Helical Structure of Recombinant Melittin - The Journal of Physical

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Helical Structure of Recombinant Melittin Lisa S. Ramirez, Jayanti Pande, and Alexander Shekhtman J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b08424 • Publication Date (Web): 20 Dec 2018 Downloaded from http://pubs.acs.org on December 23, 2018

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The Journal of Physical Chemistry

1

Helical Structure of Recombinant Melittin

Lisa S. Ramirez,1 Jayanti Pande,1* and Alexander Shekhtman1,* 1Department

of Chemistry, State University of New York at Albany, NY 12222, USA

*Correspondence: [email protected] or [email protected]

ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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2 Abstract Melittin is an extensively studied, 26-residue toxic peptide from honey-bee venom. Because of its versatility in adopting a variety of secondary (helix or coil), and quaternary (monomer or tetramer) structures in various environments, melittin has been the focus of numerous investigations as a model peptide in protein folding studies, as well as in studies involving binding to proteins, lipids and polysaccharides. A significant body of evidence supports the view that melittin binds to these macromolecules in a predominantly helical conformation, but detailed structural knowledge of this conformation is lacking. In this report, we present nuclear magnetic resonance (NMR)-based structural insights into the helix formation of recombinant melittin in the presence of TFE - a known secondary structure inducer in peptides. These studies were performed at neutral pH, with micromolar amounts of the peptide. Using Nuclear Overhauser effect (NOE)-derived distance restraints from 3D NMR spectra, we determined the atomic resolution NMR solution structure of recombinant melittin bearing a TFE-stabilized helix. To circumvent the complications with structure determination of small peptides with high conformational flexibility, we developed a workflow for enhancing proton NOEs by increasing the viscosity of the medium. In the TFEcontaining medium, recombinant monomeric melittin forms a long, continuous helical structure, which consists of the N-and C-terminal α-helices and the non-canonical 310-helix in the middle. The non-canonical 310-helix is missing in the previously solved X-ray structure of tetrameric melittin and the NMR structure of melittin in methanol. Melittin's structure in TFE containing medium provides insights into melittin’s conformational transitions, which are relevant to the peptide’s interactions with its biological targets.


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3 Introduction The toxic peptide melittin is the primary component of honey bee venom.1 The hemolytic activity and antimicrobial properties of melittin have been extensively studied in the last few decades.2-4 Melittin has also been examined as a model peptide for studies on protein folding, in part due to its structural plasticity.5,

6

Depending on solution conditions, melittin may form a

monomer or a tetramer, and may be helical or random coil.6-9 Melittin is known to bind to membranes, polysaccharides, and proteins. The structure of melittin bound to these macromolecules has been found to be predominantly helical.10-13 Among proteins, melittin has been shown to bind to calmodulin14, staphylokinase,15 centrin,12,

16

and

crystallin.17, 18 The work of Farahbakhsh et al18 suggests that melittin may assume helical or βstrand secondary structures while bound to crystallin, although the authors note that melittin does not reside in a hydrophobic cavity on crystallin as it does in membranes. In view of the observations of Sharma et al17 that the substrate mimic, melittin, binds to a functionally important region on crystallin, the possible involvement of the helical form of the peptide in such binding assumes greater significance. Previous studies have described the folding/unfolding of melittin in various media, notably TFE-water mixtures.5, 19, 20 TFE is known to stabilize secondary structures, such as α-helices and β-sheets, in proteins and peptides.21-26 It is thought that TFE induces α-helical structure through a variety of mechanisms, including promotion of intramolecular hydrogen bonding, perturbation of hydrophobic interactions, and molecular crowding.21-26

Studies on TFE-induced structural

changes on melittin carried out by molecular dynamics (MD) simulations,22 circular dichroism (CD),19 fluorescence spectroscopy,5 and NMR20, 27 spectroscopy suggest that TFE stabilizes the



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4 helical form of melittin. However, even with the substantial effort directed towards describing TFE-melittin associations, previous studies have not produced a Protein Data Bank (PDB) entry for melittin’s three-dimensional (3D) structure solved in the presence of the TFE co-solvent. In addition to X-ray crystal structures of tetrameric melittin (PDB ID: 2MLT) and centrin-bound melittin (PDB ID: 3QRX), only two solution NMR, 3D structures of melittin are currently available in the PDB: the structure of monomeric melittin (PDB ID: 2MW6) labeled with a ruthenium-containing organometallic fragment,28 and the solution NMR structure of D-Pro14melittin (PDB ID: 1BH1).29 As mentioned earlier, melittin’s structure is generally dependent on solution conditions.6-9 In particular, structural changes on melittin have been observed by varying pH, ionic strength, temperature, and concentration.6,

