NMR relaxation and structural elucidation of peptides in the presence and absence of trifluoroethanol illuminates the critical molecular nature of integrin αvβ6 ligand specificity

Abstract

Integrin αvβ6 is an important emerging target for both imaging and therapy of cancer that requires specific ligands based on Arg-Gly-Asp (RGD) peptides. There remains little correlation between integrin-RGD ligand specificity despite studies suggesting an RGD-turn-helix ligand motif is required. Here, we describe the application of ¹⁵N NMR relaxation analyses and structure determination of αvβ6 peptide ligands in the presence and absence of trifluoroethanol (TFE) to identify their critical molecular nature that influences specificity, interaction and function. Two linear peptides; one known to demonstrate αvβ6 specificity (FMDV2) and the other based on a natural RGD ligand (LAP2), were compared to two additional peptides based on FMDV2 but cyclised in different positions using a disulphide bond (DBD1 and DBD2). The cyclic adaptation in DBD1 produces a significant alteration in backbone dynamic properties when compared to FMDV2; a potential driver for the loss in αvβ6 specificity by DBD1. The importance of ligand dynamics are highlighted through a comprehensive reduced spectral density and ModelFree analysis of peptide ¹⁵N NMR relaxation data and suggest αvβ6 specificity requires the formation of a structurally rigid helix preceded by a RGD motif exhibiting slow internal motion. Additional observations include the effect of TFE/water viscosity on global NMR dynamics and the advantages of using spectral density NMR relaxation data to estimate correlation times and motional time regimes for peptides in solution.

Fig. 1

Structures closest to the mean for each peptide and NOE contacts, hydrogen bond restraints assigned for DBD1(a), DBD2 (b), FMDV2 (c) and LAP (d) in the presence of 30% (v/v) TFE. Key residues of the RGDLXXL/I motif are shown as sticks on each structure.

Fig. 2

Parametric curves showing the dependence of J(ω_N) on J(ω_h) for individual ¹⁵N nuclei from (a) DBD1-TFE, (b) DBD2-TFE, (c) FMDV2-TFE, (d) LAP2-TFE, (e) DBD1+TFE, (f) DBD2+TFE, (g) FMDV2+TFE and (h) LAP2+TFE. Defined structure regions from Fig. 1 are coloured blue for RGD-turn and red for the helix region for each peptide. The continuous curved line represents the dependence of theoretical Lorentzian spectral density functions with variable correlation time. The large-dashed line (–– –) is a least-squared fit to all the data points in (a-h), the small-large-dashed line (- – - – ) is a fit to only RGD-helix data points in (a-d) and the small-dashed line (----) is the motional extremes as defined from the data. Motional times defined as the intercept of each dashed line with the theoretical curve are shown on individual plots.

Fig. 3

Parametric curves showing the dependence of J(ω_h) on J(0) for individual ¹⁵N nuclei from (a) DBD1-TFE, (b) DBD2-TFE, (c) FMDV2-TFE, (d) LAP2-TFE, (e) DBD1+TFE, (f) DBD2+TFE, (g) FMDV2+TFE and (h) LAP2+TFE. Defined structure regions from Fig. 1 are coloured blue for RGD-turn and red for the helix region for each peptide. The continuous curved line represents the dependence of theoretical Lorentzian spectral density functions with variable correlation time. The large-dashed line (– – – –) represents a least-squared fit to all the data points in (a-h). Motional times defined as the intercept of each dashed line with the theoretical curve are shown on individual plots.

Fig. 4

Column plots describing the results of model-free Lipari-Szabo analysis of (a), (e) and (i) DBD1+TFE, (b), (f), (j) and (m) DBD2+TFE, (c), (g), (k) and (n) FMDV2+TFE and (d), (h), (l) and (o) LAP2+TFE with amino acid sequence. Data reporting within TFE defined structure regions from Fig. 1 are coloured blue for RGD-turn and red for the helix region for each peptide.

Figure 5

Column plots describing the order parameter S² results of model-free Lipari-Szabo analysis of (a) DBD1-TFE, (b) DBD2-TFE, (c) FMDV2-TFE and (d) LAP2-TFE with amino acid sequence. Data reporting within TFE defined structure regions from Fig. 1 are coloured blue for RGD-turn and red for the helix region for each peptide.