Design of a stapled peptide that binds to the Ebola virus matrix protein dimer interface

Abstract

The Ebola virus (EBOV) is a filamentous lipid-enveloped RNA virus that can cause viral hemmorhagic fever and has a high fatility rate. EBOV encodes seven genes including the lipid-binding matrix protein, VP40, which lies beneath the lipid-envelope. VP40 is a 326 amino acid protein with a N-terminal domain (NTD) harboring a high affinity dimer interface and a C-terminal domain (CTD) critical to plasma membrane lipid interactions. Disruption of VP40 dimer formation via mutagenesis inhibits assembly and budding of VP40. A series of conformationally constrained mimics of the VP40 α2 helix were designed based on the crystal structures of the VP40 dimer. A thermal shift assay was used to screen constrained and native peptides for significant alterations in VP40 stability. The most meritorious peptides were then confirmed to directly bind VP40 using microscale thermophoresis and isothermal titration calorimetry. A constrained VP40 peptide mimetic with a di-cysteine staple emerged with micromolar affinity for the VP40 dimer. This peptide was able to shift the VP40 dimer-monomer equilibrium as evidenced by size exclusion chromatography and bound near the NTD α-helix dimer interface. This study provides the first evidence of a designed small molecule induced disruption of VP40 dimer-monomer equilibrium.

Fig. 1. (A) Helical wheel diagram of VP40_106–120 derived from the X-ray crystal structure of the VP40 dimer (PDB 4LDB). (B) Non-interface residue sidechains (green) in the structure of VP40_106–120 bound to the opposing dimer protomer. (C) VP40_106–120 lead sequence and designed linear di-Cys substrates for stapling. (D) Diversity-oriented stapling with 4 dibromide electrophiles.

Fig. 2. A library of compounds was screened based on changes in melting profile of the VP40 dimer. (A) Ebolavirus VP40 dimer was purified and subjected to thermal melting with absorbance measured at 330 nm and 350 nm. The ratio of absorbance (350 nm/330 nm) was plotted for every degree change in temperature giving sigmoidal melting profile for VP40 with two inflection temperatures (Ti₁ – 55 °C, Ti₂ – 70 °C). (B) The library of compounds was individually assessed for altering the VP40 melting profile by addition of individual peptides to VP40 dimer. Inflection temperature 1 (C) and inflection temperature 2 (D) of VP40 with each of the peptides was compared to vehicle control DMSO. Statistics represent one way ANOVA with multiple comparisons to DMSO ****p < 0.0001. Each experiment was repeated three times.

Fig. 3. Circular dichroic (CD) spectra of select peptides. (A) Selected macrocyclic variants (1b, 2b, and 3b) with a p-xyl staple and the WT peptide (4) were used to collect CD spectra in 1 : 1 MeCN : H₂O. (B) Different CD spectra were detected for peptides of the same sequence with different stapling chemistry (3a–3d).

Fig. 4. Peptide 3b destabilizes both wild type and di-cysteine mutant of VP40 as thermal titration of 3b shows similar results for wild type and di-cysteine double mutant of VP40. (A) Peptide 3b was titrated with wild type VP40 and melting profiles for each concentration is plotted. (B) The inflection temperature 1 (Ti₁) and inflection temperature 2 (Ti₂) of wild type VP40 with each concentration is shown by bar graphs. (C) Peptide 3b was titrated with C311A/314A dimer and melting profiles of di-cysteine double mutant are plotted. (D) The inflection temperature 1 (Ti₁) and inflection temperature 2 (Ti₂) of C311A/314A with each concentration of peptide is shown by bar graphs. Each experiment was repeated three times.

Fig. 5. Peptide 3b binds VP40 dimer in ITC studies. (A) Peptide with xylene linker (3b) was titrated into VP40 dimer for 20 injections and heat readings for each injection was plotted. Heat of titration indicates binding and saturation of peptide. Heat of dilution of peptide was calculated by titrating equivalent amount of DMSO with VP40 dimer over 20 injections. (B) Enthalpy of binding of peptide (3b) was calculated by subtracting enthalpy of DMSO from enthalpy of peptide and plotted with mole ratio of peptide to the dimer. Each experiment was repeated three times, giving an apparent K_D of 60 ± 2 μM. Error bars indicate standard deviation of separate replicate experiments. (C) Peptide with no linker (5) was titrated into VP40 dimer for 20 injections and heat readings for each injection was plotted. Heat of titration indicate no changes in heat in initial injections and heat is reduced only at high concentrations. This pattern of heat titration is similar to that of DMSO. (D) Corresponding enthalpy of DMSO was subtracted from enthalpy of peptide 5 and plotted to give a binding curve. Each experiment was repeated three times and subtracted enthalpy indicate little to no binding of peptide 5 to VP40 dimer. Error bars indicate standard deviation from replicate experiments where each experiment was repeated three times.

