Structural and Dynamic “Portraits” of Recombinant and Native

Jul 27, 2017 - Refinement of NMR structures was performed with Rosetta version 3.3.(27) PDBStat version 5.10 was used to convert restraints from CYANA...
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Structural and Dynamic “Portraits” of Recombinant and Native Cytotoxin I from Naja oxiana: How Close Are They? Peter V. Dubovskii,*,† Maxim A. Dubinnyi,† Anastasia G. Konshina,† Ekaterina D. Kazakova,‡ Galina M. Sorokoumova,‡ Tatyana M. Ilyasova,† Mikhail A. Shulepko,† Rita V. Chertkova,† Ekaterina N. Lyukmanova,†,⊥ Dmitry A. Dolgikh,†,⊥ Alexander S. Arseniev,†,∥ and Roman G. Efremov†,∥,§ †

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklukho-Maklaya str., Moscow 117997, Russia ‡ Moscow Technological University, 86 Vernadsky pr., Moscow 119571, Russia § Higher School of Economics, 20 Myasnitskaya, Moscow 101000, Russia ∥ Moscow Institute of Physics and Technology (State University), 9 Institutskiy per., Dolgoprudny, Moscow Region 141700, Russia ⊥ Biological Faculty, Lomonosov Moscow State University, 119234 Moscow, Russia S Supporting Information *

ABSTRACT: Today, recombinant proteins are quite widely used in biomedical and biotechnological applications. At the same time, the question about their full equivalence to the native analogues remains unanswered. To gain additional insight into this problem, intimate atomistic details of a relatively simple protein, small and structurally rigid recombinant cardiotoxin I (CTI) from cobra Naja oxiana venom, were characterized using nuclear magnetic resonance (NMR) spectroscopy and atomistic molecular dynamics (MD) simulations in water. Compared to the natural protein, it contains an additional Met residue at the N-terminus. In this work, the NMR-derived spatial structure of uniformly 13C- and 15N-labeled CTI and its dynamic behavior were investigated and subjected to comparative analysis with the corresponding data for the native toxin. The differences were found in dihedral angles of only a single residue, adjacent to the N-terminal methionine. Microsecond-long MD traces of the toxins reveal an increased flexibility in the residues spatially close to the N-Met. As the detected structural and dynamic changes of the two CTI models do not result in substantial differences in their cytotoxicities, we assume that the recombinant protein can be used for many purposes as a reasonable surrogate of the native one. In addition, we discuss general features of the spatial organization of cytotoxins, implied by the results of the current combined NMR and MD study.

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main component of honey bee venom,6 and latarcins from spider venom.5 Their cysteine-rich counterparts from snake venom are cardiotoxins, or cytotoxins (CTs).7 CTs are considered as prospective cytotoxic agents for anticancer therapy,8,9 as the activity of a number of CTs exceeds that of the traditional drug, cisplatin.10 For this purpose, it is desirable to produce CTs recombinantly. The most cost-effective route for direct expression in Escherichia coli produces the cytotoxin, elongated by an extra Met residue at the N-terminus.11 The consequences of this modification have not yet been studied in atomic detail for CTs, although an attempt to investigate this has been made.1 As far as CT is concerned, modification of a single N-terminal Leu residue can result in drastic changes in the structure and activity of the molecule.12 The cytotoxic activity of recombinant cytotoxin I from Naja oxiana against rat glioma cells was indeed found to be somewhat lower, compared to that of the native analogue.11 A similar observation was made for another representative of

harmacologically useful polypeptides are often produced in their recombinant forms using genetic engineering. This brings about the necessity of introducing specific mutations and/or grafting tags into the native amino acid sequences. However, the identity of the structure and dynamics of native and recombinant proteins remains usually unexplored. Only a few examples of a comparison of this kind are available in the literature.1−3 They do show that, e.g., grafting of additional residues at the N-terminus of a protein can affect hydrogen bond networks, in which residues in the native protein are involved. Apparently, the effect might strengthen in the case of structurally rigid peptides, cross-linked with a network of disulfide and hydrogen bonds. In the work presented here, we examine polypeptides of this kind. The venoms of certain species of insects and snakes not only are rich in highly specific proteins targeting particular receptors but also contain polypeptides acting on the lipid bilayer of cell membranes. These proteins are often termed cytolytic proteins.4,5 As a rule, cytolytic peptides exhibit membrane activity in experiments with model lipid membranes in vitro and display cytotoxicity against a variety of living cells.5 The wellknown examples of linear cytolytic peptides are melittin, the © 2017 American Chemical Society

