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FEBS Letters 585 (2011) 3126–3132

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Selective and specific ion binding on at physiologically-relevant concentrations ⇑ Linlin Miao a, Haina Qin a, Patrice Koehl a,b, Jianxing Song a,c,

a Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore b Department of Computer Science and Genome Center, University of California, Davis, CA 95616, USA c Department of , Yong Loo Lin School of Medicine and National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore

article info abstract

Article history: Insoluble proteins dissolved in unsalted water appear to have no well-folded tertiary structures. Received 30 July 2011 This raises a fundamental question as to whether being unstructured is due to the absence of salt Revised 25 August 2011 ions. To address this issue, we solubilized the insoluble ephrin-B2 cytoplasmic domain in unsalted Accepted 29 August 2011 water and first confirmed using NMR spectroscopy that it is only partially folded. Using NMR HSQC Available online 6 September 2011 titrations with 14 different salts, we further demonstrate that the addition of salt triggers no signif- Edited by Miguel De la Rosa icant folding of the within physiologically relevant ion concentrations. We reveal however that their 8 anions bind to the ephrin-B2 protein with high affinity and specificity at biologically-rel- evant concentrations. Interestingly, the binding is found to be both salt- and residue-specific. Keywords: Insoluble protein Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Ephrin-B2 Intrinsically unstructured protein Protein–ion interaction NMR spectroscopy

1. Introduction reconcile these fundamental discrepancies, a high-resolution view of ion-protein interactions is needed. Ion-specific effects, first described by Franz Hofmeister in 1888 Many proteins have been found to be insoluble and their solubi- [1], are ubiquitous in a myriad of chemical and biological systems. lization usually requires the addition of denaturant and/or deter- The Hofmeister series ranks the relative influence of ions on a gent. Recently however, we discovered that these ‘‘insoluble’’ broad spectrum of phenomena ranging from colloidal assembly proteins could in fact be solubilized in unsalted water with the solu- to protein folding/stability, as well as enzymatic activity; this tion pH several units away from the pI of the protein [14,15].We seminal work is often considered as important as Mendel’s studies note that this method is now being used by other groups to study in genetics [1–4]. However, despite extensive efforts for more than insoluble proteins [16,17]. One remarkable finding from the studies a century, the underlying mechanisms for ion-specific effects on by us and other groups is that these buffer-insoluble proteins all lack biological systems still remain largely elusive. For example, well-folded tertiary structures in unsalted water [14–17]. This raises protein-ion interactions are commonly believed (with some exper- a fundamental question as to whether being unstructured for insol- imental evidences) to be predominantly non-specific, electrostatic uble proteins is due to the absence of salt ions in the unsalted water interactions at physiologically relevant ion concentrations environment. We have previously addressed this question by titrat- (<100 mM) [2,5–7,9,10,12]; a previous study using NMR spectros- ing buffer-insoluble proteins solubilized in unsalted water with copy even suggested that ion-protein interactions are very weak, NaCl and have demonstrated that the addition of NaCl triggered no showing no saturation with salt concentrations up to the molar significant formation of well-folded structures [18,19]. NaCl how- range [8]. Yet there are numerous reports describing ion-specific ever is generally considered to be neutral in the Hofmeister series effects on a variety of protein properties such as stability, solubility and therefore it is of fundamental interest to evaluate the effects and self-association at these concentration ranges [2,4,9–23].To of other salts. In the present study, we extended our previous inves- tigation by titrating the 83-residue cytoplasmic domain of ephrin- B2 with 14 salts whose anions are located in the middle, on the left ⇑ Corresponding author at: Department of Biochemistry, Yong Loo Lin School of Medicine and National University of Singapore, 10 Kent Ridge Crescent, Singapore and right sides of the Hofmeister series (these 14 salts are: Na2SO4, 119260, Singapore. Fax: +65 67792486. NaF, NaSCN, Na2H2PO4, NaCl, NaBr, NaNO3, NaI, MgCl2, KCl, CaCl2, E-mail address: [email protected] (J. Song). GdmCl, LiF, and KCl). The titrations were conducted up to 100 mM,

