NMR Structural Determinants of Eosinophil Cationic Protein Binding to Membrane and Heparin Mimetics

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INSIGHTS INTO THE STRUCTURAL DETERMINANTS OF EOSINOPHIL CATIONIC PROTEIN BINDING TO MEMBRANE AND HEPARIN MIMETICS BY

NMR. María Flor García-Mayorala, Mohammed Moussaouib, Beatriz G. de la Torrec, David Andreuc, Ester Boixb, M. Victòria Noguésb, Manuel Ricoa, Douglas V. Laurentsa and

Marta Bruixa

a Instituto de Química-Física “Rocasolano”, CSIC, b Departament de Bioquímica i Biología Molecular, Facultat de Biociències, Universitat Autònoma de Barcelona, cDepartament de

Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Running Title: ECP interactions with membrane and heparin mimetic

Address correspondence to: Marta Bruix, IQFR-CSIC, Serrano 119, 28006 Madrid, Spain. [email protected], Fax:+34 91 564 24 31

Eosinophil cationic protein (ECP) is a highly stable protein which participates in innate immune defense against parasites but also kills human cells. Both its beneficial and harmful cytotoxic actions are chiefly due to its ability to enter and disrupt membranes, a capacity that can be modulated by heparin binding. The residues involved in these processes are not completely known and the physiological role of the protein’s exceptionally high conformational stability is not clear. Here, we have used NMR spectroscopy to characterize the binding of ECP to membrane and heparin mimetics. Three Arg-rich surface loops and Trp 35 are crucial for membrane binding. The N-terminal α-helix, the third loop, the first β-strand and the C-terminal β-strand are key for heparin binding. These sets of residues are not mutually exclusive, and ECP can likely bind human membranes and heparin simultaneously. ECP is homologous to RNase A and the heparin binding residues also constitute the vestigial binding sites for RNA. An ECP N-terminal fragment consisting of the first 45 residues (ECP1-45) was found by NMR spectroscopy to retain the capacity to bind membrane and heparin mimetics but only partial helical structure in aqueous solution. The helical population increases in membrane-like environments and the second helix extends to incorporate residues which are in a loop conformation in native ECP. Considering that ECP1-45 is largely unfolded and was shown to maintain some of ECP’s cytotoxic actions, we conclude that the extraordinarily high stability is not required for cytotoxicity.

Eosinophil cationic protein (ECP, also known as human Ribonuclease 3) is present in large amounts in eosinophil granules. The 3D structure of this 133 residue protein, has been determined with (1) and without ligands by X-ray crystallography (2,3), and recently by NMR spectroscopic methods (4). ECP’s tertiary structure closely resembles that of bovine pancreatic Ribonuclease A (RNase A), although the loops in ECP are significantly longer. ECP is quite rich in Arg (19) and Pro (12) residues. ECP has two Trp residues one of which, at position 35, is hyper-exposed.

ECP is released by activated eosinophils and at low levels by activated neutrophils, and plays a role in host defense against parasites such as helminths (5,6). ECP also inactivates virus (7) and kills both Gram-negative and Gram-positive bacteria at low μM concentrations (8,9). However, ECP can be regarded as a double-edged sword as it is toxic to host epithelial tissues (10,11). In fact, ECP is implicated in asthma (12), the most common childhood disease in developed countries. ECP levels in the blood are well correlated with asthma severity and are widely used to monitor the effectiveness of asthma treatments (13). We are interested in understanding the molecular basis of ECP’s activities. Previous studies have established that ECP’s cytotoxic actions are mediated chiefly, if not entirely, by its ability to bind and disrupt membranes (14,15), rather than its ribonucleolyitc activity, which is 100-fold lower than that of RNase A (16). More recent studies employing Trp fluorescence and site directed mutagenesis have demonstrated the importance of Trp 35, the residues of the third loop 32-39, and the Arg residues in binding

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membrane components (15,17,18). These studies, though valuable, are limited to the environment of the Trp residue and the positions mutated, and site directed mutagenesis may cause structural changes which affect the binding of membrane components in unexpected ways. NMR spectroscopy can, in principle, uncover the participation of all residues in binding without perturbing them.

The main objective of this paper is to characterize more completely the interactions of ECP with a membrane mimetic, dodecylphosphorylcholine (DPC) micelles, and a disaccharide mimetic of heparin using NMR spectroscopy. DPC micelles were chosen because they have zwitterionic character, which is common among lipids present on the outer leaflet of human membranes (19,20). Heparin is a linear, highly sulfated glycosaminoglycan which is released by activated mast cells during inflammatory processes. The tight binding of heparin by ECP may modulate ECP’s cytotoxicity.

ECP is an exceptionally stable protein; it is about 3.5 kcal/mol more stable than RNase A (4,21,22). This high conformational stability has been assumed to play a role in ECP’s remarkable biological activities. Work with both an ECP-like protein from chickens (23), and more recently human ECP (24), however, has shown that peptides derived from these proteins also possess potent cytotoxic activities even though the stability of the structure they adopt in the full length proteins is expected to be drastically reduced. Therefore, to better understand the relationship between ECP’s outstanding stability and its remarkable biological activities, we have also studied the conformation of a peptide composed of the first 45 residues of ECP, called ECP1-45, in water, TFE and DPC micelles, and its binding to the heparin disaccharide.

