Design of the Chemical Modifications in CREKA
The bioactive conformation proposed for natural CREKA on the basis of theoretical calculations5
is depicted in . In this structure, the backbone defines a β type turn motif. The functionalized side chains of the central residues (Arg, Glu and Lys) face the same side of the molecule, and the backbone Cys CO and Lys NH groups are hydrogen bonded. This structural motif was identified in the global minimum of the free peptide, when inserted in a phage display protein, and in many of the accessible minima. Inspection of the side chains reveals salt bridges involving the negatively charged carboxylate group of Glu and the contiguous positively charged side chains of Arg and Lys. These side-chain interactions are made possible by the peptide backbone turn conformation. An extended backbone would place the positively and negatively charged side chains pointing towards opposite sides (). The salt-bridges presented multiple interaction patterns, with the specific atoms involved being dependent on the chemical environment considered, i.e.
the peptide in the free state, attached to a nanoparticle or inserted in a phage display protein. Accordingly, the salt bridges identified in many of the significant minima were found to differ in the atoms involved in these interactions.
The structural features observed5
for CREKA therefore lie in the β-turn conformation adopted by the peptide backbone. In this turn motif, Arg occupies the i
+1 position. Proline (Pro) is known to exhibit a high propensity to induce turns (of either the β- or the γ-type) and, in particular, to occupy position i
+1 of β-turns.34-36
Moreover, the cyclic structure of Pro, which is unique among naturally-occurring amino acids, is known to impart stability against enzymatic hydrolysis.37-40
Thus, replacing the Arg in CREKA by a proline-like derivative of this amino acid could lead to an increase in stability with a simultaneous retention, or even enhancement, of the propensity to adopt a folded turn-like conformation.
We therefore considered the replacement of Arg in CREKA by a proline derivative bearing the guanidinium group present in the Arg side chain at the γ-position of the pyrrolidine ring. Proline derivatives of this type may be synthetically accessible by transformation of γ-hydroxyproline, a starting material available in enantiomerically pure form from commercial sources. The exact chemical structure of the newly designed amino acid needs however to be selected: an Arg side chain incorporated at the Cγ in proline can exhibit either a cis or a trans conformation with respect to the carbonyl function. In addition, the guanidinium group may be attached to the Cγ atom of the pyrrolidine ring through a variable number (n) of methylene units, whose optimal value for formation of salt-bridge interactions with the proximal Glu side chain should be determined.
For this purpose, graphical molecular modeling was performed using the proposed bioactive conformation5
of natural CREKA () as a template. This qualitative analysis provided the best fitting when the Arg side chain was arranged in cis
with respect to the proline carbonyl group and for a chain length corresponding to n
= 2 (). The amino acid thus designed was denoted as (Pro)hArg, where hArg stands for homo
arginine, that is, the Arg homologue containing one more methylene unit. shows the structures of Arg, hArg and their corresponding cis
proline-like derivatives (Pro)Arg and (Pro)hArg, respectively, for comparison.
Structure of arginine (Arg) and its homologue containing an additional methylene group (hArg), as well as that of their respective proline-like derivatives considered in this work.
Conformational Profile and Force-Field Parametrization
Before analyzing the conformational impact derived from the incorporation of (Pro)hArg into CREKA, parametrization of this non-proteinogenic residue is necessary. For this purpose, the conformational properties of its N-acetyl-N'-methylamide derivative, Ac-(Pro)hArg-NHMe (), have been analyzed. Given the large number of dihedral angles in this molecule and the subsequent huge number of starting geometries to be considered, a simplified methodology has been applied. Thus, the minimum energy conformations of this compound have been derived from those obtained for the residue containing one less methylene unit, Ac-(Pro)Arg-NHMe ().
Figure 3 Dihedral angles used to identify the conformations of the N-acetyl-N'-methylamide derivatives of (Pro)Arg (a) and (Pro)hArg (b) studied in this work. The dihedral angles ω0, , ψ and ω are defined using backbone atoms, (more ...)