7

In deciding the solution conditions used for elucidating

melittin’s 3D structure, we took into consideration the fact that most structural studies on melittin have focused on ‘high’ melittin concentrations, spanning the millimolar range ~1 mM - 40mM, 2, 7

whereas even very ‘low’, micromolar concentrations of melittin were found to be toxic.3, 30-33

Here we have determined the atomic resolution NMR structure of melittin at micromolar concentrations which are consistent with bioassays on melittin-induced hemolysis and inhibition of bacterial growth. 3, 30-33 Acquisition of high-quality 3D NMR spectra for micromolar amounts of melittin in this work is facilitated by the use of uniformly isotope-labeled (13C- and

15N-)

recombinant melittin.27 In general, the NMR-based solution structure determination of small peptides is challenging due to their high conformational flexibility and fast tumbling in solution.34-36 For a small peptide, the interconversion among various conformers is usually fast on the NMR time scale, and this may result in NMR observables, such as chemical shifts and NOE cross peaks,


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5 which only provide information on the ‘average’ structure of multiple conformers.34-36 Another common problem is weak NOEs due to unfavorable values of the rotational correlation time, τc.34, 37, 38

Moreover, using low (micromolar) peptide concentrations exacerbates the problem of weak

NOEs. Altogether, these difficulties hamper NMR solution structure calculations, which require an abundance of NOE-derived distance restraints. Therefore, in the present study, we employed a systematic approach to enhance the nuclear Overhauser effect of melittin by adjusting the viscosity of the solvent. The restraints derived from the NOEs were used to solve the 3D solution NMR structure of recombinant melittin in its TFE-stabilized helical form (PDB ID: 6DST), which serves as the only solution NMR structure in the PDB for monomeric melittin without organometallic labels and without modification of the Pro14 residue.

Methods Preparation of NMR Samples Deuterated glycerol (D8, 99%), 15N-ammonium chloride (15N, 99%), and 13C-glucose (U-13C6, 99%) were purchased from Cambridge Isotope Laboratories. Deuterated 2,2,2-trifluoroethanol (D3, 99.5%) was purchased from Sigma-Aldrich. Unless otherwise stated, all NMR samples were prepared with deuterated glycerol and deuterated TFE. Recombinant [U-13C,15N]-melittin was expressed and purified using a previously published protocol.27 Briefly, Escherichia coli (E. coli) strain C41(DE3) was transformed with a plasmid encoding a fusion protein engineered with a poly-histidine tag at its N-terminus followed by methionine and the melittin sequence (GIGAVLKVLTTGLPALISWIKRKRQQ). Colonies expressing the melittin-containing fusion protein were cultured and grown in M9 medium containing

15NH

4Cl

and [U-13C]-glucose to produce [U-13C,15N]-labeled fusion protein.



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6 Overexpression was induced using 1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG). Following expression, cells were pelleted and lysed. The fusion protein was purified by nickel affinity chromatography under denaturing conditions. The fusion protein was cleaved using cyanogen bromide to release the recombinant melittin fragment, and the melittin peptide was purified by reversed phase high performance liquid chromatography (HPLC). The mass of purified recombinant melittin determined by electrospray ionization mass spectrometry (ESI-MS) was 2847 Da without uniform labeling and 3000 Da with uniform 13C- and 15N-labeling. Recombinant melittin of at least 95% purity was lyophilized and stored at -80 oC until it was used in NMR experiments. Screening of Optimal Conditions for Acquiring NOESY Spectra NMR spectra were acquired using Bruker Ascend 600 MHz or Avance II 700 MHz NMR spectrometers equipped with ultrasensitive cryoprobes. Standard triple resonance experiments (HNCA, HNCOCA, HNCACB), 3D 1H-15N nuclear Overhauser effect spectroscopy (NOESY), 3D 1H-13C NOESY, 2D 1H-15N heteronuclear single quantum coherence (HSQC), 2D 1H-13C HSQC, 2D 1H-1H NOESY, and 2D 1H-1H rotating frame Overhauser effect spectroscopy (ROESY) were acquired with the WATERGATE pulse sequence for water suppression.39 The NOESY mixing time was 200 ms for 3D 1H-13C NOESY, 3D 1H-15N NOESY, 2D 1H-1H NOESY and 2D 1H-1H ROESY. NOESY experiments for generating distance restraints were recorded at 285 K. Data were processed with TOPSPIN 3.1 and analyzed using CARA.40 In a typical 3D NMR experiment, 50 μM [U-13C,15N]- melittin was dissolved in the NMR buffer, 10 mM potassium phosphate buffer at pH 7.0 with 10% v/v D2O. Immediately after dissolving melittin, 30% v/v deuterated TFE was added to the sample, followed by either 0% or 10% w/v deuterated glycerol. Freshly reconstituted recombinant melittin was used for all 3D NMR