Received: May 11, 2017 Revised: July 26, 2017 Published: July 27, 2017 4468

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assignments were made and cross-peak integrations performed with Cara version 1.8.4 (http://cara.nmr.ch). For calculation of the structure of the “minor” form, only nuclear Overhauser effects (NOEs) unique for this form were used. They were collected mainly from 15N-detected NOESY spectra featuring non-overlapped cross-peaks for the “major” and “minor” forms. Refinement of NMR structures was performed with Rosetta version 3.3.27 PDBStat version 5.10 was used to convert restraints from CYANA to Rosetta format.28 Analysis of the calculated structures was performed with the PROSESS Web server.29 To obtain heteronuclear assignments for nCTI, twodimensional (2D) 15N−1H HSQC and 13C−1H HSQC spectra were acquired at 30 °C and pH 6.0 at the natural abundance of the nuclei. The protein was dissolved in H2O/D2O (90/10), except for the acquisition of the 13C−1H HSQC spectrum of nCTI. In this case, the protein was dissolved in 100% D2O. The acquisition parameters were identical to those already reported in the literature.25 The sample concentration was 12 mg/mL. The acquisition times of the 15N−1H HSQC and 13C−1H HSQC spectra of nCTI were 18 and 12 h, respectively. For comparison, the acquisition times of the 15N−1H HSQC and 13 C−1H HSQC spectra for [U-13C,15N]rCTI were 20 and 15 min, respectively. The same suite of programs for processing and pick-picking of the acquired spectra was used for nCTI. Analysis of the chemical shifts of Pro residues was performed with PROMEGA.30 Molecular Dynamics. All MD calculations were performed in the all-atom AMBERff99SB-ILDN force field.31 Each protein molecule was placed in a rectangular box with periodic boundary conditions. The minimal distance from any protein atom to the edges of the box was 12 Å. The number of water molecules added was not fewer than ∼5000. The TIP3P water model32 was used. To achieve electrical neutrality of the system, chloride counterions were added. To equilibrate the protein− water system, a step-by-step minimization of its energy was performed using the steepest descent method. First, coordinates of all heavy atoms, then all backbone atoms, and finally only Cα atoms were consecutively fixed. Then, the energy of the whole system was minimized without constraints. After that, the system was heated from 5 to 300 K over 200 ps in an NVT ensemble. The configuration obtained was used as a starting one for MD runs in an NPT ensemble. Electrostatic interactions were treated via the particle mesh Ewald (PME) algorithm33 with a 10 Å cutoff. van der Waals interactions were calculated using a spherical cutoff function with parameters of 10 and 12 Å. The integration step was equal to 2 fs. To keep the temperature and the pressure constant (in an NPT ensemble), a V-rescale thermostat34 and a Berendsen barostat35 were used. A set of structures extracted from the MD trajectory (except for the stage of system minimization and heating) was used for further analysis. MD calculations and data processing were performed with the use of the GROMACS package (version 4.5.2 and newer)36 and in-house utilities. For MD calculations of nCTI, the starting model was that obtained by NMR in aqueous solution [Protein Data Bank (PDB) entry 5NPN, model number 1 from 20 calculated].37 In the case of rCTI, there were as many as three starting models, including a surrogate one (see details in Molecular Dynamics: nCTI versus rCTI). For the purpose of comparison, a single start from a model of 1CDT (CT VII4 Naja mossambicca) was also performed. The duration of each trajectory amounted to ≥1 μs. PyMol (http://pymol.org/) was used to build and

the three-finger family of toxins, WTX. The recombinant mutant WTX analogue, which possesses an additional Nterminal methionine residue, demonstrated an altered pharmacological profile and weakened binding of orthosteric antagonist N-methylscopolamine to human M1- and M2mAChRs (muscle acetylcholine receptor). Thus, a concern about the equivalence of the recombinant toxin with the native one is justified and determines the goal of this work. CTs are purely β-structural polypeptides.8,13,14 The data on their interaction with model lipid membranes evidence that CTs destabilize the bilayer and anionic lipids strengthen the effect.15 It is assumed that the plasma membrane (or that of an organelle within the cell), containing either glycolipid sulfatide,16 phosphatidylserine,17 cardiolipin,18,19 or phosphatidylglycerol,20,21 is a target of attack by CTs in a living cell. We compare the spatial organization and dynamics of the native cytotoxin I (CTI) from N. oxiana venom with those of its recombinant analogue containing an extra Met residue at the N-terminus. CTI is a suitable example because it has rather rigid three-dimensional (3D) fold but can modulate its activity against lipid membranes because of finely tuned local conformational changes. Therefore, the role of even a single residue can become important, thus requiring careful analysis at the atomic level. We obtained isotopically 13C- and 15Nenriched recombinant toxin.11 Finally, we compared dynamics of the both toxins in aqueous solution, using all-atom molecular dynamics (MD) simulations. Because of the moderate impact of the N-Met residue on the structure and dynamics of the remaining part of the cytotoxin molecule, we were able to assign heteronuclear 13C−1H and 15N−1H HSQC spectra of native CTI (nCTI), acquired at the natural abundance of these nuclei. Thus, for the first time, we obtained nearly full heteronuclear chemical shifts of a native cytotoxin. In addition, we discuss the general features of the spatial organization of CTs, revealed by our combined nuclear magnetic resonance (NMR) and MD study of recombinant and native cytotoxins.

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MATERIALS AND METHODS Origin of the Toxins. The production and refolding of rCTI were performed as described in ref 11. The natural CTI (nCTI) was isolated via fractioning of the whole N. oxiana cobra venom, obtained as described elsewhere.22 NMR Spectroscopy. To obtain nearly complete signal assignments in the heteronuclear NMR spectra of uniformly 13 C- and 15N-labeled rCTI (H2O/D2O, 95:5, pH 6.0), the same set of 3D NMR spectra was acquired, as for the 13C- and 15Nlabeled recombinant neurotoxin (also possessing an additional N-Met residue), investigated previously.23 All spectra were acquired with an Avance spectrometer (Bruker), featuring a 1H NMR frequency of 700.13 MHz. For determination of the spatial structure of the toxin, 15N-detected NOESY spectra (mixing times of 100 and 150 ms) and a 13C-detected NOESY spectrum (mixing time of 70 ms) were obtained. Processing of the spectra was performed with TOPSPIN version 2.0.a, provided by the manufacturer of the NMR spectrometer. The spatial structure of rCTI was calculated with CYANA,24 performing simulated annealing in the torsion angle space, using protocols described previously.25 In addition, chemical shifts of [U-13C,15N]rCTI were transferred into dihedral angle restraints, using TALOSN.26 The obtained list was edited manually to determine the correspondence to the ranges of variation of dihedral angles, obtained from the local structure analysis, using subroutine HABAS of CYANA. Spectral 4469

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Figure 1. Spectral features of the conformational equilibrium in aqueous solution of [U-13C,15N]rCTI (pH 6.0, 30 °C, H2O/D2O, 95/5). Portions of (a) 15N−1H HSQC and (b and c) 13C−1H HSQC spectra. The cross-peaks are marked with the names of residues using the single-letter abbreviations. A lowercase m is added to those corresponding to the “minor” conformer. “Major” and “minor” cross-peaks are connected with a double-headed arrow, when they are well-resolved. The HD/CD and HA/CA regions of the 13C−1H HSQC spectrum are represented in panels b and c, respectively. The similarly named cross-peaks in panel b correspond to non-equivalent δ-protons.