The backbone (ω0
,ψ,ω) and side chain (χi
, endocyclic; ξi
exocyclic) dihedral angles considered for Ac-(Pro)Arg-NHMe and Ac-(Pro)hArg-NHMe are defined in . The minimum energy conformations of these compounds have been denoted using a three-label code describing the backbone conformation, the puckering of the five-membered ring and the conformation of the exocyclic substituent. Specifically, the first label identifies the backbone conformation, defined by the
,ψ dihedral angles, using Perczel's nomenclature.41
Accordingly, nine different backbone conformations can be distinguished in the potential energy surface E=E
,ψ) of an α-amino acid: γD
. In the case of proline, only the γL
(γ-turn or C7
(α-helical), and εL
(polyproline II) conformations are possible because
is fixed around - 60°.34-36
puckering of the five-membered pyrrolidine ring is next indicated using the [u] and [d] labels, respectively. These conformational states are defined as those in which the Cγ
atom and the carbonyl group of proline (or the proline-like residue) lie on the same or opposite sides, respectively, of the plane defined by the Cδ
, N and Cα
atoms. In particular, the down
ring puckering is identified when χ1
are positive while χ2
are negative. Conversely, the up
ring puckering is characterized by negative values of χ1
and positive values of χ2
. Finally, the last set of letters indicates the gauche+
) or gauche-
) arrangement of each exocyclic dihedral angle ξi
In a first step, the intrinsic conformational properties of Ac-(Pro)Arg-NHMe () were investigated, using DFT calculations at the B3LYP/6-31+G(d,p) level. The conformational search was performed considering that this compound retains the restrictions imposed by the five-membered pyrrolidine ring in proline. Thus, the three minimum energy conformations previously characterized for Ac-Pro-NHMe42
with all trans
amide bonds (ω0
and ω ≈ 180°), namely γL
[u] and αL
[u], were used to generate the starting structures of Ac-(Pro)Arg-NHMe. The arrangement of the side group in (Pro)Arg is defined by the flexible dihedral angles ξ1
(), which are expected to exhibit three different minima: trans
(60°) and gauche-
(-60°). Accordingly, 3 (minima of Ac-Pro-NHMe) × 3 (minima of ξ1
) × 3 (minima of ξ2
) = 27 minima were anticipated for the potential energy hypersurface (PEH) E =E(
) of Ac-(Pro)Arg-NHMe. All these structures were used as starting points for subsequent full geometry optimizations.
lists the geometric parameters and the relative energies (ΔEgp) of the five minima characterized for this compound in the gas phase, which are displayed in . In the lowest energy minimum (γL[u]g+t, ), the backbone acetyl CO and methylamide NH sites form an intramolecular hydrogen bond defining a seven-membered cycle (γ-turn or C7 conformation), while the pyrrolidine ring exhibits an up puckering and the exocyclic dihedral angles ξ1 and ξ2 are arranged in gauche+ and trans, respectively. This side chain disposition allows formation of a strong hydrogen bond involving the (Pro)Arg carbonyl oxygen and the NH moiety in the guanidinium group. Similar backbone···backbone and backbone···side chain hydrogen-bond interactions are present in the third minimum (γL[d]s+s+, ) although, in this case, a down puckering of the pyrrolidine ring and a skew+ conformation of both ξ1 and ξ2 are required, and this side chain rearrangement is associated with an energy penalty of 3.6 kcal/mol.
Table 1 Dihedral anglesa,b of the backbone and the exocyclic side group, pseudorotational parametersa of the pyrrolidine ring (A and P), and relative energyc (ΔEgp) of the minimum energy conformations characterized for Ac-(Pro)Arg-NHMe at the B3LYP/6-31+G(d,p) (more ...)
Figure 4 Minimum energy conformations of Ac-(Pro)Arg-NHMe obtained from B3LYP/6-31+G(d,p) calculations: (a) γL[u]g+t; (b) γL[d]ts-; (c) γL[d]s+s+; (d) εL[d]g-s+; (e) γL[d]g-s+ (see for geometries). Distances (H···O) (more ...)
Indeed, for a γL backbone conformation, the most favorable backbone···side chain interaction when the pyrrolidine ring is down-puckered seems to involve the NH2 rather than the NH site in the guanidinium side chain. This is inferred from the geometry of the second conformer in (γL[d]ts-, ), which is destabilized with respect to the global minimum by only 0.4 kcal/mol. Interestingly, deviation of the ψ angle in this second conformer to values around 100° results in a new minimum energy structure (γL[d]g-s+, ) where the backbone···backbone and backbone···side chain hydrogen-bond interactions are retained. However, the large ψ angle leads to a much less favorable geometry for the hydrogen bond stabilizing the γ-turn conformation and this results in a ΔEgp value of 6.4 kcal/mol.