Experimental Procedures

Materials- Dodecylphosphorylcholine-d38 (98% atom D) (DPC) was an Isotec product obtained from Sigma-Aldrich. The heparin sulfate disaccharide, 2S sulfated Iduronic acid (IdoA) 1-4 linked to NS sulfated Glucosamine (GlcN), which represents the shortest

repeating unit of this oligosaccharide, was obtained from Sigma-Aldrich (heparin disaccharide I-S). 2,2,2-Trifluoroethanol (TFE) (>99.5 % pure, NMR spectroscopy grade) was purchased from Sigma-Aldrich. Peptide synthesis- Two peptide samples containing the first 45 and 19 N-terminal residues of ECP (ECP1-45 and ECP1-19) were chemically synthesized by Fmoc solid phase procedures and purified by HPLC to 95% purity. Cys residues at positions 23 and 37 in the native sequence were replaced by Ser to avoid potential formation of inter- or intramolecular disulfide bridges. The identity and sequence of the peptides were confirmed by MALDI-TOF mass spectrometry and NMR spectroscopy. Purification of recombinant 15N-ECP- The protein was expressed in E. coli BL21(DE3) cells in minimum medium with 15NH4Cl as the sole source for nitrogen and purified from inclusion bodies as described previously (25). NMR samples- All NMR samples were recorded at 25 ºC, pH 4.5 and contained 50 μM of DSS as the internal chemical shift reference. ECP samples- A ∼0.5 mM sample of 15N-ECP was initially prepared in 90%H2O/10%D2O. Analogous aqueous solution samples of 15N-ECP were then prepared in deuterated DPC micelles, heparin sulfate disaccharide, and in the presence of both DPC and heparin sulfate. The final concentrations were ∼0.5 mM 15N-ECP and ∼40 mM DPC for the sample containing DPC; ∼0.25 mM 15N-ECP and ∼0.25 mM heparin sulfate (1:1 complex) for the sample with the disaccharide; and ∼0.5 mM 15N-ECP, ∼40 mM DPC, and ∼0.5 mM heparin sulfate for the sample with the three compounds present. ECP1-45 peptide samples- The initial sample of ∼1.1 mM ECP1-45 peptide was prepared in aqueous solution of 90%H2O/10%D2O. Three subsequent samples of ∼1.1 mM ECP1-45 peptide in the same aqueous solution were prepared in different environments as mentioned above. These samples contained 40% deuterated TFE, 20 mM deuterated DPC micelles, and 20 mM deuterated DPC plus ∼1mM heparin sulfate disaccharide, respectively. A fourth sample of ECP1-45 in 1:1 complex with heparin sulfate disaccharide

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alone was prepared at concentration of ∼2.5 mM. Another ∼1.1 mM sample of the peptide was prepared in D2O solution to evaluate amide proton exchange rates with the solvent. The final pH reading for this sample was adjusted to 4.5, with no correction for the deuterium isotope effect. ECP1-19 peptide sample- The ECP1-19 peptide sample was prepared in 90%H2O/10%D2O with a final concentration of ∼1.0 mM and pH 4.5. Heparin sulfate disaccharide sample- A 1.6 mM sample of heparin sulfate disaccharide in D2O was used to record NMR spectra for the assignment of this molecule. NMR experiments- Series of 2D phase-sensitive COSY spectra and 2D 1H-1H NOESY and TOCSY spectra were acquired in Bruker Avance 800 MHz spectrometer equipped with a z-gradient cryoprobe at 25ºC. Standard pulse sequences were used to assign the backbone and side-chain resonances of the peptides. Mixing times were 150 ms and 60 ms for NOESY and TOCSY experiments recorded for the ECP1-45 and ECP1-19 samples, and 100 ms and 60 ms respectively for the 15N-ECP sample. Pulse sequences with 15N decoupling squemes during acquisition were needed in this case. The Watergate module was used for solvent suppression.

The same set of experiments were recorded in the heparin sulfate disaccharide sample for the spectral assignment with mixing times of 200 ms and 60 ms for 2D 1H-1H NOESY and TOCSY spectra, respectively.

The spectra were processed with Topspin 1.3 software (Bruker, Germany) and transferred to Sparky (26) for further analysis. MolMol program was used for molecular display (27).

RESULTS

The 1H resonances of ECP and the peptide ECP1-45 in the different environments, TFE, DPC, and heparin sulfate disaccharide were assigned using the standard procedure of protein NMR spectroscopy (28). Spin system identification was obtained from the analysis of 2D 1H-1H TOCSY spectra, and sequential connectivities were established from 2D 1H-1H NOESY spectra. The assignments are essentially complete except

for a few isolated residues in the presence of DPC due to spectral overlap or broadening.

ECP1-45 in water solution. We have used conformational chemical shifts (CCS) for 1Hα protons to assess the secondary structure content of the peptide in the different environments tested (29). As shown in Fig. 1A, the peptide in aqueous solution is mainly unstructured and displays a low propensity to form two α-helices spanning approximately residues R7-S17 and residues I25-R45. The boundaries of the first helix match those of native helix α1 of ECP. However, the second helix has remarkably lengthened at its C-terminus by incorporating residues in loop 3 and strand β1 of native ECP. Based on the 1Hα Δδ value of + 0.39 ppm for 100% helix content (29), the helix populations are calculated to be 47% and 21% for the first and second helices, respectively. Very few and weak sequential 1HN-1HN NOEs were observed. The chemical shifts of the 1H side chains are in the range of unstructured peptides and no evidences for native or non-native clusters are seen. Similar experimental data were obtained for the peptide ECP1-19, a shorter version of ECP1-45, indicating that it retains identical secondary structure in the equivalent region (supplementary Fig. 1A and 1B). This suggests that any stabilizing interactions between the first and second helices in ECP1-45 are small or insignificant.