lacks a cavity in loop 2, where exchange of tightly bound water molecules takes place. The MD trajectory was started from this structure to reveal whether such a cavity in loop 2 can be formed during MD simulation. On the basis of these data, we were able to conclude that rCTI possesses all the features of the spatial organization, which are specific to the native cardiotoxins. Signal Assignments in the NMR Spectra of [13C,15N]rCTI. Inspection of the 15N−1H HSQC spectrum of [U-13C,15N]rCTI reveals that nearly each cross-peak has a twin with a lower intensity (Figure 1a). This implies the presence of “minor” and “major” forms, identified for CTII from N. oxiana.38 Using a set of 3D NMR spectra and a traditional sequential signal assignment protocol, nearly complete 1H, 13C, and 15N assignments for both forms of rCTI were obtained. As compared to the wild-type toxin, the bacterially expressed rCTI possesses an additional methionine residue at the N-terminus. For the sake of simplicity, we designated this Met residue as Met0 and started numbering from Leu1. Interestingly, the 13 C− 1H HSQC spectrum of [U- 13C,15N]rCTI exhibits twinning of δ-protons and the α-proton for only one of the Pro residues (Pro8) (Figure 1b,c). The difference in the

optimize one of them. MD simulations of rCTI were performed using the same computational protocol that was used for nCTI.

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RESULTS AND DISCUSSION The overall flowchart of the current study is as follows. The spatial structure of nCTI has recently been determined with a high degree of precision.37 Therefore, in this study, we focused on determination of the spatial organization of rCTI. We obtained uniformly 13C- and 15N-labeled CTI11 and used a standard protocol of 3D NMR spectroscopy for determination of its spatial structure. The obtained ensemble of structures was computationally refined to a high quality. Then, we performed MD simulations of rCTI and nCTI starting from their resulting spatial models and observed local differences in their dynamics. In addition to the primary goal of the work, i.e., comparison of the structure and dynamics of the recombinant and native CTI, we studied general features of the spatial organization of cytotoxins. For the first time, we obtained heteronuclear chemical shifts of nCTI and compared them with available incomplete assignments of α-carbon chemical shifts of several cardiotoxins. Also, we analyzed the shape of loop II in the spatial structures of cardiotoxins. One of the models (1CDT) 4470

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Figure 2. NOE data and spatial organization of recombinant CTI. (a) Combination of NOE, disulfide bonds, and chemical shift data, used for calculation of the structure of rCTI. For the S−S bonds, the cysteine residues forming disulfide bonds are connected with arrows; the Cys residue numbers are indicated on the ends of the arrows. NOE contacts for [U-13C,15N]rCTI (major form) in aqueous solution (pH 6.0, 30 °C). Sequential d connectivities were obtained from the 15N-detected NOESY (mixing time of 100 ms) spectrum. Instead of missing amide protons, Hδ protons are used for Pro residues. Information about the secondary structure of the toxin, obtained via analysis of NOE data, is presented below the d connectivity section with boxes. The start and end residues of the β-strands are indicated above the boxes. The probability of the formation of βstrands (predSS), obtained with TALOS-N26 via analysis of the chemical shifts, is presented in the next histogram. Model-free order parameters (S2), calculated from the random coil index (RCI), are given in the next graph. The calculation was performed with TALOS-N, according to the procedure described in ref 52. Rosetta-optimized structures of the (b) “major” and (c) “minor” forms of rCTIm, superimposed on the backbone atoms of residues 1−60 of the first model from the set of 20 NMR-calculated structures (PDB entry 5NPN) of nCTI (thick red backbone). One of the models is shown in ribbon representation to indicate the positions of the β-strands. These residues are marked with their numbers. From all the side chains, the Met0 residue is shown to indicate its proximity to the so-called loop IV (residues 15−18). The tips of the loops are marked with roman numerals. I, II, and III correspond to residues 5−10, 27−34, and 46−48, respectively. In panel c, the first model from the “major” ensemble (thick blue backbone) is superimposed on the ensemble of the minor form of rCTI. The orientation of the models and thus the positioning of the loops and β-strands are identical to those shown in panel b. The coordinates of the ensembles shown in panels b and c have been deposited in the PDB as entries 5T8A and 5LUE, respectively. The chemical shifts were deposited in the BioMagResBank as entries 30172 and 34039, respectively.

chemical shifts between the “major” and “minor” forms of rCTI displays its maximum near Pro8. Analysis of the chemical shifts of proline residues in these forms of rCTI reveals the cis configuration of the Val7−Pro8 bond in the “minor” form of this toxin. Thus, a slow equilibrium exists between the two forms of rCTI in aqueous solution because of the cis−trans isomerism of the Val−Pro peptide bond in loop I. It is of interest to compare chemical shifts of [U-13C,15N]rCTI and nCTI. For this purpose, heteronuclear 13C−1H and

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N−1H HSQC spectra of nCTI were acquired at the natural abundance of these nuclei. The difference in the chemical shifts for the “major” forms of both toxins reaches the maximum upon approaching the Met attachment site. Most probably, these differences can be attributed to deviation of the coordinates of a few N-terminal residues of rCTI, compared with their values in the native analogue, nCTI (PDB entry 5NPN for the major form). This was clarified by analysis of the 4471

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Figure 3. MD data for recombinant vs native CTI. (a) Root-mean-square fluctuation (RMSF) for the atoms of the polypeptide backbone for rCTI and nCTI. The curves for rCTI (black, blue, and green) and nCTI (red; data points, filled circles) are superimposed on the same graph. The curves for rCTI, displayed in different colors, correspond to MD runs started from the surrogate model (black), the NMR-calculated model (green), and the Rosetta-optimized NMR model (blue). Data points on these graphs have been omitted. (b) Statistics of the exchange of water molecules within the tip of loop II of nCTI (red) and rCTI (green). The total number of water molecules that entered the cavity of this loop is given on the Y-axis. Always, only one water molecule is situated in the cavity and forms at least three hydrogen bonds with the protein backbone atoms. The MD simulation time is plotted on the X-axis.