Fig. 6. Peptide 3b binds to intact VP40 dimer. (A) Representative figure of VP40 octamer with residues 106–120 highlighted in red for each protomer (shown in blue and cyan). (B) Wild type VP40 was recombinantly expressed and purified using Ni-NTA chromatography and subjected to a size exclusion column. Wild type VP40 shows predominant dimer and octamer peaks. Octamer fractions were collected and concentrated for ITC studies. (C) Peptide 3b was titrated with wild type VP40 octamer for over 20 injections and heat of titration for each concentration were measured. As described previously, heat of dilution was measured by titrating an equivalent amount of DMSO with VP40 octamer. Enthalpy of binding of peptide was plotted by subtracting enthalpy of DMSO from enthalpy of peptide 3b with mole ratio of peptide to VP40 octamer. Experiments were repeated three times with error bars representing standard deviation and binding affinity K_D of 51 ± 3 μM. (D) Representative image indicates mutation of isoleucine to arginine at position 307 abrogates CTD–CTD interactions. (E) VP40 with I307R mutation was purified as above and subjected to size exclusion chromatography indicating the mutated protein predominantly forms octamer. (F) Peptide 3b was titrated with I307R octamer and binding curve was plotted by subtracting enthalpy of DMSO from the enthalpy of peptide. Each experiment was replicated three times giving K_D of 29 ± 7 μM. Error bars indicate standard deviation of replicate experiments. (G) Representative image indicates mutation of leucine to alanine at position 117 disrupts the interprotomer hydrogen bonding required for stabilization of dimer. (H) Recombinant expression and purification of L117A mutant shows predominant octamer formation. (I) Peptide 3b was titrated for 20 injections with L117A mutant octamer and enthalpy of binding was determined by subtracting enthalpy of DMSO from the enthalpy of peptide and plotted with mole ratio of peptide to L117A mutant. Experiments were repeated three times with error bars indicating standard deviation. No binding was observed for peptide 3b with L117A mutant octamer. Each experiment was repeated three times.

Fig. 7. Peptide 3b binds to Sudan Ebolavirus VP40 dimer. (A) Conserved sequence of dimerization interface between different Ebolavirus strains. (B) Sudan VP40 dimer was recombinantly expressed and purified and peptide 3b was titrated for 30 injections with the dimer. Heat of dilution was calculated by titrating equivalent amount of DMSO for 30 injections. Enthalpy was plotted by subtracting enthalpy of DMSO from enthalpy of peptide. Experiment was repeated three times giving a K_D of 43 ± 5 μM with error bars indicating standard deviation between experiments. (C) Similarly, peptide 5 was titrated with Sudan VP40 dimer for 20 injections and subtracted enthalpy from DMSO was plotted. Each experiment was repeated three times, with error bars indicating standard deviation and the experiments showed no binding of peptide 5 to the Sudan VP40 dimer. Each experiment was repeated three times.

Fig. 8. Peptide 3b slightly shifts equilibrium of VP40 dimer. (A) Peptide 3b was added to purified VP40 dimer and incubated at 4 °C overnight. The sample was injected in a Superdex 200 10/300 GL column to separate the oligomeric forms of VP40. Elution profile of VP40 with peptide 3b is shown in red and it indicates a dimer peak and a smaller peak at 22 mL elution volume. As a vehicle control, VP40 dimer was treated with equivalent DMSO concentration and subjected to size exclusion chromatography (in black) showing a dimer peak. (B) 1 mL fractions were collected and immunoblotted for VP40 using anti-6xHistidine antibody. VP40 was detected in all the fractions from 11–24 mL for the peptide while DMSO samples show presence of VP40 from 12–21 mL and less amounts in 23 and 24 mL fractions. There was no protein detected in the 22 mL fraction in case of DMSO while there is presence of protein in case of peptide 3b indicating peptide induced formation of smaller order oligomers of VP40 which are eluted at higher volumes.

Fig. 9. Structure and dynamics of VP40–3b complex. (A) Structure of the VP40–3b complex at 0 ns. The N-terminal tail (indicated by a small arrow) is pointing downward and not interacting with 3b. (B) As in the VP40 dimer structure, 3b has significant hydrophobic contacts with VP40 at the dimer interface. The hydrophobic side chains involved in the interactions are shown in stick whereas the hydrophobic surface is shown as a transparent white surface. The N-terminal tail (indicated by a small arrow) wraps the peptide during the simulation. (C) Residues involved in hydrogen bond interactions at the VP40–3b interface. (D) The total number of hydrogen bonds between VP40 and 3b during the simulation. (E) Specific VP40–3b hydrogen bond pairs that are observed to form at least 10% of the time during the 1 μs simulation. These simulations were run three different times.