The 1D 1H NMR spectrum recorded at 25ºC, pH 4.5 in a D2O sample, revealed essentially complete exchange of all amide hydrogens. Among the NH groups in ECP1-45, those of I13 and A26 have the slowest intrinsic H/D exchange rates (kex = 0.29 min-1 at 25ºC and pD = 4.9) (30). The resonances of these NH groups are not observed in the first 1D 1H spectrum recorded about 15 minutes after the peptide was dissolved in D2O, which indicates that their exchange rates are slowed by a factor of three or less. In comparison, the folded helical structure of full length ECP slows the NH exchange rates of I13 and A26 by factors of 2.8 x 106 and about 5 x 104, respectively (4). Thus protection afforded by helical structure of ECP1-45 against exchange is very weak; this is in agreement with the low helical populations detected by the CCS analysis.

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ECP1-45 in TFE. Next, the conformation of ECP1-45 was studied in 40% TFE. The 40% TFE solution bears some resemblance to membrane environments, since both TFE molecules and the aliphatic chains of lipid molecules, are less efficient at forming hydrogen bonds to peptide groups than water. When dissolved in 40% TFE, the 1Hα Δδ values reveal a significant increase in the α-helical propensity. The boundaries for the two helices are comparable to those in water solution, and notably the second helix is also longer in 40% TFE. The estimated helical populations are 76% for the first helix and 63% for the second (Fig. 1B), values which represent a 1.5 and 3-fold increase, respectively, to their populations in aqueous solution. In addition to the 1Hα Δδ values, the much higher tendency to form the two helices is reflected by: 1) changes in 1HN Δδ >0.15 ppm for most residues distributed along the sequence, 2) an increase in the number and intensities of sequential HN-HN NOEs, and 3) the observation of some unequivocal non-overlapped Hα-HN (i, i+3) NOEs characteristic of helical conformation such as those between A8-F11, W10-I13 in the first helix, and between R28-N31, I30-Y33, W35-K38 in the second helix. Finally, 4) a large number of HN-HN connections have been assigned especially in the segments Q4-I16 and T24-L44.

ECP1-45 in DPC micelles. To mimic the phospholipidic membrane environment in a more realistic way, the peptide’s conformation was studied in a 20 mM DPC solution. DPC has been commonly used to study membrane peptides and proteins because it forms small uniform micelles that reorient rapidly enough for solution NMR spectroscopy (31). DPC micelles contain an average of 40 DPC monomers and have an approximate mean mass of 17 kDa. Therefore, our solution is expected to contain about 0.5 mM of DPC micelles and about 1 mM of peptide. As in 40% TFE, the 1Hα Δδ values (Fig. 1C) of ECP1-45 in the presence of DPC micelles also reveal a significantly increased tendency to form the two α-helices as compared to the aqueous medium, with estimated helical populations of 71% and 47% for the first and second helices, respectively. Thus, the population of the first

helix in the presence of DPC is similar to that seen in 40% TFE, with that of the second helix being slightly lower than in 40% TFE. In the presence of DPC, residues N32-L44 also adopt a helical conformation which extends helix α2. The population of the second helix, as measured by the 1Hα Δδ values, is intermediate between the low amount seen in water and the higher quantity observed in 40% TFE. Despite the spectral line broadening induced by decreased mobility of the complex, the backbone assignment is complete with the exception of the Hα of R22. The poorer quality of the spectra made it more difficult to identify (Hα-HN i, i+3) helical NOEs, however, those between W10-I13 in the N-terminal helix, and between N31-R34 and N32-W35 in the central part of the C-terminal helix could be unambiguously assigned. A good number of HN-HN NOE connections are observed between R7-A8, A8-Q9, Q9-W10, F11-A12, Q14-H15, H15-I16, I30-N31, R34-W35, R36-S37, Q40-N41, N41-T42, T42-F43, and F43-L44. The chemical shift perturbation (CSP) analysis reveals changes >0.15 ppm for the HN protons of R7, Q9, F11, H15, I16, S17, L18, A26, A29, I30, R34, W35, K38, N39 and F43 (Fig. 2A), and the Hα of T6, I13, Q14, L18, S23, T24, I25, A26, I30, Y33 (Fig. 2B). Some side chain protons also experience important Δδ (0.15-0.35 ppm), for example, those of W10, Q14, H15, N32, and W35 to mention a few examples; Arg side chains are also greatly perturbed.

ECP1-45 interaction with Heparin mimetic. Previous studies have established the ability of ECP to interact with glycosaminoglycan structures, in particular heparin (32) that could play an important role in ECP's immunomodulating properties. We have decided to study this interaction by NMR spectroscopy, using heparin sulfate disaccharide and CSP analysis, to reveal in more detail the residues involved. Initially, the disaccharide resonances were assigned on the basis of 2D 1H-1H-NOESY and TOCSY spectra. Two sets of resonances are observed for the GlcN unit of the disaccharide as a consequence of the α−β conformational equilibrium. The chemical shifts measured were in agreement with those previously reported (33).