0014-5793/$36.00 Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2011.08.048 L. Miao et al. / FEBS Letters 585 (2011) 3126–3132 3127 as higher concentrations led to signs of precipitation of the protein HSQC-TOCSY and HSQC-NOESY spectra were recorded on a 15N- (which is expected, as the protein is deemed insoluble). We found labeled sample at a protein concentration of 800 lM. Sequential that the addition of these 14 salts triggered no formation of the assignment was achieved with the well-established procedure well-folded structure of the protein. Very surprisingly, however, [26]. All NMR data were processed with NMRPipe [27] and subse- 2À 1 we were able to show that the 8 anions of these 14 salts (SO4 , quently analyzed with NMRView [28]. The H chemical shift was ref- À À À À À À À F , SCN ,H2PO4 ,Cl ,Br ,NO3 ,I ) bind selectively and specifically erenced directly to 2,2-dimethyl-2-silapentanesulfonic acid (DSS), to the protein, with apparent dissociation constants (Kd) usually less whereas the 15N chemical shifts were indirectly referenced to DSS than 50 mM, i.e., in their physiological ranges. These results empha- (see [19] for details). size the importance of salts in modulating protein functions. For NMR characterization of the interactions of the ephrin-B2 cytoplasmic domain to 14 salts, NMR samples were prepared by 2. Materials and methods dissolving the protein powder in Milli-Q water (electric conductiv- ity of 0.78 ls) to a final concentration of 300 lM (electric conduc- 2.1. Cloning, expression and purification of the ephrin-B2 cytoplasmic tivity of 363 ls). The solution pH was measured to be 4.0 without 1 15 domain further adjustment. A series of two-dimensional H- N HSQC spectra were acquired on the 15N-labeled domain at a concentra- The DNA fragment encoding the human ephrin-B2 cytoplasmic tion of 300 lM in the absence or in the presence of different salts domain (83 residues) was amplified by the PCR reaction from a at varying salt concentrations, as we previously conducted with full-length ephrin-B2 cDNA by using two designed primers; it NaCl [18]. The pH values were measured for the samples without was subsequently cloned into a modified pET32a vector (Novagen). and with the different salts and we found that the presence of salts, The vector was transformed into Escherichia coli BL21 (DE3) cells even at 100 mM, resulted in no significant change of the pH value. (Novagen) for protein expression. The recombinant protein was found in the inclusion body. The pellets were dissolved in a buffer 2.4. Data fitting containing 8 M urea and first purified by Ni2+-affinity chromatog- raphy (Qiagen) under denaturing condition in the presence of By superimposing the HSQC spectra, the shifted HSQC peaks 8 M urea. The eluted fraction was further purified by reverse-phase could be identified and further assigned to the corresponding res- 1 15 HPLC on a C18 column and lyophilized. As the protein samples pre- idues. The differences in H and N chemical shifts between the cipitate upon addition of thrombin, the 16-residue His tag was not HSQC peaks of the domain in the absence of salt and in presence removed; as we previously reported for the all other insoluble pro- of different salts at varying concentrations were calculated. 1 15 teins [14,18,19]. We separately fitted the shift tracings of H and N chemical The production of the isotope-labeled proteins for NMR studies shift changes by using the one binding site model [29] to obtain followed a similar procedure except that the bacteria were grown residue-specific dissociation constants (Kd); these values are sum- 15 15 in M9 medium with the addition of ( NH4)2SO4 for N labeling or marized in Table S1. 15 13 15 13 ( NH4)2SO4 and [ C]glucose for N and C double labeling [18,19]. The purity of all protein samples was verified by measuring 3. Results their molecular weights with a Voyager STR matrix-assisted laser desorption ionization time of flight mass spectrometer (Applied Bio- 3.1. Solution conformation of the entire ephrin-B cytoplasmic domain systems). The concentration of protein samples was determined by spectroscopic methods in the presence of denaturant [20]. Previously the entire domain consisting of 83 residues was found to be insoluble and consequently its structure remains 2.2. Calculation of the electrostatic potentials unknown. However, the NMR structure of its soluble C-terminal subdomain containing the last 33 residues has been previously The extended structure for the first 50 residues of the ephrin-B2 determined [22,23], with the first 22 residues of the sub-domain cytoplasmic domain plus the 16-residue His-tag was generated adopting a well-packed b-hairpin followed by largely unstructured with the CYANA program [21] while the structure of the last 33 11 residues. Here we confirmed that the entire domain is indeed residues was previously published [22,23]. buffer-insoluble but subsequently we succeeded in solubilizing it PDB2PQR [24] was first used to convert the two structures from in unsalted water at pH 4.0 by following our previous discovery PDB format to PQR format. Subsequently these PQR files were [14,15]. Preliminary characterizations by far-UV CD (Fig. 1A) and given as input into APBS (Adaptive Poisson–Boltzmann Solver) NMR 1H-15N HSQC spectroscopy (Fig. 1B) indicate that the entire program [25] to generate their electrostatic potentials at pH 4.0 domain is largely unstructured without tight tertiary packing in (http://www.poissonboltzmann.org/apbs/citing-your-use). solution. By further analyzing heteronuclear NMR spectra includ- ing a pair of triple resonance experiments HNCACB and CBCA 2.3. CD and NMR experiments (CO)NH, 15N-edited HSQC-TOCSY and HSQC-NOESY, we achieved the sequential assignment of the whole domain; the chemical Far-UV CD spectra of the ephrin-B2 cytoplasmic domain were shifts for the Ha support the conclusion that the domain is largely acquired on a Jasco J-810 spectropolarimeter equipped with a ther- disordered (Fig. 1C). As such, the entire cytoplasmic domain of mal controller as described previously [14] at a protein concentra- ephrin-B2 can be categorized as an intrinsically unstructured tion of 20 lMat25°C, using 1 mm path length cuvette with a protein although it behaves different from other members in being 0.1 nm spectral resolution. Data from five independent scans were buffer-insoluble [30,31]. added and averaged. On the other hand, we did observe that the last 33 residues of All NMR data were collected at 25 °C on an 800 MHz Bruker the entire domain have relatively large conformational shifts, Avance spectrometer equipped with a shielded cryoprobe as which are almost identical to those found for the isolated segment described previously [18]. To achieve sequential assignments, of last 33 residues with the exception of Cys67 as this residue triple-resonance experiments, including HNCACB and CBCA(CO)NH, becomes the N-terminal residue in the isolated ephrinB-33 subdo- were conducted on a doubly labeled sample at a protein concentra- main [22,23]. This strongly indicates that the last 33 residues adopt tion of 800 lM. To obtain the Ha chemical shifts, 15N-edited a similar conformation both in the entire domain and as an isolated 3128 L. Miao et al. / FEBS Letters 585 (2011) 3126–3132