The only conformer in with a backbone structure other than γL is the fourth minimum (εL[d]g-s+, ), which corresponds to a polyproline II conformation. Despite the presence of a strong interaction involving the (Pro)Arg carbonyl and one guanidinium NH2 moiety, this conformer is unfavored by 5.6 kcal/mol, due to the absence of hydrogen bonds linking the backbone amide groups.
The free energies in the gas phase (ΔGgp) calculated for the five minimum energy conformations of Ac-(Pro)Arg-NHMe are displayed in . As can be seen, consideration of the ZPVE, thermal and entropic corrections for the transformation of ΔEgp into ΔGgp represents relative variations lower than 0.2 kcal/mol in all cases with the exception of the γL[d]ts- disposition, for which these statistical corrections produce a destabilization of 1.2 kcal/mol. Accordingly, γL[u]g+t is the only conformation significantly populated in the gas phase at room temperature according to a Boltzmann distribution since all the local minima exhibit ΔGgp values higher than 1.5 kcal/mol.
Table 2 Relative free energya in the gas phase (ΔGgp) and in carbon tetrachloride, chloroform and aqueous solutions (ΔGCC14,ΔGCHC13 and ΔGH2O, respectively) for the minimum energy conformations of Ac-(Pro)Arg-NHMe at the B3LYP/6-31+G(d,p) (more ...)
In order to obtain an estimation of the solvation effects on the relative stability of the different minima, single point calculations were conducted on the optimized structures using the PCM method. includes the relative free energies in carbon tetrachloride, chloroform and water solutions (ΔGCCl4, ΔGCHCl3 and ΔGH2O, respectively). The solvent introduces significant changes in the relative stability of the different minima characterized for Ac-(Pro)Arg-NHMe. Carbon tetrachloride was found to considerably stabilize theγL[d]ts-, εL[d]g-s+ and γL[d]g-s+ conformations, to the point that the former becomes the most stable structure and the two latter are stabilized by 3.3 and 2.3 kcal/mol, respectively, with respect to the gas phase. The higher polarity of chloroform results in a further stabilization of the εL[d]g-s+ conformation, even though the lowest energy structure remains γL[d]ts-, as in carbon tetrachloride. Finally, εL[d]g-s+ becomes the most stable structure in aqueous solution, the ΔGH2O values of the other four conformers being higher than 1.5 kcal/mol. The stabilization detected for this conformation, which increases significantly with the polarity of the solvent, should be attributed to the accessibility of the peptide bonds to the solvent. Thus, the amide groups are better solvated when the backbone adopts a εL conformation than when it is arranged in γL. Furthermore, the strength of the attractive amide···solvent interactions increases with the polarity of the solvent, which should be attributed to the crucial role played by the electrostatic contribution. On the other hand, the relative stabilities of the γL[d]s+s+ and γL[d]g-s+ remains essentially unaltered upon salvation.
The γL[u]g+t and εL[d]g-s+ arrangements, corresponding to the lowest free energy conformations of Ac-(Pro)Arg-NHMe in the gas phase and in aqueous solution, respectively (), were used as starting structures for the conformational study of Ac-(Pro)hArg-NHMe (). It is worth noting that this decision was taken on the basis of the following considerations: (i) the εL[d]g-s+ was the only conformation found for Ac-(Pro)Arg-NHMe with a non-negligible population in aqueous solution, which is the environment that will be considered in the study of the pentapeptide; and (ii) consideration in the force-field parametrization process of the two backbone conformations identified in and , γL and εL, is a priori highly desirable. Thus, the starting geometries for the latter compound were prepared by including an additional methylene group in the side chain of (Pro)Arg for such two representative structures. The conformation of the new methylene group is defined by the dihedral angle ξ3 (), for which three different arrangements were considered: gauche+, trans and gauche-. Accordingly, the conformational search of Ac-(Pro)hArg-NHMe was carried out considering 2 [representative minima of Ac-(Pro)Arg-NHMe] × 3 (minima of ξ3) = 6 starting geometries. Energy minimizations at the B3LYP/6-31+G(d,p) level led to five different minimum energy structures, the three most stable being given in and . The remaining two are not included since their relative energies were found to be above 10 kcal/mol and therefore were not considered representative.