spatial organization of rCTI using NOE and chemical shift data, as reported in the next section. Spatial Organization of rCTI. The combination of NOE (from both 13C- and 15N-edited NOESY spectra) and chemical shift data used for structure calculation of the “major” form of rCTI is presented in Figure 2a. The secondary chemical shift data, in combination with model-free order parameters estimated from the random coil index (RCI39) and calculated from backbone 1H, 13C, and 15N chemical shifts, suggest that the whole toxin molecule is well-ordered, including loop regions (residues 4−10, 13−18, 27−34, and 39−48, interconnecting β-strands). The resulting set of rCTI structures is presented in Figure 2b. For comparison, structure number 1 from the set of NMR models of nCTI (PDB entry 5NPN) is added to the bundle shown in Figure 2b. The differences are

located primarily within the position of the N-terminus. Its coordinates are closer to loop IV (residues 15−18) in rCTI than in the nCTI molecule. This draws the side chain of the Met0 residue more closely to this loop. A similar procedure, followed by structural refinement, was used to calculate the “minor” form of rCTI. 15N NOE data were predominant, as in 15N−1H HSQC, but not in 13C−1H HSQC, spectra many resonances of the “major” and “minor” forms do not overlap (Figure 1a). The refined ensemble of 20 structures of the “minor” form of rCTI is presented in Figure 2c. Indeed, the Val7−Pro8 peptide bond features a “cis” configuration, resulting in a distinctly different shape of the tip of loop I compared with that of the “major” form (Figure 2b). It is noteworthy that we did not use any 13C NOE data for the Pro8 residue, because the Cα/Hα cross-peak of this residue in the 4472

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Figure 4. Comparison of Cα chemical shifts of cardiotoxins 3 (CT3Na) and 2 (CT2Na) from Naja atra and CTI from N. oxiana (CT1No). The graphs are colored deep blue for CT3Na (data points are diamonds), magenta for CT2Na (squares), and cyan for CT1No (triangles). The amino acid sequences of CTs are shown below the graph in the respective color. Identical amino acid residues are enclosed in boxes. The residues forming the β-sheet structure are indicated by the arrows above with the numbers of starting and finishing residues in each strand. The corresponding residues in the graph are enclosed in transparent gray boxes. The chemical shifts of CT3Na were thought to be correctly referenced (BMRB entry 4966). Constant values were added to chemical shifts of two remaining CTs to minimize deviations in Cα chemical shifts of residues forming βstructure. 13

C−1H HSQC spectrum is hidden by the water signal under the conditions used for structural calculation. The well-resolved cross-peaks of Val7 and Ile9 residues in 15N−1H HSQC spectra of the “minor” form gave enough restraints to allow structural reconstruction. In the calculated set of models, the distance between α-protons of Val7 and Pro8 residues does not exceed 2.5 Å, a typical feature of the cis configuration of this bond.38 Thus, both chemical shift and NOE data agree that the difference between the two forms of rCTI is due to slow (on the NMR time scale, ∼s−1) cis−trans isomerism of the Val7− Pro8 peptide bond. A similar conformational equilibrium exists in an aqueous solution of CTII from N. oxiana.38 Molecular Dynamics: nCTI versus rCTI. Until now, only the dynamic behavior of functionally different three-finger proteins, cardiotoxins and short-chain neurotoxins, had been explored.40 In this work, for the first time we undertake atomistic MD simulations of two CTs differing by a single amino acid residue. We studied the effect of the starting structure on the MD simulations (Figure 3a). First, before we obtained a high-quality structure of rCTI, we used a surrogate model that was built by a simple attachment of a Met residue to the N-terminus of a model taken from the NMR ensemble of nCTI (PDB entry 5NPN). In this model, the dihedral angles of Leu1 and subsequent residues are identical to those in the native cytotoxin (model 5NPN). This surrogate model was stable for ∼750 ns of the MD trajectory. However, a gradual decrease in the population of several hydrogen bonds in the neighborhood of the Met0 residue (Cys14 NH···CO Leu1, Lys18 NH···CO Pro15, and Arg58 NH···CO Lys2) was noted. After all of the hydrogen bonds mentioned above had been lost, the structure collapsed (our unpublished observations). Apparently, the Met0 residue should be appropriately

accommodated within the structure. According to NMR data (Figure 2b), this is achieved via modification of dihedral angles of the Leu1 residue. Thus, attachment of the N-Met residue to the CTI molecule has an only local effect. MD trajectories that started from the NMR model of nCTI and the NMR model or Rosetta-optimized model of rCTI revealed the structural stability of the proteins over the course of the simulations. The deviations of the backbone geometry from the starting one [root-mean-square deviation (RMSD) over the backbone atoms] amounted to 1.2 ± 0.1, 1.3 ± 0.1, and 1.1 ± 0.1 Å, respectively. These values for the stable part of the MD trajectory of the surrogate model amounted to 1.5 ± 0.2 Å. The mobility of the toxins’ backbone expressed in terms of the root-mean-square fluctuation (RMSF) over backbone atoms of each residue is presented in Figure 3a. It is clear that the residues located in the spatial proximity of Met0 in rCTI (15−17 and 55−57) demonstrate higher RMSFs in all trajectories of rCTI, compared with their characteristics in nCTI. Thus, addition of the Met0 residue to nCTI is a perturbing mutation, causing local structural distortion and an increased number of local fluctuations within residues, which are spatially close to the added Met0 residue (15−17 and 55− 57). This resembles the situation for the recombinant toxin WTX (rWTX), also belonging to the family of three-finger folded proteins and containing an additional Met0 residue.3 The minor changes in the backbone conformation apparently caused by the introduction of the N-terminal Met are observed in the three regions of rWTX: Lys13−Glu21, Cys42−Cys46, and Cys63−Arg65. These fragments belong to the toxin’s “head” and C-terminal tail and are spatially close to strand β1.3 The residues, which are well apart of the Met0 residue in rCTI, exhibit similar structural dynamic properties, as in nCTI. Let us illustrate this with the tip of loop II (residues 27−34). In 4473