Fig. 1. Solution conformation. (A) Far-UV CD spectrum of the entire ephrin-B2 cytoplasmic domain at a protein concentration of 20 lM (pH 4.0) at 25 °C. (B) Two-dimensional 1H-15N NMR HSQC spectrum at a protein concentration of 300 lM (pH 4.0) at 25 °C. (C) Ha conformational shifts of the entire ephrin-B2 cytoplasmic domain (83 residues) with a 16-residue His-tag (blue bars) and of the isolated last 33 residues called ephrinB-33 (red bars) previously reported [20]. The first 7 residues of the His-tag could not be assigned due to missing side-chain resonances. Lys17 is the starting residue of the cytoplasmic domain while Cys67 is the starting residue of ephrinB-33. fragment (Fig. 2A). We generated an extended model for the first We subsequently examined the effects of salts on the protein 50 residues (plus an N-terminal 16-residue His-tag). We calculated conformation by NMR spectroscopy. We acquired 1H-15N HSQC the electrostatic potentials for both extended model and NMR spectra of the entire domain solubilized in unsalted water at pH structure of the last 33 residues at pH 4.0 using APBS [25]. The 4.0 with gradual addition of the salts at different concentrations. 33-residue C-terminal subdomain has a relatively neutral surface We observed no significant increase of the spectral dispersions (Fig. 2B) while the N-terminal fragment up to Arg43 has a contin- but only shifts of HSQC peaks (see Fig. S2). These results are consis- uous and very positive electrostatic surface (Fig. 2C). tent with those obtained by CD, suggesting no significant folding upon adding different ions with concentrations up to 100 mM. 3.2. Effects of salts on the conformation of EphrinB2 in solution Therefore, for the ephrin-B2 cytoplasmic domain, being unstruc- tured is not a result of absence of salt ions, but most likely an We have assessed the effects of 8 salts with the same cation intrinsic property that is well established for other buffer-soluble (sodium) but different anions on the solution conformation of intrinsically unstructured proteins [30,31]. the domain solubilized in unsalted water by both CD and hetero- nuclear NMR HSQC spectroscopy. For far-UV CD characterization, 3.3. Selective binding of salts as visualized by NMR spectroscopy we collected data at five different salt concentrations: namely 0 mM, 1 mM, 10 mM, 50 mM and 100 mM for 8 salts, namely The binding between ions and the protein triggers environmen-