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Spectra of ECP 1-45 in the presence of the disaccharide are quite similar to those in water solution. Again, the CCSs (Fig. 1D) indicate a low tendency to form two α-helices. The percentages of the helical contents are comparable to those obtained in aqueous solution, with values of 50% and 24% for the first and second helices, respectively. The boundaries for both helices are similarly retained. Despite these similarities, the NMR data provide conclusive evidence for the binding of the peptide and the disaccharide: 1) partial sample precipitation is observed upon complex formation, 2) the sign of the disaccharide intramolecular NOEs changes from positive in the spectra registered in the absence of ECP1-45 to negative in its presence. Surprisingly enough, no significantly large CSPs are detected for the peptide resonances in the presence of the disaccharide or for the disaccharide resonances in the presence of the peptide. No intermolecular NOEs could be detected in the spectra probably because the complex formed is an ensemble with a diverse set of disaccharide conformations or binding sites or both. The two sets of resonances corresponding to the α and β conformers of the GlcN unit are still observed in the presence of the peptide, indicating that no preferential conformer is trapped when the complex is formed. CSPs are small for HN and Hα protons; values larger than 0.025 ppm are observed for the HN of F5, T6, R7, Q9, W10, F11, A12, Q14, H15, I16, S17, L18, R28, I30, N32, and K38, and the Hα of F5, R7, F11, I13, Q14, H15, I16, I30, and N32 (Fig. 2C, 2D). Residues from R34 to N39 have been suggested to participate in the interaction of native ECP with heparin based on characteristic patterns of basic and hydrophobic residues observed in glycosaminoglycan interacting proteins and also identified in loop 3 of EDN (34). In addition, residues from the N-terminal helix of ECP and Q14 at the active site have been proposed to mediate the interaction based on docking simulations*. The chemical shifts of the aromatic side chains of F5, F11, Y33, and W35 are similar to those in water solution lacking the heparin disaccharide, as are the side chains of the remaining residues in the segment R34-N39. In contrast, the Hε3 proton

of W10 is clearly shifted towards higher field about 0.05 ppm, and the 1H chemical shift values of Qγ, Hε21, and Hε22 of Q14 are shifted upfield about 0.08, 0.13, and 0.04 ppm, respectively, suggesting the implication of these side chains in the interaction.

ECP1-45 in DPC micelles and heparin sulfate disaccharide. To investigate whether the high affinity interaction of ECP1-45 with heparin sulfate disaccharide displaces the interaction with DPC, which would indicate common/partially superimposing interfaces, we added the disaccharide to our previous sample in DPC solution. The spectra showed the broad resonances characteristic of the peptide-micelle complex, and the spectra could be easily assigned using ECP1-45’s assignments in DPC micelles. The Hαs of Q14 and R22 could not be identified. The CCSs are similar to those in the DPC medium alone (percentages of 71% and 47% for both helices, Fig. 1E), thus the increased helix population induced by DPC is not reduced by adding the heparin mimetic. The chemical shift differences provoked in ECP1-45 by the disaccharide in the presence of DPC are similar to those detected in the absence of DPC. In general, the CSPs are small, with chemical shift changes greater than 0.025 ppm occurring for HN protons of residues Q4, F5, A8, W10, F11, I13, H15, T24, I30, and N32, and Hα of P3, Q4, F5, T6, R7, A12, I13, H15, S17, and N19. Significant chemical shift changes are found for the Hε1, Hδ1, and Hε3 of W10 (0.05, 0.05, 0.09 ppm, respectively). These changes match quite well those observed with the disaccharide alone suggesting that the interaction surfaces with DPC and heparin are not mutually exclusive.

ECP interaction with DPC micelles. Next, 15N-HSQC spectra of native, full length, N15–labeled ECP in aqueous solution (Fig. 3A) and in DPC micelles (Fig. 3B) were recorded and assigned based on recently published data (4) and corroborated at these solvent conditions using 3D 15N-NOESY-HSQC spectra. Using the CSPs induced in 1HN and 15N chemical shifts upon binding to DPC, we have mapped the residues most affected on the molecule surface (Fig. 4A and 4B). The interaction surface is well defined and constituted by residues belonging to different loops, Q4-T6 in loop 1, N32-R36 in

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loop 3, T46 in the segment connecting strand β1 with helix α3, and N87, A90-I93, C96, R97 in loop 6. The Hε1 side chain proton of W35 experiences one of the largest variations observed in the HSQC spectrum corroborating its crucial role in the process. These three protein loops project outwards on the same face of the molecule with residues in loop 3 stacked in between loops 1 and 6. W35 side chain is hyper-exposed, which facilitates its insertion into the micelle and stabilization of the complex interface environment (35).

ECP interaction with heparin mimetic in the presence of DPC micelles. ECP strongly interacts with heparin sulfate oligosaccharides (34). At concentrations suitable for NMR spectroscopy, we observed here that the ECP sample precipitated instantly upon adding the heparin sulfate disaccharide, which thwarted efforts to characterize their complex under these conditions. The 15N-HSQC spectrum of this sample (Fig. 3C) indicates that no native ECP is left in solution. Therefore, the disaccharide was added to a sample containing ECP and DPC. No precipitation was observed, and the effects of the addition of the disaccharide were analyzed by the chemical shift changes induced in the 15N-HSQC spectrum of ECP recorded in these conditions with respect to the DPC solution alone (Fig. 3D). We used again the average 1HN and 15N chemical shift values to map the interaction surface (Fig. 4C and 4D). In this case, the biggest perturbations affect residues Q4 in loop 1, A8 and F11-Q14 in helix α1, Y33-R36 in loop 3, Q40-L44 in strand β1, C62, H64, R66 in loop 4, R75, R77 in loop 5, C83, L85 in strand β3, and H128-D130 in strand β6. The side chain Hε1 protons of W10 and W35 are also considerably perturbed. A large number of Arg residues with perturbed chemical shifts are detected, which is a common feature in heparin binding domains due to their hydrogen bonding propensity and strong electrostatic interactions with sulfate groups (36). Although residues in loop 3 are common to the interaction surface with DPC, here residues in the C-terminal part of the first helix rather than loop 1 are affected; indeed, the largest perturbation occurs for the catalytically relevant Q14 residue at the end of the first helix.

