Conformational Dynamics in Extended RGD-Containing Peptides

Abstract

RGD is a prolific example of a tripeptide used in biomaterials for cell adhesion, but the potency of free or surface-bound RGD tripeptide is orders-of-magnitude less than the RGD domain within natural proteins. We designed a set of peptides with varying lengths, composed of fragments of fibronectin protein whose central three residues are RGD, in order to vary their conformational behavior without changing the binding site's chemical environment. With these peptides, we measure the conformational dynamics and transient structure of the active site. Our studies reveal how flanking residues affect conformational behavior and integrin binding. We find that disorder of the binding site is important to the potency of RGD peptides and that transient hydrogen bonding near the RGD site affects both the energy landscape roughness of the peptides and peptide binding. This phenomenon is independent of longer-range folding interactions and helps explain why short binding sequences, including RGD itself, do not fully replicate the integrin-targeting properties of extracellular matrix proteins. Our studies reinforce that peptide binding is a holistic event and fragments larger than those directly involved in binding should be considered in the design of peptide epitopes for functional biomaterials.

Figure 1.

The structure of RGD-containing fibronectin fragments. Fibronectin fragments studied are highlighted in purple. RGD sites of each peptide are highlighted in yellow. (a) FMP9 includes the RGD site and three flanking residues on either side. (b) FMP15 includes the RGD site and six flanking residues on either side. (c) FMP21 includes the RGD site and nine flanking residues on either side. (d) FMP27 includes the RGD site and 12 flanking residues on either side.

Figure 2.

Dynamic behavior at the RGD site changes discontinuously with length. (a) Arrhenius plots of rotational diffusion of TOAC peptides in PBS buffer, generated from EPR spectral analysis. (b) Arrhenius plots of rotational diffusion determined by EPR in 25% DMSO/75% buffer. The DMSO denaturant prevents secondary structure formation. (c) Activation energy of rotational diffusion in buffer (black data points) and 25% DMSO as a denaturant (red data points).

Figure 3.

Molecular dynamics simulations illustrate intrachain hydrogen bond formation in FMP peptides and fibronectin protein. (a) Hydrogen bond probability maps of FMP peptides and the corresponding region of fibronectin, determined by MD simulations. Weak hydrogen bonding between residues near to the RGD site (appearing near the y = x line) is observed. Strong hydrogen bonding between flanking chains is observed in FMP27 and fibronectin, but the residues involved in these intramolecular hydrogen bonds differ between FMP27 and fibronectin. (b) Intramolecular hydrogen bonding occurring more than 4% of the time is depicted in purple. A transient hydrogen bonding pattern appears along the y = x line for FMP15, FMP21, and FMP27, which has not fully evolved in FMP9.

Figure 4.

Distributions determined by DEER (black) and MD simulations (red) describe the distances between the glycine of RGD and the N-terminus of each FMP peptide. FMP9 shows a narrow distribution centered at 1.9 nm, and FMP15, FMP 21, and FMP27 each show broader distance distributions centered at 2.3, 2.9, and 3.4 nm, respectively.

Figure 5.

Displacement of fluorescently labeled fibronectin, bound to cellular αVβ3, by FMP peptides. Normalized mean fluorescence intensity (MFI) of integrin-bound Alexa647-labeled hFN10 protein is presented as a function of FMP peptide concentration. The longer peptides, relative to FMP9, exhibit a ~5× improvement in their ability to bind to activated integrins. In the longest peptide (FMP27), this improvement is eliminated due to misfolding. The half-maximal inhibitory concentrations (IC₅₀s) observed are FMP9, 1030 ± 208 nM; FMP15, 172 ± 30 nM; FMP21, 218 ± 52 nM; and FMP27, 2740 ± 1070 nM.