Table 3 Dihedral anglesa,b of the backbone and the exocyclic side group, pseudorotational parametersa of the pyrrolidine ring (A and P), and relative energyc (ΔEgp) of the minimum energy conformations characterized for Ac-(Pro)hArg-NHMe at the B3LYP/6-31+G(d,p) (more ...)
Figure 5 Minimum energy conformations of Ac-(Pro)hArg-NHMe obtained from B3LYP/6-31+G(d,p) calculations: (a) γL[d]tg+g-; (b) εL[d]g-g+g+; (c) γL[d]g-tt (see for geometries). Distances (H···O) and angles (N-H···O) (more ...)
The lowest energy conformation (γL[d]tg+g-, ) corresponds to a γ-turn conformation stabilized by a hydrogen bond linking the backbone terminal CO and NH sites. The (Pro)hArg CO group and one of the NH2 moieties in the guanidinium side chain are also involved in a strong hydrogen-bond interaction. The latter is lost in the γL[d]g-tt minimum (), which explains its high relative energy. Conversely, the second conformer in (εL[d]g-g+g+, ) exhibits no backbone···backbone hydrogen bond, as expected for an εL conformation, while being stabilized by a strong side chain···backbone interaction. Accordingly, it is destabilized by 5.5 kcal/mol with respect to the lowest energy minimum.
lists the ΔGgp, ΔGCCl4, ΔGCHCl3 and ΔGH2O values calculated for the three Ac-(Pro)hArg-NHMe minima described above. Notably, the ZPVE, thermal and entropic corrections stabilize the γL[d]g-tt structure in the gas phase by 3.5 kcal/mol. In spite of this, the γL[d]tg+g- remains the only accessible conformation both in the gas phase and in carbon tetrachloride solution. In contrast, γL[d]g-tt becomes the most stable structure in the presence of chloroform or water. This significant variation should be attributed to the charged nature of the side group. Accordingly, the impact of the environment on the arrangement of the side chain increases with the polarity of the solvent and, therefore, the largest change is detected in aqueous solution.
Table 4 Relative free energya in the gas phase (ΔGgp) and in carbon tetrachloride, chloroform and aqueous solutions (ΔGCC14,ΔGCHC13 and ΔGH2O, respectively) for the minimum energy conformations of Ac-(Pro)hArg-NHMe at the B3LYP/6-31+G(d,p) (more ...)
These results indicate that (Pro)hArg retains the most important structural features of proline,42
reflecting a high tendency to induce peptide turns. It should also be noted that, even if the γL
conformations are characterized by different values of the ψ dihedral angle, both of them correspond to turn-like backbone conformations, namely the i
+1 position of a γ- and a βII-turn, respectively.34-36
The stretching, bending, torsion and van der Waals parameters used in the AMBER force-field30
to describe Pro and Arg were directly transferred to (Pro)hArg. Atomic centered charges for the minimum energy conformations listed in were calculated by fitting the UHF/6-31G(d) quantum mechanical and the Coulombic molecular electrostatic potentials (MEPs) to a large set of points placed outside the nuclear region. It should be noted that the electrostatic parameters derived at this level of theory are fully compatible with the current AMBER force-field.30
On the other hand, electrostatic force-field parametrization using a strategy based on weighted multiple conformations through a Boltzmann distribution in the gas phase, which was originally proposed by different authors,43
has been demonstrated to be especially successful for non-proteinogenic residues.43b,44
Accordingly, parameters for (Pro)hArg () have been obtained considering the atomic charges of the lowest energy minimum only (γL
, ) since the other two local minima are unfavored by more than 5.1 kcal/mol and, therefore, their contribution to a normalized Boltzmann distribution can be considered negligible.
Electrostatic parameters determined for the (Pro)hArg residue.