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Figure 5. Typical structure of loop II of CTs and related proteins, together with a tightly bound water molecule. (a) True cardiotoxins with an Ωshape of loop II. (b) Atypical Π-shape of loop II of a CT. (c) Shape of loop II of a CLBP (cardiotoxin-like basic protein). CT’s name, snake species name, and PDB code of the structure are shown in parentheses at the bottom of each panel. The NH groups, CO groups, backbone, hydrogen bonds, and water molecule are colored blue, red, green, yellow, and gray, respectively. Residues whose atoms form hydrogen bonds with the water molecule are labeled.

both nCTI and rCTI, it adopts an Ω-shape (Figure 2b) and combines into a single hydrophobic motif the tips of the neighboring loops I and III. There is a cavity within the tip of this loop, where a water molecule can enter and exit.38,41 This is observed in MD calculations of both nCTI and rCTI (Figure 3b). The lifetimes of a water molecule within this site fall in the range of 10−200 ns for both toxins. Such water molecules form three hydrogen bonds: with the NH proton of the Met26 residue and with the CO groups of the Asp29 and Ile32 residues. A similar shape of the tip of loop II and the characteristics of water exchange within the site suggest intactness of this region in the rCTI molecule, compared to that in nCTI. The tip of loop II together with those of loops I and III forms the membrane binding motif of cardiotoxins.15 The shapes of the loops and their tips remain generally similar for rCTI and nCTI along the respective MD trajectories (Figure 3a). Thus, the activities of the toxins, arising because of their membrane partitioning, should also be similar for recombinant and native analogues. This has already been proven for the cytotoxic activity of rCTI and nCTI against rat C6 glioma cells.11 Because of the conservation of the spatial organization of CTs, we assume that this conclusion is valid for all of them, and hence, the recombinant CTs can be broadly used for pharmacological and other needs. Heteronuclear Chemical Shifts of nCTI. Comparison of 13 C−1H and 15N−1H HSQC spectra (acquired at the natural abundance of the nuclei for the native toxin) of the native and recombinant toxins showed that the chemical shifts for both toxins are very similar, except for those of the few residues at the N-terminus. After the three-dimensional NMR spectra acquired for recombinant [U-13C,15N]CTI had been assigned and 1H spectral assignments for native CTI (BMRB entry 5989) had been taken into account, it was rather straightforward to transfer assignments from the recombinant toxin to the native one. 13C chemical shifts of carbonyl groups were omitted from this analysis. The resulting heteronuclear chemical shifts of nCTI were deposited as BMRB entry 27086. It is interesting to compare the chemical shifts of nCTI with those of other cardiotoxins (Figure 4). Available in the literature are Cα chemical shifts of a pair of CTs, cardiotoxin 3 (CT3Na)41 and cardiotoxin 2 (CT2Na)42 from Naja atra (Figure 4). Cα chemical shifts of the residues forming the β-sheet differ between these CTs minimally, because these residues are conservative and assume the same type of secondary structure. The exceptions to this rule are

likely due to improper assignments (Leu20 and Tyr51 for CT2Na and Ile39 for CT3Na). One can also see that the differences between the Cα chemical shifts of the non-β-sheet residues are mainly caused by the amino acid type. These residues (9, 10, 16, 27−32, and 45−47) are located in more flexible loop regions of CTs.7 This conforms to a view that all CTs feature a similar spatial organization, whose three-finger scaffold is determined by β-sheet-forming residues with others forming more flexible loop moieties.7 Spatial Organization of CTs. Analysis of the available spatial models of CTs shows that these molecules exhibit differences in the spatial organization of the tips of loops I and II, i.e., residues 5−10 and 27−35.7 These data have been obtained by X-ray crystallography. With regard to the tip of loop I, all CTs are divided into those containing a pair of Pro residues in loop I (group I) and a single Pro residue within this loop (group II).43 Structural NMR data are available mainly for CTs of group II. With regard to group I, the NMR data remain elusive, because NMR spectra of the respective CTs in aqueous solution lack assignments of a number of proton signals43,44 and the respective NMR models suffer from poor quality.45 Today, the problem persists, and no exhaustive NMR data have yet been obtained for group I of CTs to compare them with the respective X-ray data. The situation differs for the tip of loop II. A majority of the spatial structures of CTs determined to date exhibit a similar shape of loop II (Figure 5a). It is noteworthy that one of them, N. atra CT A1, has the same amino acid sequence in loop II (residues 24−34) that CTI does. The spatial structures of CTI and A1 are quite similar, despite the fact that the structure of A1 has been determined without taking into account the bound water molecule.46 As a result, no water forming the same three hydrogen bonds can be placed in loop II without introducing substantial clashes. Protein sequence similarity and the common Ω-shape of the loops of these two toxins suggest that a tightly bound water molecule should be located here. All available structures of the P-type CTs feature the Ω-shape loop II (Figure 5a). The presence of a tightly bound water molecule in the major and minor forms of N. oxiana CT II was proven by NMR NOESY/ROESY experiments in aqueous solution38 and in dodecylphosphocholine (DPC) micelles.47 The reported structures were calculated by taking into account only the hydrogen bond with HN Met26. However, two additional hydrogen bonds with the CO Ala29 and CO Val32 atoms are also likely present. The toxin molecule in the DPC micelle is 4474

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Biochemistry

perturbation with the tips of the loops; however, a stronger attenuation of the activity is expected, when more complicated activity mechanisms, requiring overall stability and rigidity of the molecule, are implicated.

