Design of a Peptide-Based Model Leads for Scavenging Anions

Among several peptide-based anion recognition motifs, the “CαNN” motif containing C–1α, N0, and N+1 of three consecutive residues is unique in its mode of interaction. Having a spatial geometry of βαα or βαβ, this motif occurs in the N terminus of a helix and often found at the functional interface of a protein, mediating crucial biological significance upon interaction with anion(s). The interaction of anion(s) with chimeric peptide sequences containing the naturally occurring “CαNN” motif (CPS224Ac, CPS226, and CPS228) reported in our previous attempts strongly confirms that the information regarding the interaction is embedded within the local sequences of the motif segment. At these prevailing circumstances, an effort has been pursued to design novel scaffolds based on the “CαNN” motif for achieving better recognition of anion(s). Exploring the existing data set of the “CαNN” motif available in the FSSP database, four novel peptide-based scaffolds have been designed (DS1, DS2, DS3, and DS4), and preliminary screenings have been performed using computational approaches. Our initial work suggests that two (DS1 and DS3) out of the four scaffolds are potential candidates for better anion recognition. By employing biophysical characterization using both qualitative and quantitative measures, in this present study, we report the interaction of sulfate and phosphate ions with these two designed scaffolds, in which there is much better recognition of anions by these scaffolds than the natural sequences, justifying their logical engineering. Our observation strongly suggests that these designed scaffolds are better potential candidates than those of the naturally occurring “CαNN” motif in terms of anion recognition and could be utilized for the scavenging of anion(s) for different purposes

Distribution of H-bond distance and angle constraints obtained from interaction of anion(s) and ‘experimental NMR’ structure of CPS224Ac.

<p>(a) Distribution of (X)H—O distance for interaction with sulfate ion; (b) distribution of α₋₁/N₀/N₊₁). The mean (μ) value, standard deviation (σ) value and goodness of the fit (adjusted R²) of each distribution are mentioned. The ratio of σ/μ is shown to emphasize that there is very little spread about the mean value.</p

MD simulations (for 40 ns at 276K) of the sulfate ion-bound conformer of the ‘experimental NMR structure’ of CPS224Ac.

<p>a) Partial representation (first 1 ns of total 40 ns simulation) of sulfate ion interaction with the C^αNN′ motif segment of CPS224Ac (blue lines indicate weak interaction while red line indicate strong H-bond) along with the detail monitoring of backbone dihedral angles (φ, ψ) of Lys3 with respect to time in the MD trajectory; b) Ramachandran plot of the backbone dihedral angle distributions (φ, ψ) of all the residues during the MD simulation (40 ns) showing perturbation takes place only in the C^αNN′ motif segment; c) Distribution of backbone dihedral angles (φ, ψ) of Lys3 residue as Ramachandran plot during the 40 ns MD-simulation, emphasizing its existence in helical conformation only during interaction of sulfate ion with C^αNN′ motif segment peptide.</p

Conformational Preference of ‘C^αNN’ Short Peptide Motif towards Recognition of Anions

<div><p>Among several ‘anion binding motifs’, the recently described ‘C^αNN’ motif occurring in the loop regions preceding a helix, is conserved through evolution both in sequence and its conformation. To establish the significance of the conserved sequence and their intrinsic affinity for anions, a series of peptides containing the naturally occurring ‘C^αNN’ motif at the N-terminus of a designed helix, have been modeled and studied in a context free system using computational techniques. Appearance of a single interacting site with negative binding free-energy for both the sulfate and phosphate ions, as evidenced in docking experiments, establishes that the ‘C^αNN’ segment has an intrinsic affinity for anions. Molecular Dynamics (MD) simulation studies reveal that interaction with anion triggers a conformational switch from non-helical to helical state at the ‘C^αNN’ segment, which extends the length of the anchoring-helix by one turn at the N-terminus. Computational experiments substantiate the significance of sequence/structural context and justify the conserved nature of the ‘C^αNN’ sequence for anion recognition through “local” interaction.</p> </div

Computational design of model scaffold for anion recognition based on the `(CNN)-N-alpha' motif

The `novel phosphate binding `CaNN' motif', consisting of three consecutive amino acid residues, usually occurs in the protein loop regions preceding a helix. Recent computational and complementary biophysical experiments on a series of chimeric peptides containing the naturally occurring `CaNN' motif at the N-terminus of a designed helix establishes that the motif segment recognizes the anion (sulfate and phosphate ions) through local interaction along with extension of the helical conformation which is thermodynamically favored even in a context-free, nonproteinaceous isolated system. However, the strength of the interaction depends on the amino acid sequence/conformation of the motif. Such a locally-mediated recognition of anions validates its intrinsic affinity towards anions and confirms that the affinity for recognition of anions is embedded within the `local sequence' of the motif. Based on the knowledge gathered on the sequence/structural aspects of the naturally occurring `CaNN' segment, which provides the guideline for rationally engineering model scaffolds, we have modeled a series of templates and investigated their interactions with anions using computational approach. Two of these designed scaffolds show more efficient anion recognition than those of the naturally occurring `CaNN' motif which have been studied. This may provide an avenue in designing better anion receptors suitable for various biochemical applications

Interaction parameters between the sulfate/phosphate ion with the related ‘C^αNN’ segment of the chimeric peptides in a context free system (250 docked structures of the individual conformation) are described in terms of X-H—O (where X = C^α₋₁/N₀/N₊₁) distances(<b>Å</b>) and angles(°) indicating the nature of H-bond formation (mean value of the parameters in parenthesis).

<p>The ranges of the parameters comply well with those obtained for respective crystal structures and described by Denessiouk et al., in 2005. The estimated binding free energy gives a measure of relative stability of interaction (relative affinity for the anion) which largely depends on the conformational status of the ‘C^αNN’ segment.</p

Details of NH—NH_(i,i+1) distance(s) around the ‘C^αNN’ region during the MD simulation.

<p>a) For 40 ns and b) first 8 ns; showing the destabilization of helical conformation at the ‘C^αNN’ segment in the MD trajectory.</p

Details of designed peptide sequences used for computational work.

a<p>PDB IDs from where the ‘C^αNN’ motif segment residues are taken, residue number of PDB IDs are in parenthesis; the</p>b<p>‘C^αNN’ motif segment is underlined;</p>c<p>anchor helix (ABGY) sequence: -Ala-Aib-Ala-Lys-Ala-Aib-Lys-Ala-Lys-Ala-Aib-Gly-Gly-Tyr-NH₂.</p