DISCUSSION

In spite of the rapid progress made during the last years, we are still far from a complete understanding of the mechanism of action of cytotoxic RNases. The elucidation of their activities requires structural studies with targets. To characterize the structural bases of ECP’s cytotoxic activity related to its interaction with membranes, we have undertaken a complete NMR study of the behavior of ECP and the N-terminal-derived peptide ECP1-45 in water and in media that mimic the membrane environment of eukaryote cells. ECP1-45 was chosen as a good model since it retains most of ECP’s membrane-destabilizing and antimicrobial activities (24).

ECP1-45 conformational preferences and binding to DPC membrane mimetic. Secondary chemical shifts and rapid hydrogen/deuterium exchange data provide clear evidence that the ECP1-45 peptide, though mainly unstructured in water, forms two partially populated α-helices separated by coil residues L18-T24. Moreover, the presence of two consecutive Pro residues (P20 and P21) favors this helix-hinge-helix arrangement, as their conformational rigidity disrupts helical structure (37,38). The first helical fragment (R7-S17) coincides with helix α1 of ECP, the second helical fragment (I25-R45) includes native helix α2 (C23-N31) and is extended to residues corresponding to loop 3 (N32-N39) and strand β1 (Q40-R45). In native ECP C23 and C37 form disulfide bridges with C83 and C96, respectively. These two residues have been replaced with Ser in ECP1-45. Since the conformational restraint imposed by the disulfide bridge C37-C96 of native ECP is not present in the peptide, the substitution of C37 by S37 may allow the second helix to become longer.

The second helix is less populated in all the environments studied, although its helical content rises substantially in 40% TFE and in DPC micelles. An overall increase in helical structure has also been observed by CD spectroscopy for ECP1-45 and ECP1-19 in the presence of SDS micelles or bacterial membrane components (24). The helical content for ECP1-45 in SDS was gauged to be about 70% by CD, which is similar to the

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value reported here by NMR in DPC micelles. Interestingly, this helix-turn-helix motif is common in many peptides which interact with membranes, including a variety of antimicrobial peptides (39-42). It has been suggested that the interhelical hinge confers flexibility and might facilitate its adsorption onto the curved surface of micelles (43).

Many studies have already identified some residues in the fragment 1-45 as important for the interaction with membranes. In particular, the highly solvent-exposed W35 and nearby arginine residues, R34 and R36, participate actively in the interaction of ECP. In the peptide, the 1HN chemical shifts of residues R34 and W35 are modified by DPC. However, the pattern of perturbed residues is wider and unevenly distributed throughout the first and second α helices. The analysis is further complicated by possible chemical shift variations arising from conformational changes when ECP1-45 is transferred from the water-soluble state to the lipid-associated state. Two of the more perturbed residues in the first helix, the positively charged H15 and R7, are placed in the same face of the helix, as well as the hydrophobic F11 side chain. This observation is in agreement with the proposed contribution of the first part of the ECP sequence in membrane association and destabilization (24) .

Models for the lengthened helix 2 place A26, A29 and I30 on one face and R34, W35, K38, N39, F43 on the other. Thus both charged and aromatic side chains are in position to form electrostatic and hydrophobic interactions with the membrane (44).

Insights into ECP’s capacity to bind and disrupt membranes. The interaction surface of ECP with DPC micelles delimited by CSP analysis is well-defined and is composed of residues from loops 1, 3 and 6 (Fig. 4A, 4B). These findings corroborate the previously established roles proposed for W35 and nearby arginines (R34 and R36) in the interaction (15,17,18). The residues from loop 3 are sandwiched by loops 1 and 6, which can also contribute to the affinity of the interaction. Some Gln and Asn side chains (Q4, N32, Q91) and many Arg side chains have high degrees of solvent exposure and are likely to be involved in hydrogen bonding formation. For example, R97 is also solvent exposed and could assist arginines in loop 3

in binding to DPC micelles. Interestingly, R97 corresponds to the only polymorphism found in the ECP coding sequence. In fact, the Arg 97 to Thr substitution implies a new glycosylation site and the R97T variant is markedly less cytotoxic (35). Upon deglycosylation, the R97T variant recovers its cytotoxicity, suggesting that glycosylation can block a key region for the protein cytotoxic activity (45). Recent studies suggest that ECP activity is enhanced by deglycosylation during eosinophil activation (46). Another post-translational modification recently identified for eosinophil secretion proteins is the Tyr-nitration, a process associated to the eosinophil maturation, which takes place for ECP only on Y33 (47) and may modify its interacting properties. In contrast to the hyper-exposed side chain of W35, the aromatic ring of Y33 is expected to contribute less to the interaction with membranes as it is buried in some NMR solution structures. Nitration likely favors the more exposed conformation of Y33 seen in other NMR solution structures and could position it to interact favorably with positively charged choline moieties in membranes.

The groove between helix α1 and loop 3 of ECP can accommodate heparin mimetics. A recent study has demonstrated the role of mammalian cell surface glycosaminoglycans, specifically heparin sulfate proteoglycans, in ECP binding and endocytosis (34). Heparan sulfate chains contain heparin regions that are good models to study these interactions. We chose the disaccharide, 2S sulfated Iduronic acid (IdoA) 1-4 linked to NS sulfated Glucosamine (GlcN), as the shortest heparin repeating unit.