perturbed by the micellar environment, and no hydrogen bond with the CO Ala29 atom can be formed because the distance from water protons to the CO atom exceeds 4 Å. Another hydrogen bond with the CO Met26 atom is consistent with the protein structure. A single structure of an S-type CT VII4 from N. mossambicca, determined so far by X-ray, deserves a special comment48 (Figure 5b). The toxin molecule in the both asymmetric subunits does not reveal a Ω-shape of the tip of loop II. This region is more extended and sharp, and we will refer to it as a Π-shape. Several water molecules are found in the vicinity of this loop. However, they do not exhibit any hydrogen bonds with the protein. The HN Leu2···CO Val32 H-bond is formed without participation of any water molecule, being found between these atoms. Therefore, the β-sheet extends to these residues. The CO Lys29 atom is too far from residues 26 and 32 to form H-bonds with water molecules. Some authors classify toxin A5 from N. atra as a cardiotoxin49 (Figure 5c). This toxin possesses an additional residue in loop II and thus features a specific spatial organization. Loop II incorporates either a single or a pair of bound water molecules in the two asymmetric crystal subunits. The single water molecule is held by the three H-bonds with atoms NH Leu27, CO Leu32, and CO Phe34. The presence of an additional residue within loop II and the special shape of the latter argue that A5 should be not classified as a cardiotoxin, but a cardiotoxin-like basic polypeptide (CLBP).50,51 Our further efforts were directed at elucidating the conformational abilities of the loop II structure of CTs. Taking N. oxiana CTI and N. mossambicca CT VII4, we performed their long full-atom MD simulations in aqueous solution to elucidate possible interconversions in loop II. MD Simulation of N. mossambicca CT VII4. This toxin features a Π-shape of the tip of loop II (Figure 5b). During the 1 μs long trajectory, we observed that this conformation of loop II is very stable. Only a few events in which the water molecule penetrated this loop were detected. However, in this case, the conformation of the tip of the loop was different from one to which an Ω-shape was ascribed (Figure 5a) and observed during the MD simulation of CTI (see the preceding section). We conclude that this structure contradicts NMR data, supporting the view that a long-living water molecule resides within the tip of this loop. Thus, care should be taken to use this model as a template for calculation of spatial models of other CTs. The spatial model of this CT molecule (1CDT) was probably obtained under conditions that distorted the shape of the tip of loop II.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.7b00453. Four figures illustrating chemical shift differences between major and minor forms of [U-13C,15N]rCTI, superimposed 13C−1H and 15N−1H HSQC spectra of [U-13C,15N]rCTI and nCTI, chemical shift differences for major forms of these toxins, an illustration of sequential resonance assignments with the HNCA spectrum of [U-13C,15N]rCTI, and a table summarizing the restraints for the structural calculation of both forms of rCTI and their structural characteristics (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, 16/10 Miklukho-Maklaya str., 117997 Moscow, Russia. Telephone: +7-330-56-83. Fax: +7-495-335-50-33. E-mail: peter@ nmr.ru. ORCID

Peter V. Dubovskii: 0000-0003-2289-6023 Funding

This work was supported by the Russian Foundation for Basic Research (Grants 13-04-02128, 16-04-01479, and 16-0400578) and by the RAS Program “Molecular and Cellular Biology”. Access to computational facilities of the Supercomputer Center “Polytechnical” at the St. Petersburg Polytechnic University is appreciated. R.G.E. is grateful to Russian Academic Excellence Project ‘5-100’ for support. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS CTs, cytotoxins; CTI, cytotoxin I from N. oxiana; rCTI, recombinant CTI; nCTI, native CTI, isolated from the venom of N. oxiana; H-bond, hydrogen bond.

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REFERENCES

(1) Kumar, T. K., Yang, P. W., Lin, S. H., Wu, C. Y., Lei, B., Lo, S. J., Tu, S. C., and Yu, C. (1996) Cloning, direct expression, and purification of a snake venom cardiotoxin in Escherichia coli. Biochem. Biophys. Res. Commun. 219, 450−456. (2) Gorbatyuk, V. Y., Tsai, C. K., Chang, C. F., and Huang, T. H. (2004) Effect of N-terminal and Met23 mutations on the structure and dynamics of onconase. J. Biol. Chem. 279, 5772−5780. (3) Lyukmanova, E. N., Shenkarev, Z. O., Shulepko, M. A., Paramonov, A. S., Chugunov, A. O., Janickova, H., Dolejsi, E., Dolezal, V., Utkin, Y. N., Tsetlin, V. I., Arseniev, A. S., Efremov, R. G., Dolgikh, D. A., and Kirpichnikov, M. P. (2015) Structural Insight into Specificity of Interactions between Nonconventional Three-finger Weak Toxin from Naja kaouthia (WTX) and Muscarinic Acetylcholine Receptors. J. Biol. Chem. 290, 23616−23630. (4) Bernheimer, A. W., and Rudy, B. (1986) Interactions between membranes and cytolytic peptides. Biochim. Biophys. Acta, Rev. Biomembr. 864, 123−141.

CONCLUSIONS Taking all the above data into account, we conclude that the recombinant CTI possesses all features of the spatial organization and dynamic behavior of the native cardiotoxins. (1) Loop dynamics is identical for both proteins. The loop I features slow interconversion between cis and trans isomers of Val7−Pro8 peptide bonds. Loop II manifests an overall Ωshape because of the presence of a cavity, where exchange of water molecules takes place. (2) rCTI exhibits higher flexibility in the fragments, which are spatially close to the added N-Met side chain (residues 15− 17 and 55−57). Apparently, this attenuates the overall rigidity and stability of rCTI, compared to those of the native protein. (3) The functional activity of rCTI seems to be close to that of nCTI, if the activity mechanism is based on membrane 4475