Na2SO4,Na2H2PO4, NaF, NaCl, NaBr, NaNO3, NaI and NaSCN. We tal changes on the protein surface and consequently alters the observed that the addition of salts at low concentrations did not chemical shifts of the 1H and 15N nuclei in the binding area induce any significant folding. High quality CD spectra were only [32,33]. Strikingly, as shown by HSQC titrations (Fig. S2), the salt obtained in the presence of Na2SO4, NaH2PO4 and NaF at 4 differ- effects are not uniform over the protein. By superimposing the ent salt concentrations; these CD spectra show little to no changes HSQC spectra at different salt concentrations, we mapped out the compared to the corresponding reference spectrum in the absence residue-specific changes of 1H and 15N chemical shifts upon salt of salt (Fig. S1), suggesting that no significant folding had occurred. titrations (Figs. 3 and S3). We found that upon the addition of salts, Data on the other salts were less conclusive due to high noise level. most of the residues with HSQC peaks shifts are located over the L. Miao et al. / FEBS Letters 585 (2011) 3126–3132 3129

N-terminal half of the protein. Interestingly the patterns of the chemical shift changes are highly diverse for three salts whose an- ions are located on the left side of the Hofmeister series including

Na2SO4, NaH2PO4 and NaF (4) while they are largely uniform for those anions that are on the right side of the series including NaCl,

NaBr, NaNO3, NaI and NaSCN. To determine the relative contribu- tion of cations and anions to the HSQC peak shifts induced by salts, we monitored the shifts by titrating chloride and fluoride salts

with different cations, including MgCl2, KCl, CaCl2, Guanidinium chloride (GdmCl), LiF and KF. The different chloride salts caused very similar patterns of HSQC peak shifts at low concentrations, suggesting that the observed effects are mostly due to the chloride anion (Fig. S4). At higher concentrations (>50 mM), chloride salts with different cations did cause slightly differential shifts, in par- ticular for the last residue Val99. Similar results were also observed for the different fluoride salts (Fig. S5). These results suggest that the HSQC peak shifts observed here are mostly triggered by the asymmetric binding of different anions to the protein residues. Fig. 2. Electrostatic properties. (A) Three-dimensional structure of ephrinB-33 Nevertheless, it cannot be excluded that at high concentrations previously determined by NMR spectroscopy [22]. (B) The electrostatic potential the cation type may also be relevant to binding. The latter effect calculated with APBS at pH 4.0 (see Supporting Information for details) is visualized is probably indirect, as specific cations are known to affect the at the level of the accessible surface of the protein, with blue and red corresponding to positive and negative potential values, respectively. (C) The electrostatic clustering of anions in solution [34]. In addition, we believe that potential at the level of the accessible surface of an extended model structure for the unusual behavior detected at Val99 is a consequence of a the first 50 residues of the ephrin-B2 cytoplasmic domain (plus the N-terminal strong interaction between its free carboxyl group and the cations His-tag). of the salt considered.