The oligomerization of heparin-binding proteins on the heparin chain is a common event (48,49). We observed this phenomenon when ECP was mixed with a solution containing the heparin sulfate disaccharide. This precluded the study of the interaction between full length ECP and the heparin disaccharide. Instead, ECP1-45 plus disaccharide, and ECP in DCP micelles plus this disaccharide were studied. The CSP data recorded on the ECP1-45 peptide are in general agreement with those observed for ECP in DPC solution containing the disaccharide, although we find important

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differences. Residues in the stretch Q9-H15 are also the most perturbed and this effect extends to L18. In contrast, no significant changes are detected in the peptide for residues belonging to loop 3 of ECP. The backbone and side chain protons of W35 are not perturbed with the interaction, instead, the aromatic protons of F5, W10, and F11 are affected, and particularly the Hε3 of W10. The fact that the native secondary and tertiary structure of residues around W35 break down in the peptide suggests that structure is important for the recognition of heparin by ECP. The residues that we find to be most perturbed on the peptide (R7, W10, F11, Q14, H15) are mainly clustered in one face of the first helix. It is possible that W10, assisted by F11 and H15, forms a hydrophobic platform in one side of the helix, and together with F5 would substitute for W35 of native ECP. R7 would be important for the electrostatic contribution of the interaction.

Concerning ECP, it is well known that certain sequence motifs in heparin binding proteins (50) are responsible for the interaction. Using site-directed mutagenesis and synthetic peptides containing the loop 3 sequence of ECP it was shown that the segment 34-38 (RWRCK) serves as a specific heparin binding site (34). Nevertheless, the specific ECP heparin binding site has not been structurally characterized yet; this led us to undertake the NMR study reported here. The residues identified to be involved directly or indirectly in heparin binding can be grouped into four main groups shown in Fig. 4C and 4D: (1) A8-Q14 in helix α1; (2) Y33-R36 in loop 3; (3) Q40-L44 in strand β1, and (4) H128-D130 in strand β6. Residues are not included in these groups when their perturbations are likely due to indirect effects. For example, H64 is close to H128 and D130 and C83 is likely to be affected by F43.

Whereas the interaction of residues in loop 3 of ECP with heparin mimetics was previously described (34), those in the other 3 groups have been identified here for the first time. Examining these results in the context of the recently determined solution structure of the protein we are able to propose a model for the interaction. The ECP structure reveals that residues in loop 3, are spatially close to the second half of helix α1, and these two

regions face each other defining a groove that would accommodate the sugar moiety. This groove is bound on top by residues in strand β1. The opposite face of the helix α1 is close to residues of strand β6.

Two Trp residues (W10 and W35) are found in the groups mentioned above. Our data indicate that the HN protons of these two residues are not disturbed with heparin binding, however, the side chain Hε1 protons move notably, and especially that of W35. As this Trp residue is highly solvent-exposed while W10 is mainly buried, it is expected that its contribution to the binding affinity will be higher. The two Arg residues adjacent to W35 probably contribute to the binding affinity by ion-pairing spatially with negatively charged sulfate or carboxylate groups of the disaccharide. Such interactions are common for Arg-rich membrane penetrating peptides (51). The side chains of W10, Q14, and Y33 are near each other and are oriented towards the interior of the cavity. Actually, the perturbation observed for the HN of Q14 is one of the largest in the spectra suggesting that this residue is involved in the binding process. In the structure, Q14 is also in close proximity to N41 and F43; this would explain the perturbations observed for residues in strand β1. F11 is on the side of the helix pointing away from the cavity and towards residues in strand β6, and in particular its side chain is close to the side chain of L129, which is also near strand β1. These contacts suggest an explanation for the changes in this region of the structure.

Interestingly the set of residues in the protein surface that are in contact with heparin disaccharide only partly overlap that involved in DPC interactions (Fig. 4E and 4F). ECP can bind to membranes and to heparin simultaneously, and this could have implications for ECP’s efficient internalization in membranes (51), host cytotoxicity and its ability to modulate the immune system. The residues binding DPC micelles, which mimic the outer leaflet of human membranes, are largely those which are also implicated in binding to bacterial membrane and cell wall components (52). This suggests that it will be difficult to convert ECP, a double-edged sword killing

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both parasites and host cells, into a single edged sable.

In summary, we have shown that the N-terminal peptide ECP1-45 is largely unfolded in aqueous solution, although it conserves two partially populated helices in similar positions to helices α1 and α2 of full-length native ECP. The second helix is elongated to include residues N32-R45. The helical content increases substantially in membrane-like environments. Both hydrophobic and electrostatic interactions are important for ECP binding to DPC and heparin mimetics. The main interaction segment with DPC-containing membranes includes residues N32-R36 (particularly W35 and Args 34 and 36) in loop 3, with contributions from residues in loops 1 and 6. Residues in loop 3 are also common to the binding site of ECP with heparin mimetics, but additionally, residues in helix α1 (particularly Q14), and strands β1 and β6, which are not involved in DPC binding, play a crucial role in the interaction with heparin. Importantly, we have provided evidence that the interaction surface of ECP with heparin is extended with respect to that described in previous works (fragment 34-38), and have discovered the role of three additional regions of the protein in the complex formation. Thus, our results confirm and extend previous knowledge about these interactions. Some of

the residues that we have found to participate in ECP’s binding to heparin disaccharide (Q14, Q40, T42, L129) are located at RNA substrate binding sites. Among the catalytic residues, K38 and H15 do not seem to be perturbed, although H128 is affected. Consequently some of the residues involved in ECP’s ribonucleolytic activity also participate in ECP binding to heparin, and this can account for the decreased ECP RNase activity when the heparin concentration increases*.