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7 experiments, so that only the trans-Pro14 isomer of melittin was observed in the NMR spectra. In a previous report, we determined that the cis-trans isomerization of the proline residue in recombinant melittin occurs slowly in the NMR buffer without TFE at 298 K, and the population of the cis isomer was virtually absent in a 1-day old solution of recombinant melittin, but gradually increased to ~20-27% of the total melittin population after two weeks of storage in the NMR buffer.27 For 2D 1H-1H NOESY and 1H-1H ROESY experiments, freshly reconstituted synthetic melittin (Genscript, Piscataway NJ) was used. Melittin concentration was determined at 280 nm using a molar extinction coefficient of 5,500 M-1cm-1. Homonuclear NMR experiments were also carried out on ‘blank’ samples at 285 K which contained 30% v/v TFE in the NMR buffer, and a combination of 30% v/v TFE and 10% w/v glycerol in NMR buffer to determine spectral artifacts due to incompletely deuterated TFE and glycerol. To evaluate the effect of decreasing temperature and increasing viscosity on the transverse relaxation rate (R2), one-dimensional jump-return Hahn echo experiments were performed as described previously.41 The pulse sequence included gradient purge pulses for the efficient water suppression. The signal intensities of the amide 1HN envelope from spectra of melittin recorded at two echo delays,   ms and   6.1 ms, were measured. The ratio of signal intensities in the two spectra, I2/I1, is related to R2 in the following equation:42 R2 = {-1/[2(ln(I2/I1)

(1)

The reciprocal of R2 is equal to T2, the transverse relaxation time constant.42 Chemical shift perturbations (CSPs) were calculated using a previously described method.27 CSPs are the weighted average chemical shift deviations for each backbone 1H/15N pair in the 1H15N

HSQC spectra of melittin in the low-viscosity condition (melittin at 298 K without glycerol)

and the high-viscosity condition (melittin at 285 K with 10% w/v glycerol). Chemical shift index



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8 (CSI) analysis43 version 3.0 was used to calculate the helical probability scores (abbreviated Hprob) of melittin residues in the high-viscosity and low-viscosity conditions. Assignments of backbone 15N, 13C, and 1H were uploaded to the CSI 3.0 web server at http://csi3.wishartlab.com. Backbone assignments of melittin in the low-viscosity condition were taken from our previous report,27 while those for melittin in the high-viscosity condition were taken from BMRB entry 30481. Resonance assignment of recombinant melittin in a TFE/water/glycerol environment The extent of assignment is 96% for the peptide backbone (N, HN, Cα, Hα, and Cβ) and 95% for the amino acid side chains (Biological Magnetic Resonance Bank, BMRB accession number: 30481). Sequence-specific resonance assignments for the

15N, 13C,

and 1H nuclei of the

polypeptide backbone of [U-13C,15N]-melittin were obtained through analysis of 3D NMR spectra recorded at 298 K, as detailed in a previous report.27 Side chain assignments were completed by recording HCCH-Total Correlation SpectroscopY, TOCSY spectra at 298 K and in the presence of 10% w/v glycerol.42 All assignments made at 298 K were transferred to the corresponding NOESY spectra recorded at 285 K (Figure S3). 1H-13C HSQC spectra of the [U-13C,15N]-melittin sample with 10% w/v glycerol recorded at various temperatures (298 K, 295 K, 292 K, 290 K, and 285 K) were used as guides in transferring side chain assignments at 298K to spectra recorded at 285K. Notably, for the P14 residue, the 13Cβ chemical shift is ~31 ppm, corresponding to the trans conformation of the L13-P14 peptide bond.44 Structure Calculations CYANA45 version 3.98 was used to calculate the 3D structure of melittin in a TFE/water/glycerol environment. The input consisted of a chemical shift list obtained from the


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9 resonance assignment, a 3D 1H-13C NOESY (optimized for the aliphatic 13C region), and a 3D 1H15N

NOESY. The NOE peaks were assigned automatically and converted into distance constraints using the

standard CYANA protocol with seven cycles of NOE assignment and simulated annealing in torsion angle space. The CISPROCHECK routine of CYANA 3.98 classified the P14 conformation as trans, based on the average value and standard deviations for the difference between the chemical shifts of Cβ and Cγ.46 Backbone Φ and Ψ dihedral angle constraints were determined using TALOS+47 version 3.80. These constraints were used as input data in each cycle of the structure calculation. To determine the optimal geometry of W19, a 2D 1H-1H NOESY spectrum was recorded in D2O solvent. The lack of inter-residue NOEs involving aromatic protons was used to exclude structures showing W19 aromatic protons in close proximity (

Helical Structure of Recombinant Melittin - The Journal of Physical

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