Proline cis - trans isomerization and its implications for the dimerization of analogues of cyclopeptide stylostatin 1: a combined computational and experimental study

Cis and trans proline conformers are often associated with dramatic changes in the biological function of peptides. A slow equilibrium between cis and trans Ile-Pro amide bond conformers occurs in constrained derivatives of the native marine cyclic heptapeptide stylostatin 1 (cyclo-(NSLAIPF)), a potential anticancer agent. In this work, four cyclopeptides, cyclo-(NSTAIPF), cyclo-(KSTAIPF), cyclo-(RSTAIPF) and cyclo-(DSTAIPF), which are structurally related to stylostatin 1, are experimentally and computationally examined in order to assess the effect of residue mutations on the cis-trans conformational ratio and the apparent capacity to form dimeric aggregates. Primarily, cyclo-(KSTAIPF) and cyclo-(RSTAIPF) showed specific trends in circular dichroism, MALDI-TOF and HPLC purification experiments, which suggests the occurrence of peptide dimerization. Meanwhile, the NMR spectrum of cyclo-(KSTAIPF) indicates that this cyclopeptide exists in the two slow-exchange families of conformations mentioned above. Molecular dynamics simulations combined with quantum mechanical calculations have shed light on the factors governing the cis/trans conformational ratio. In particular, we have found that residue mutations affect the internal hydrogen bond pattern which ultimately tunes the cis/trans conformational ratio and that only trans conformers are capable of aggregating due to the shape complementarity of the two subunits.

Table S1.NOE signals between peptide NH protons from 1 H-1 H NOESY 2D spectrum (Figure 4) for both conformers: trans c(KSTAIPF) (top) and cis c(KSTAIPF) (bottom).trans c(KSTAIPF), major conformation in NMR experiments (green labels and circles in Figure 4) Source  a Distance from carbonyl oxygen of residue i and the nitrogen of NH unit of residue i+2 (γ-turn) and i+3 (βturn), shown in bold.
Table S3.Number of conformations selected (at least of 75 % of the conformational space covered) from 100 ns MD simulations for the cis and trans isomers of each cyclopeptide in order to compute by semiempirical and DFT methods the relative Gibbs free energies between both isomers.256 conformations in total.a Distance between C α carbons of residues i and i+2 (γ-turn) or i+3 (β-turn).

Cyclopeptides
b Distance from carbonyl oxygen of residue i and the nitrogen of the peptide unit of residue i+2 (γ-turn) and i+3 (β-turn).See Table S4 for the values of other cyclopeptides.Upon temperature increase from room temperature to 45°C the most relevant peaks coalesce into a single peak.This might be explained by the existence of different species of c(KSTAIPF) in water.This could be interpreted as the existence of different conformational states, such as an equilibrium between the monomeric cis and trans species and the dimeric form, when one considers these data in light of the experimental and theoretical results reported in this work.Nevertheless, the chromatogram also reveals the existence of other minor peaks, near the main peak, which could reflect the existence of a lowly populated conformations; and also far away from it, which could perhaps be attributed to impurities generated during the synthesis of the cyclic peptide.The spectrum was recorded in the 525-4000 cm -1 range with 32 scans at a resolution of 4 cm -1 .The IR spectrum is an averaged spectrum of the cis and trans conformations since the synthesis of c(KSTAIPF) yields a mixture of the two.However, the overall shape of the amide-I band of both conformations should be very similar as both contain intramolecular hydrogen bonds formed of peptide carbonyl groups and also peptide carbonyl extending out into the solvent; see Figure 5  Here 25 and 24 conformations of (left) cis and (right) trans c(KSTAIPF), respectively, are represented.This is an illustrative example of the conformational space explored by the 256 conformations of the five cis and trans conformers of cyclopeptides, see Table S3.Backbone as bold sticks, side chain as thin sticks.Hydrogens are omitted.Green, red and blue for carbon, oxygen and nitrogen atoms, respectively.
cis c(KSTAIPF) trans c(KSTAIPF) . Computational protocol for structural elucidation of the trans,trans dimeric form of c(KSTAIPF; see Experimental section for more details).The name of the five relative orientations denotes the residues of both peptides that are placed facing each other in the initial structure.Identical protocols were applied to the cis,cis and cis,trans dimeric forms, but in this latter case after step 4 all MD simulations led to an unbound structure.

Figure S5 .
Figure S5.IR spectrum of a solid sample of c(KSTAIPF) registered using a Thermo Scientific Nicolet iZ10 FTIR system.

Figure S6 .Figure S7 .
Figure S6.ROESY experiments showing in circles the exchange of signals for specific residues in the trans (_t; green) and cis (_c; purple) conformers.

Figure S8 .
Figure S8.Conformational ensemble optimized at the M06-2X/6-31G(d)/SMD level of computation for cis and trans conformers of c(KSTAIPF) used to determine the relative Gibbs free energies between both conformers.Here 25 and 24 conformations of (left) cis and (right) trans c(KSTAIPF), respectively, are represented.This is an illustrative example of the conformational space explored by the 256 conformations of the five cis and trans conformers of cyclopeptides, see TableS3.Backbone as bold sticks, side chain as thin sticks.Hydrogens are omitted.Green, red and blue for carbon, oxygen and nitrogen atoms, respectively.

Figure S10 .Figure S11 .
Figure S10.Conformational analysis of both monomers (A and B) of the trans,trans dimeric cyclopeptides.The plot shows the contour map (isodensity lines) of the projection of the backbone cartesian coordinates of MD simulations of both monomers (a and b, respectively) of all trans,trans dimer cyclopeptides over the same conformational space of trans monomeric c(KSTAIPF) taken as a reference by principal component analysis.The same procedure as explained in Figure6is applied here.

Table S2 .
Distances of and -turns in cis and trans cyclopeptides.a

Table S4 .
Relative energies of cis conformers versus trans conformers for all studied cyclopeptides, in kcal/mol.a

Table S5 .
Gibbs free energy calculated at M06-2X/6-31G(d)/SMD level for all selected conformations for all studied cyclopeptides (conformation number, Gibbs free energy in hartree and % population according to the Boltzmann distribution)

Table S6 .
Geometry optimization at M06-2X/6-31G(d,p)/SMD level of theory for the 67 most important conformations for all studied cyclopeptides (conformation number, Energy in hartree and % population according to the Boltzmann distribution)

Table S9 .
Average intramonomeric and intermonomeric relevant distances (Å) of the trans,trans dimeric cyclopeptides.a Distance from the nitrogen of the NH unit of Pro residue in monomer A and the analogous of monomer B. c

Table S10 .
Phi and psi backbone torsional angles (degrees) and distances (Å) of and -turns in the dimeric species of trans,trans c(KSTAIPF).a