Finally, the fact that the peptide ECP1-45 maintains ECP’s membrane disruption and heparin binding capacities indicates that the highly stable native structure of ECP is not required for these functions. Then, what is the physiological role of ECP’s remarkably high conformational stability? The observation that some different residues in folded ECP compared to partly unfolded ECP1-45 are involved in membrane binding suggests that some residues may be sequestered inside the folded protein under physiological conditions to prevent undesirable interactions with the membrane. Future studies of the interactions of ECP variants and ECP derived peptides with mimetics of the eosinophil granule membrane could test this plausible explanation.

REFERENCES

1. Mohan CG, Boix E, Evans HR, Nikolovski Z, Nogués MV, Cuchillo CM, and Acharya KR. (2002) Biochemistry 41, 12100-12106

2. Boix E, Leonidas DD, Nikolovski Z, Nogués MV, Cuchillo CM, and Acharya KR. (1999) Biochemistry 38, 16794-16801

3. Mallorquí-Fernández G, Pous J, Peracaula R, Aymamí J, Maeda T, Tada H, Yamada H, Seno M, de Llorens R, Gomis-Rüth FX, and Coll M. (2000) J Mol Biol 300, 1297-1307

4. Laurents DV, Bruix M, Jiménez MA, Santoro J, Boix E, Moussaoui M, Nogués MV, and Rico M. (2009) Biopolymers 91, 1018-1028

5. McLaren DJ, Peterson CG, and Venge P. (1984) Parasitology 88, 491-503 6. Hamann KJ, Gleich GJ, Checkel JL, Loegering DA, McCall JW, and Barker RL. (1990)

J Immunol 144, 3166-3173 7. Domachowske JB, Dyer KD, Adams AG, Leto TL, and Rosenberg HF. (1998) Nucleic

Acids Res 26, 3358-3363 8. Lehrer RI, Szklarek D, Barton A, Ganz T, Hamann KJ, and Gleich GJ. (1989) J

Immunol 142, 4428-4434 9. Boix, E., Torrent, M., Sánchez, D., and Nogués, M. V. (2008) Current Pharm Biotec. 9,

141-152 10. Fredens K, Dahl R, and Venge P. (1982) J Allergy Clin Immunol 70, 361-366 11. Young JD, Peterson CG, Venge P, and Cohn ZA. (1986) Nature 321, 613-616

10

12. Munthe-Kaas MC, Gerritsen J, Carlsen KH, Undlien D, Egeland T, Skinningsrud B, Torres T, and Carlsen KL. (2007) Allergy 62, 429-436

13. Koh CG, Shek LP, Goh DY, Van Bever H, and Koh DS. (2007) Respir Med 101, 696-705

14. Navarro S, Aleu J, Jiménez M, Boix E, Cuchillo CM, and Nogués MV. (2008) Cell Mol Life Sci 65, 324-337

15. Carreras E, Boix E, Rosenberg HF, Cuchillo CM, and Nogués MV. (2003) Biochemistry 42, 6636-6644

16. Sorrentino S, and Glitz DG. (1991) FEBS Lett 288, 23-26 17. Carreras E, Boix E, Navarro S, Rosenberg HF, Cuchillo CM, and Nogués MV. (2005)

Mol Cell Biochem 272, 1-7 18. Torrent M, Cuyás E, Carreras E, Navarro S, López O, de la Maza A, Nogués MV,

Reshetnyak YK, and Boix E. (2007) Biochemistry 46, 720-733 19. Matsuzaki K. (2009) Biochim Biophys Acta 1788, 1687-1692 20. Verkleij AJ, Zwaal RF, Roelofsen B, Comfurius P, Kastelijn D, and van Deenen LL.

(1973) Biochim Biophys Acta 323, 178-193 21. Maeda T, Mahara K, Kitazoe M, Futami J, Takidani A, Kosaka M, Tada H, Seno M,

and Yamada H. (2002) J Biochem 132, 737-742 22. Nikolovski Z, Buzón V, Ribó M, Moussaoui M, Vilanova M, Cuchillo CM, Cladera J,

and Nogués MV. (2006) Protein Sci 15, 2816-2827 23. Nitto T, Dyer KD, Czapiga M, and Rosenberg HF. (2006) J Biol Chem 281, 25622-

25634 24. Torrent M, de la Torre BG, Nogués MV, Andreu D, and Boix E. (2009) Biochem J 421,

425-434 25. Boix E, Nikolovski Z, Moiseyev GP, Rosenberg HF, Cuchillo CM, and Nogués MV.

(1999) J Biol Chem 274, 15605-15614 26. Goddard TD, and Kneller DG. (2005) Sparky 3, University of California, San Francisco 27. Koradi R, Billeter M, and Wüthrich K. (1996) J Mol Graph 14, 51-55, 29-32 28. Wüthrich K. (1986) Wiley-Interscience, John Wiley & Sons 29. Wishart DS, Sykes BD, and Richards FM. (1991) J Mol Biol 222, 311-333 30. Bai Y, Milne JS, Mayne L, and Englander SW. (1993) Proteins 17, 75-86 31. McDonnell PA, and Opella SJ. (1993) J Magn Reson series B 102, 120-125 32. Maeda T, Kitazoe M, Tada H, de Llorens R, Salomon DS, Ueda M, Yamada H, and

Seno M. (2002) Eur J Biochem 269, 307-316 33. Mulloy B, and Johnson EA. (1987) Carbohydrate Research 170, 151-165 34. Fan TC, Fang SL, Hwang CS, Hsu CY, Lu XA, Hung SC, Lin SC, and Chang MD.