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Article

Biochemistry (5) Dubovskii, P. V., Vassilevski, A. A., Kozlov, S. A., Feofanov, A. V., Grishin, E. V., and Efremov, R. G. (2015) Latarcins: versatile spider venom peptides. Cell. Mol. Life Sci. 72, 4501−4522. (6) Moreno, M., and Giralt, E. (2015) Three valuable peptides from bee and wasp venoms for therapeutic and biotechnological use: melittin, apamin and mastoparan. Toxins 7, 1126−1150. (7) Konshina, A. G., Dubovskii, P. V., and Efremov, R. G. (2012) Structure and dynamics of cardiotoxins. Curr. Protein Pept. Sci. 13, 570−584. (8) Gasanov, S. E., Dagda, R. K., and Rael, E. D. (2014) Snake Venom Cytotoxins, Phospholipase As, and Zn-dependent Metalloproteinases: Mechanisms of Action and Pharmacological Relevance. J. Clin. Toxicol. 4, 1000181. (9) Dubovskii, P. V., and Utkin, Y. N. (2015) Antiproliferative activity of cobra venom cytotoxins. Curr. Top. Med. Chem. 15, 638− 648. (10) Ebrahim, K., Shirazi, F. H., Mirakabadi, A. Z., and Vatanpour, H. (2015) Cobra venom cytotoxins; apoptotic or necrotic agents? Toxicon 108, 134−140. (11) Shulepko, M. A., Lyukmanova, E. N., Shenkarev, Z. O., Dubovskii, P. V., Astapova, M. V., Feofanov, A. V., Arseniev, A. S., Utkin, Y. N., Kirpichnikov, M. P., and Dolgikh, D. A. (2017) Towards universal approach for bacterial production of three-finger Ly6/uPAR proteins: Case study of cytotoxin I from cobra N. oxiana. Protein Expression Purif. 130, 13−20. (12) Wu, C. Y., Chen, W. C., Ho, C. L., Chen, S. T., and Wang, K. T. (1997) The role of the N-terminal leucine residue in snake venom cardiotoxin II (Naja naja atra). Biochem. Biophys. Res. Commun. 233, 713−716. (13) Dufton, M. J., and Hider, R. C. (1988) Structure and pharmacology of elapid cytotoxins. Pharmacol. Ther. 36, 1−40. (14) Dubovskii, P. V., and Utkin, Y. N. (2014) Cobra cytotoxins: structural organization and antibacterial activity. Acta Naturae 6, 11− 18. (15) Dubovskii, P. V., Konshina, A. G., and Efremov, R. G. (2014) Cobra cardiotoxins: membrane interactions and pharmacological potential. Curr. Med. Chem. 21, 270−287. (16) Wu, P.-L., Chiu, C.-R., Huang, W.-N., and Wu, W.-G. (2012) The role of sulfatide lipid domains in the membrane pore-forming activity of cobra cardiotoxin. Biochim. Biophys. Acta, Biomembr. 1818, 1378−1385. (17) Konshina, A. G., Boldyrev, I. A., Utkin, Y. N., Omel’kov, A. V., and Efremov, R. G. (2011) Snake cytotoxins bind to membranes via interactions with phosphatidylserine head groups of lipids. PLoS One 6, e19064−e19064. (18) Batenburg, A. M., Bougis, P. E., Rochat, H., Verkleij, A. J., and de Kruijff, B. (1985) Penetration of a cardiotoxin into cardiolipin model membranes and its implications on lipid organization. Biochemistry 24, 7101−7110. (19) Gasanov, S. E., Shrivastava, I. H., Israilov, F. S., Kim, A. A., Rylova, K. A., Zhang, B., and Dagda, R. K. (2015) Naja naja oxiana Cobra Venom Cytotoxins CTI and CTII Disrupt Mitochondrial Membrane Integrity: Implications for Basic Three-Fingered Cytotoxins. PLoS One 10, e0129248. (20) Dubovskii, P. V., Lesovoy, D. M., Dubinnyi, M. A., Konshina, A. G., Utkin, Y. N., Efremov, R. G., and Arseniev, A. S. (2005) Interaction of three-finger toxins with phospholipid membranes: comparison of Sand P-type cytotoxins. Biochem. J. 387, 807−815. (21) Dubovskii, P. V., Vorontsova, O. V., Utkin, Y. N., Arseniev, A. S., Efremov, R. G., and Feofanov, A. V. (2015) Cobra cytotoxins: determinants of antibacterial activity. Mendeleev Commun. 25, 70−71. (22) Kukhtina, V. V., Weise, C., Muranova, T. A., Starkov, V. G., Franke, P., Hucho, F., Wnendt, S., Gillen, C., Tsetlin, V. I., and Utkin, Y. N. (2000) Muscarinic toxin-like proteins from cobra venom. Eur. J. Biochem. 267, 6784−6789. (23) Bocharov, E. V., Lyukmanova, E. N., Ermolyuk, Y. S., Schulga, A. A., Pluzhnikov, K. A., Dolgikh, D. A., Kirpichnikov, M. P., and Arseniev, A. S. (2003) Resonance assignment of C-13-N-15-labeled snake neurotoxin II from Naja oxiana. Appl. Magn. Reson. 24, 247−254.