Conformational Search of the CREKA Analogue Attached to a Nanoparticle. Comparison with the Natural Peptide
The conformational preferences of the CREKA analogue incorporating (Pro)hArg as an Arg substitute, hereafter denoted as CR*EKA, have been explored using the sampling technique previously employed for the study of the natural pentapeptide.5
represents the evolution of the number of unique minimum energy conformations against the number of modified SA-MD production cycles necessary for the conformational search of CR*EKA to converge. As can be seen, the exploration is completed after seven cycles, the last one providing only 3 new structures to the list of unique conformations. Interestingly, the replacement of Arg by (Pro)hArg did not lead to a reduction in the amount of modified SA-MD cycles required to complete the conformational search, but the number of accessible low energy conformations diminished dramatically, i.e.
612 and 1305 minima were obtained for CR*EKA and CREKA, respectively. Moreover, the energetic distribution of the minima generated was also affected by this targeted replacement, as indicated in . Thus, the conformational restrictions imposed by the presence of (Pro)hArg eliminated a large number of minima with relative energies in the interval between 4 and 11 kcal/mol, which was found to be the most populated for CREKA. This effect is shown by the distribution obtained for CR*EKA, which contrasts with the Gaussian-like profile achieved for the natural peptide.
Figure 7 (a) Number of unique minimum energy conformations found for CR*EKA (grey line and diamonds) and natural CREKA (black line and circles) against the number of modified SA-MD cycles used for the conformation search. (b) Distribution of energies for the unique (more ...)
The conformational preferences of the backbone in the two pentapeptides have been compared by analyzing the virtual dihedral angles used to define the specific arrangement of each residue. Results are given in , which represents the distribution of such dihedrals through histograms. As can be seen, the incorporation of (Pro)hArg produces some changes in the general conformational profile of the peptide. The distributions obtained for the (Pro)hArg and Glu residues in CR*EKA are very similar to those found for their Arg and Glu counterparts in CREKA, while important variations are detected for the other three residues, especially Lys and Ala. Indeed, the conformational space of the two latter amino acids is significantly narrower in CR*EKA, indicating that the incorporation of (Pro)hArg reduces the conformational flexibility of the whole peptide. This is also reflected in , which compares the distribution of the
ψ backbone dihedral angles of the five residues for the minima with relative energies lower than 2 kcal/mol.
Figure 8 Comparison of the distribution of virtual dihedral angles used to define the backbone conformation of CREKA (a) and CR*EKA (b) in each unique minimum energy structure obtained. Color code for the bars: black for dihedral angle values ranging from 0° (more ...)
Ramachandran plot distribution for the five residues of CR*EKA (open circles) and CREKA (filled black circles) considering the more representative minimum energy structures, i.e. those within a relative energy interval of 2 kcal/mol.
A clustering analysis based on hydrogen bonds and salt bridges indicated that 68% of the CR*EKA minima (417 structures) present at least one such interaction, while this value reaches 82% (1092 structures) for natural CREKA. The total number of interactions (either of the hydrogen-bond or salt-bridge type) detected in these minima is 667 (1.6 interactions per structure) and 2198 (2.1 interactions per structure) for CR*EKA and CREKA, respectively, which are distributed in 106 and 448 clusters (). It is interesting to note that only 3 clusters in CR*EKA and 8 in CREKA contain more than 25 structures, respectively grouping 31% and 25% of the minima. Clusters are organized as follows: (i) backbone···backbone hydrogen bonds: 79.3% (529 interactions) for CR*EKA and 83.6% (1839 interactions) for CREKA; (ii) backbon···eside chain hydrogen bonds: 0.0% (no interaction) for CR*EKA and 4.7% (102 interactions) for CREKA; (iii) side chain···side chain salt bridges: 20.7% (138 interactions) for CR*EKA and 11.7% (257 interactions) for CREKA.
Distribution of the unique minimum energy structures generated for CR*EKA (top) and CREKA (bottom) in clusters, which have been grouped on the basis of the formation of hydrogen bonds and salt bridges.