(2008) J Biol Chem 283, 25468-25474 35. Rubin J, Zagai U, Blom K, Trulson A, Engström A, and Venge P. (2009) J Immunol

183, 445-451 36. Gandhi NS, and Mancera RL. (2008) Chem Biol Drug Des 72, 455-482 37. Blaber M, Zhang XJ, and Matthews BW. (1993) Science 260, 1637-1640 38. Strehlow KG, Robertson AD, and Baldwin RL. (1991) Biochemistry 30, 5810-5814 39. Porcelli F, Verardi R, Shi L, Henzler-Wildman KA, Ramamoorthy A, and Veglia G.

(2008) Biochemistry 47, 5565-5572 40. Lequin O, Bruston F, Convert O, Chassaing G, and Nicolas P. (2003) Biochemistry 42,

10311-10323 41. Dubovskii PV, Volynsky PE, Polyansky AA, Chupin VV, Efremov RG, and Arseniev

AS. (2006) Biochemistry 45, 10759-10767 42. Subasinghage AP, Conlon JM, and Hewage CM. (2008) Biochim Biophys Acta 1784,

924-929 43. Galanth C, Abbassi F, Lequin O, Ayala-Sanmartin J, Ladram A, Nicolas P, and Amiche

M. (2009) Biochemistry 48, 313-327 44. Vogt B, Ducarme P, Schinzel S, Brasseur R, and Bechinger B. (2000) Biophys J 79,

2644-2656

11

45. Trulson A, Byström J, Engström A, Larsson R, and Venge P. (2007) Clin Exp Allergy 37, 208-218

46. Woschnagg C, Rubin J, and Venge P. (2009) J Immunol 183, 3949-3954 47. Ulrich M, Petre A, Youhnovski N, Prömm F, Schirle M, Schumm M, Pero RS, Doyle

A, Checkel J, Kita H, Thiyagarajan N, Acharya KR, Schmid-Grendelmeier P, Simon HU, Schwarz H, Tsutsui M, Shimokawa H, Bellon G, Lee JJ, Przybylski M, and Döring G. (2008) J Biol Chem 283, 28629-28640

48. Iwasaki W, Nagata K, Hatanaka H, Inui T, Kimura T, Muramatsu T, Yoshida K, Tasumi M, and Inagaki F. (1997) EMBO J 16, 6936-6946

49. Spivak-Kroizman T, Lemmon MA, Dikic I, Ladbury JE, Pinchasi D, Huang J, Jaye M, Crumley G, Schlessinger J, and Lax I. (1994) Cell 79, 1015-1024

50. Cardin AD, and Weintraub HJ. (1989) Arteriosclerosis 9, 21-32 51. Fuchs SM, and Raines RT. (2006) Cell Mol Life Sci 63, 1819-1822 52. Torrent M, Navarro S, Moussaoui M, Nogués MV, and Boix E. (2008) Biochemistry 47,

3544-3555

FOOTNOTES

* M. Torrent, M.V. Nogués and E. Boix, unpublished observations. This paper was supported by projects CTQ2008-00080/BQU, BIO2008-04487-CO3-02, BFU2006-15543-CO2-01 and BFU2009-09371 from the Spanish Ministerio de Ciencia e Innovación. The abbreviations used are: ECP, Eosinophil Cationic Protein; NMR, Nuclear Magnetic Resonance; NOE, Nuclear Overhausser effect; DSS, 4,4-dimethyl-4-silapentane-1-sulfonic acid; TFE, 2,2,2-Trifluoroethanol; DPC, dodecylphosphorylcholine; CCS, conformational chemical shift; CSP, chemical shift perturbation.

FIGURE LEGENDS

Fig. 1. Conformational chemical shifts of ECP1-45 peptide: Δδ values for Hα protons at pH 4.5 and 25ºC are represented for the five different environments estudied, 1A) aqueous solution 1B) 40% TFE, 1C) 20 mM DPC, 1D) Heparin sulfate disaccharide (1:1 complex with ECP1-45), and 1E) 20mM DPC plus heparin disaccharide (1:1 complex with ECP1-45). Fig. 2: Effects of DPC micelles and the heparin mimetic on ECP1-45. The plots represent the Δδ for HN and Hα protons for aqueous solution versus 20 mM DPC micelles, 2A) and 2B), or a solution containing heparin sulfate disaccharide in 1:1 peptide/disaccharide molar ratio, 2C) and 2D). Fig. 3. 15N-HSQC spectra of native ECP, pH 4.5 and 25 ºC, in different conditions. 3A) aqueous solution, and the presence of 3B) 40 mM DPC, 3C) heparin sulfate disaccharide (1:1 complex), and 3D) 40 mM DPC and heparin sulfate disaccharide (1:1 complex). Fig. 4. Interaction surfaces of native ECP with DPC and heparin mimetic. 4A) Chemical shift perturbation map for DPC displayed on the surface of ECP; red Δδ >0.08 ppm, orange 0.05<Δδ<0.08. 4B) Residues involved in the interaction of ECP with DPC are coloured according to the groups described in the text, loop 1 (violet), loop 3 (coral), and loop 6 (green). 4C) Chemical shift perturbation map for Heparin disaccharide displayed on the ECP’s molecular surface, red Δδ > 0.08 ppm, orange 0.05<Δδ<0.08 ppm. 4D) Residues involved in the interaction of ECP with heparin sulfate disaccharide are coloured according to the groups described in the text, A8-Q14 (green), Y33-R36 (red), Q40-L44 (yellow), H128-D130 (blue). Other residues affected are coloured pink. 4E-F) Simultaneous representation of ECP’s interaction surface with DPC and heparin mimetic. Residues in blue are common to the

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interaction with DPC and heparin disaccharide, residues in yellow belong exclusively to the interaction surface with DPC, and residues in violet correspond exclusively to the interaction surface with heparin sulfate disaccharide.

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