fig. S5 - Structural features of CCL2 chain A in models complexed or not with CCR218-31 : data from molecular dynamics (MD) simulations; Fig. S6 - Dynamical behavior of the sulfotyrosines as observed during the MD simulations; Fig. S7 - Multiple sequence alignment of CC chemokines.

Acknowledgements This work was supported by grants from Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnológico (CNPq), Funda¸caõ de Amparo a Pesquisa do Estado do Rio de Janeiro Carlos Chagas Filho (FAPERJ) grants E-26/010.002420/2016, E-26/203.198/2016 and E26/111.307/2014. The National Institute of Structural Biology and Bioimaging (INBEB) is greatly acknowledged for NMR data acquisition. The authors also acknowledge the N´ ucleo 28


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Avan¸cado de Computa¸cão Cient´ıfica (NACAD/COPPE/UFRJ) for providing the computational resources.

Author contributions statement V.S.P. and B.A.C.H designed research. K.S.D. and V.S.P. prepared the samples, performed the NMR experiments, molecular docking and analysed the data. E.R.A.O and B.A.C.H performed the MD simulations and analysed the trajectories. E.R.A.O, B.A.C.H, A.P.V and V.S.P. wrote the manuscript. All authors reviewed the manuscript.

Additional information Competing financial interests: The authors declare no competing financial interests.

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