Fig. 3. Residue-specific chemical shift difference. Upper panel: the sequence of the ephrinB2 cytoplasmic domain (83 residues) with an N-terminal His-tag (16 residues). The residues are differentially colored: blue for positively-charged, red for negatively charged, purple for neutral polar, and green for hydrophobic. Lower panel: chemical shift difference (CSD) of the amide proton (1H) and nitrogen (15N) for residues of the ephrin-B2 cytoplasmic domain upon addition of three representative salts

(Na2SO4, NaF and NaSCN) at 50 mM (blue bars) and 100 mM (red circles). Residues with significant changes are labeled. 3130 L. Miao et al. / FEBS Letters 585 (2011) 3126–3132

3.4. Quantitative assessment of binding affinity disordered as evidenced from small NMR conformational shifts (Fig. 1c) and absence of medium- and long-range NOEs (data not We plotted the chemical shift changes induced by ions as a shown). Based on the extended model that was generated for Eph- function of salt concentration for the residues that demonstrate rinB2 (see above and Fig. 2) and ephrinB2-33 structure, we com- significant peak shifts (Figs. 4 and S6). Using the one binding site puted the accessibility of all side chains and of all amide protons. model, we separately fitted these titrations based on 1H and 15N The amide protons of the folded ephrinB2-33 sub-domain appear chemical shifts. Two representative residue-specific apparent dis- largely inaccessible (see Fig. S7): this is a likely reason for the eph- sociation constants (Kd) obtained for Na2SO4, NaF and NaSCN are rinB2-33 residues to be relatively less perturbed by the addition of shown in Fig. 4, respectively; full data for the eight salts are sum- most ions considered in this study. Accessibility however is not the marized in Table S1. Of particular significance, the majority of the main factor influencing ion binding: although the amide protons of apparent Kd values are found to be less than 50 mM, indicating the N-terminal 66 residues are all largely accessible (Fig. S7), the that all eight anions bind to the ephrinB2 cytoplasmic domain with eight anions we have studied bind mainly to the region that covers high affinity. Na2SO4 is found to be the strongest binder to most residues Arg14-His29; this region forms a continuous and posi- residues, with an average apparent Kd of 1 mM if we exclude the tively-charged surface (Figs. 2c and 3). Notice that binding is ob- outlier Val99 (Fig. 4). The results are consistent with previous stud- served on non-positive residues also, albeit with lower affinity. ies which showed that Na2SO4 has the strongest ability to stabilize To quantify this observation, we have categorized the residues of ribonuclease [9] and to reduce the effective charge of hen-egg the ephrinB2 cytoplasmic domain into 4 groups, namely positively white lysozyme [5]. charged, negatively charged, neutral polar, and hydrophobic resi- dues (Fig. 3) and subsequently calculated the average Kd values 3.5. Residue specificity of anion binding to EphrinB2 for each group and each salt. Results are shown in Table 1. As intu- itively expected, when binding occurs, it is stronger with positively Ion binding to EphrinB2 is not uniform along the sequence: we charged residue than with other types of residues. For many salts, 1 found that the majority of residues with significantly perturbed H i.e., NaCl, NaNO3, NaSCN and to a lesser extend NaBr, binding to and 15N chemical shifts are located in the N-terminal part of the residues with negatively charged side-chains and binding to protein; this region has been characterized to be predominantly hydrophobic residues occur with similar affinity; this suggests that

Fig. 4. Residue-specific apparent dissociation constants (Kd). Experimental (red dots) and fitted (blue lines) values for the 1H and 15N chemical shift changes induced by gradual addition of salts. The fitted line is computed with the one binding site model. Displayed are data for two representative residues: His25/Ser26 for Na2SO4; Ser26/ Asp76 for NaF; and His25/Ser26 for NaSCN.