(24) Guntert, P., and Buchner, L. (2015) Combined automated NOE assignment and structure calculation with CYANA. J. Biomol. NMR 62, 453−471. (25) Dubovskii, P. V., Vassilevski, A. A., Slavokhotova, A. A., Odintsova, T. I., Grishin, E. V., Egorov, T. A., and Arseniev, A. S. (2011) Solution structure of a defense peptide from wheat with a 10cysteine motif. Biochem. Biophys. Res. Commun. 411, 14−18. (26) Shen, Y., and Bax, A. (2015) Protein structural information derived from NMR chemical shift with the neural network program TALOS-N. Methods Mol. Biol. 1260, 17−32. (27) Mao, B., Tejero, R., Baker, D., and Montelione, G. T. (2014) Protein NMR structures refined with Rosetta have higher accuracy relative to corresponding X-ray crystal structures. J. Am. Chem. Soc. 136, 1893−1906. (28) Tejero, R., Snyder, D., Mao, B., Aramini, J. M., and Montelione, G. T. (2013) PDBStat: a universal restraint converter and restraint analysis software package for protein NMR. J. Biomol. NMR 56, 337− 351. (29) Berjanskii, M., Liang, Y., Zhou, J., Tang, P., Stothard, P., Zhou, Y., Cruz, J., MacDonell, C., Lin, G., Lu, P., and Wishart, D. S. (2010) PROSESS: a protein structure evaluation suite and server. Nucleic Acids Res. 38, W633−640. (30) Shen, Y., and Bax, A. (2010) Prediction of Xaa-Pro peptide bond conformation from sequence and chemical shifts. J. Biomol. NMR 46, 199−204. (31) Lindorff-Larsen, K., Piana, S., Palmo, K., Maragakis, P., Klepeis, J. L., Dror, R. O., and Shaw, D. E. (2010) Improved side-chain torsion potentials for the Amber ff99SB protein force field. Proteins: Struct., Funct., Genet. 78, 1950−1958. (32) Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W., and Klein, M. L. (1983) Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926−935. (33) Darden, T., York, D., and Pedersen, L. (1993) Particle mesh Ewald: An W log(N) method for Ewald sums in large systems. J. Chem. Phys. 98, 10089−10092. (34) Bussi, G., Donadio, D., and Parrinello, M. (2007) Canonical sampling through velocity rescaling. J. Chem. Phys. 126, 014101. (35) Berendsen, H. J. C., Postma, J. P. M., van Gunsteren, W. F., DiNola, A., and Haak, J. R. (1984) Molecular dynamics with coupling to an external bath. J. Chem. Phys. 81, 3684−3690. (36) Pronk, S., Pall, S., Schulz, R., Larsson, P., Bjelkmar, P., Apostolov, R., Shirts, M. R., Smith, J. C., Kasson, P. M., van der Spoel, D., Hess, B., and Lindahl, E. (2013) GROMACS 4.5: a highthroughput and highly parallel open source molecular simulation toolkit. Bioinformatics 29, 845−854. (37) Dubovskii, P. V., Dubinny, M. A., Volynsky, P. E., Pustovalova, Y. E., Konshina, A. G., Utkin, Y. N., Efremov, R. G., and Arseniev, A. S. (2017) Impact of Membrane Partitioning on the Spatial Structure of an S-type Cobra Cytotoxin; in preparation. (38) Dementieva, D. V., Bocharov, E. V., and Arseniev, A. S. (1999) Two forms of cytotoxin II (cardiotoxin) from Naja naja oxiana in aqueous solution: spatial structures with tightly bound water molecules. Eur. J. Biochem. 263, 152−162. (39) Berjanskii, M. V., and Wishart, D. S. (2008) Application of the random coil index to studying protein flexibility. J. Biomol. NMR 40, 31−48. (40) Gorai, B., and Sivaraman, T. (2013) Unfolding stabilities of two paralogous proteins from Naja naja naja (Indian cobra) as probed by molecular dynamics simulations. Toxicon 72, 11−22. (41) Sue, S. C., Jarrell, H. C., Brisson, J. R., and Wu, W. G. (2001) Dynamic characterization of the water binding loop in the P-type cardiotoxin: implication for the role of the bound water molecule. Biochemistry 40, 12782−12794. (42) Lee, C. S., Kumar, T. K. S., Lian, L. Y., Cheng, J. W., and Yu, C. (1998) Main-chain dynamics of cardiotoxin II from Taiwan cobra (Naja naja atra) as studied by carbon-13 NMR at natural abundance: Delineation of the role of functionally important residues. Biochemistry 37, 155−164. 4476

DOI: 10.1021/acs.biochem.7b00453 Biochemistry 2017, 56, 4468−4477

Article

Biochemistry (43) Chen, T.-S., Chung, F.-Y., Tjong, S.-C., Goh, K.-S., Huang, W.N., Chien, K.-Y., Wu, P.-L., Lin, H.-C., Chen, C.-J., and Wu, W.-G. (2005) Structural difference between group I and group II cobra cardiotoxins: X-ray, NMR, and CD analysis of the effect of cis-proline conformation on three-fingered toxins. Biochemistry 44, 7414−7426. (44) Otting, G., Steinmetz, W. E., Bougis, P. E., Rochat, H., and Wuthrich, K. (1987) Sequence-specific 1H-NMR assignments and determination of the secondary structure in aqueous solution of the cardiotoxins CTXIIa and CTXIIb from Naja mossambica mossambica. Eur. J. Biochem. 168, 609−620. (45) O’Connell, J. F., Bougis, P. E., and Wuthrich, K. (1993) Determination of the nuclear-magnetic-resonance solution structure of cardiotoxin CTX IIb from Naja mossambica mossambica. Eur. J. Biochem. 213, 891−900. (46) Jahnke, W., Mierke, D. F., Beress, L., and Kessler, H. (1994) Structure of cobra cardiotoxin CTX I as derived from nuclear magnetic resonance spectroscopy and distance geometry calculations. J. Mol. Biol. 240, 445−458. (47) Dubovskii, P. V., Dementieva, D. V., Bocharov, E. V., Utkin, Y. N., and Arseniev, A. S. (2001) Membrane binding motif of the P-type cardiotoxin. J. Mol. Biol. 305, 137−149. (48) Rees, B., Bilwes, A., Samama, J. P., and Moras, D. (1990) Cardiotoxin VII4 from Naja mossambica mossambica. The refined crystal structure. J. Mol. Biol. 214, 281−297. (49) Sun, Y. J., Wu, W. G., Chiang, C. M., Hsin, A. Y., and Hsiao, C. D. (1997) Crystal structure of cardiotoxin V from Taiwan cobra venom: pH-dependent conformational change and a novel membranebinding motif identified in the three-finger loops of P-type cardiotoxin. Biochemistry 36, 2403−2413. (50) Sivaraman, T., Kumar, T. K., Yang, P. W., and Yu, C. (1997) Cardiotoxin-like basic protein (CLBP) from Naja naja atra is not a cardiotoxin. Toxicon 35, 1367−1371. (51) Kawaguchi, Y., Tatematsu, Y., Tabata, A., Nagamune, H., and Ohkura, K. (2015) Cytolytic Activity and Molecular Feature of Cardiotoxin and Cardiotoxin-like Basic Protein: The Electrostatic Potential Field Is an Important Factor for Cell Lytic Activity. Anticancer Res. 35, 4515−4519. (52) Berjanskii, M. V., and Wishart, D. S. (2005) A simple method to predict protein flexibility using secondary chemical shifts. J. Am. Chem. Soc. 127, 14970−14971.

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