The most frequent interactions in the modified peptide are the Glu(N- H)···(O=C)Cys hydrogen bond (219 interactions; 32.8%) and the Glu···Lys salt bridge (138 interactions; 20.7%), while in natural CREKA they are the Glu(N-H)···(O=C)Ac, Lys(N-H)···(O=C)Cys and Ala(N-H)···(O=C)Arg backbone···backbone hydrogen bonds (251, 225 and 207 interactions, respectively; 11.4%, 10.2% and 9.4%, respectively) and the Arg···Glu salt bridge (187 interactions; 8.5%). These distinct interaction patterns suggest important differences between the two peptides, which are confirmed upon a cross-comparison of the frequency in which a particular interaction is observed. Thus, the backbone···backbone hydrogen bond most frequently detected in CR*EKA, Glu(N-H)···(O=C)Cys, accounts for only 3.4% of the CREKA interactions. Conversely, the Glu(N-H)···(O=C)Ac, Lys(N-H)···(O=C)Cys and Ala(N-H)···(O=C)Arg hydrogen bonds are, respectively, 0.8%, 3.6% and 5.1% of the CR*EKA interactions. Regarding side chain···side chain contacts, the frequency of the Glu···Lys salt bridge is 7.5% in CREKA, while the Arg···Glu interaction was not identified in any of the CR*EKA minima.
These results indicate that the conformational restrictions imposed by the presence of (Pro)hArg lead to some alterations in the conformational profile of the whole peptide that affect the turn type generated and the residues involved in this turn, as well as the interactions between adjacent ionized side chains. As expected, the conformational propensities of the (Pro)hArg-containing peptide seem to be more clearly defined than those of the natural sequence, as shown by the smaller variation observed for the preferred backbone···backbone and side chain···side chain interactions in CR*EKA with respect to CREKA. This is deduced from the cluster analysis described above and it becomes even clearer when the interaction schemes of the three conformations of lowest energy generated for the two peptides are compared (). Thus, up to three different β-turns are detected in these CREKA conformations, namely those centered at Arg-Glu [stabilized by a Lys(N-H)···(O=C)Cys hydrogen bond; minima # 1 and 3], Glu-Lys [stabilized by a Ala(N-H)···(O=C)Arg hydrogen bond; minimum # 2] and Lys-Ala [stabilized by a NHMe(N-H)···(O=C)Glu hydrogen bond; minimum # 2]. In comparison, all three CR*EKA conformers in share a γ-turn centered at the (Pro)hArg residue and stabilized by a Glu(N-H)···(O=C)Cys hydrogen bond. This is due to the much higher propensity of proline, when compared to other proteinogenic amino acids, in nucleating turns and therefore occupying the central turn positions.34-36
The fact that the turn observed in the lowest energy conformation in CREKA is of the β type whereas it is of the γ type in CR*EKA, suggests that the difference in the overall shapes of the molecules is rather small. In both turns the i
+1 position is occupied by either Arg (in CREKA) or its substitute (in CR*EKA) and both turn conformations orient the charged side chains of the three central residues toward the same region of the molecule. Thus, the main structural requirements considered to be essential for the bioactivity of CREKA5
are also present in CR*EKA.
Comparison between the interaction patterna of the three minima of lowest energy generated for natural CREKAb and its analogue CR*EKA. Relative energiesc (ΔE) are also given.
In spite of this similarity, the natural peptide and its analogue differ in the interactions involving the three charged side chains. Specifically, the only salt-bridge type detected in the three CR*EKA minima in involves Glu and Lys, while the natural CREKA presents multiple salt bridges Arg···.Glu ···.Lys (minimum # 1) or an Arg···.Glu only (minima # 2 and 3). The Arg···.Glu salt bridge seems to be disfavored when (Pro)hArg replaces Arg, but the biological consequences of this change are difficult to predict since this specific interaction may not be essential to bioactivity. In the peptide-receptor recognition process, the Arg, Gly and Lys side chains are likely to interact with the complementary groups in the receptor, rather than among themselves.
Overall, these results suggest that, although the targeted replacement of Arg by (Pro)hArg in CREKA induces some changes in the conformational profile of the peptide, the most important structural trends of the natural compound are retained. This general resemblance is reflected in the lowest energy conformations found for CR*EKA and for CREKA, with the backbones within 1.5 Å of each other. shows the superposition of the two structures. This shape similarity between the most stable minima suggests that the incorporation of (Pro)hArg to provide resistance against the proteolytic enzymes would not induce major alterations in the conformational features considered to be essential for the tumor-homing activity of the pentapeptide.
Lowest energy minimum obtained for CR*EKA (a) and CREKA (b) attached to a nanoparticle, and superposition of both structures (c). The surface used to mimic the nanoparticle is represented by a single green ball.