Table 1 Residue-type specificity of salt binding to EphrinB2.

Salt Residue typea Charged, positive Charged, negative Neutral, polar Hydrophobic

b c Na2SO4 1.2 (0.7) NA (NA) 1.2 (1.0) 16.8 (2.3)

NaH2PO4 5.2 (6.9) 59.6 (13.7) 8.0 (4.6) 19.6 (16.2) NaF 73.4 (13.4) 8.1 (11.3) 46.6 (15.0) 18.0 (9.1) NaCl 18.0 (16.2) NA (25.6) 10.1 (11.5) 18.6 (25.9) NaBr 10.1 (24.5) NA (18.3) 12.5 (18.6) 25.8 (30.5)

NaNO3 6.9 (10.3) 14.6 (37.3) 8.3 (13.3) 18.9 (28.9) NaI 11.0 (17.9) 20.3 (22.3) 72.1 (17.9) 14.6 (95.4) NaSCN 10.5 (15.5) 19.4 (21.6) 11.4 (14.6) 18.3 (24.4)

a Amino acids were divided into 4 categories: charged positive (K, R, H), charged negative (D, E), neutral polar (N, Q, S, T, C, M, F, W, Y), hydrophobic (G, A, V, I, L, P). b The apparent dissociation constants Kd computed from the changes of 1H and 15N chemical shifts upon salt titrations are averaged for each residue type; note that only residues for which binding was detected and quantified are considered. Results based on 15N chemical shifts are given in parenthesis. c NA: not available. L. Miao et al. / FEBS Letters 585 (2011) 3126–3132 3131 for these residues and those salts, binding is largely independent of Acknowledgements the side-chain and mainly occurs at the level of the backbone. We notice that all eight anions we have studied bind with similar affin- The study is supported by Singapore National Medical Research ities to hydrophobic residues. Council Grants R-154-000-382-213 and Ministry of Education (MOE) of Singapore Tier 2 Grant R-154-000-388-112 and 4. Discussion MOE2011-T2-1-096 to J. Song.

In the present study, we initiated a systematic assessment of Appendix A. Supplementary data the effect of 14 salts on the conformation of the insoluble eph- rin-B2 cytoplasmic domain solubilized in unsalted water. By CD Supplementary data associated with this article can be found, in and NMR characterization, we first demonstrated that in unsalted the online version, at doi:10.1016/j.febslet.2011.08.048. water, its N-terminal subdomain is largely unstructured while its C-terminal 33 residues adopt a conformation highly similar to that References of the isolated fragment in the buffer [22,23]. Strikingly, the addi- tion of 14 salts up to 100 mM induces no significant folding of the [1] Kunz, W., Henle, J. and Ninham, B.W. (2004) Zur Lehre von der Wirkung der Salze (about the science of the effect of salts): Franz Hofmeister’s historical domain, implying that being unstructured for the ephrin-B2 cyto- papers. Curr. Opin. Coll. Inter. Sci. 9, 19–37. plasmic domain is mostly resulting from its intrinsic property. [2] Baldwin, R.L. (1996) How Hofmeister ion interactions affect protein stability. Strikingly, by minimizing the interference of background buffer Biophys. J. 4, 2056–2063. ions, we reveal that the 8 anions of the 14 salts do bind to the pro- [3] Kunz, W. (2010) Specific ion effects in colloidal and biological systems. Curr. Opin. Coll. Inter. Sci. 15, 34–39. tein with high selectivity and affinity at physiologically-relevant [4] Zhang, Y. and Cremer, P.S. (2009) The inverse and direct Hofmeister series for salt concentrations, with most apparent Kd values less than lysozyme. Proc. Natl. Acad. Sci. USA 106, 15249–15253. 50 mM. It is particularly noteworthy that the binding is not uni- [5] Gokarn, Y.R., Fesinmeyer, R.M., Saluja, A., et al. (2011) Effective charge measurements reveal selective and preferential accumulation of anions, but form along the protein sequence. 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