Structural Characterization of the Cyclic Cystine Ladder Motif of θ‑Defensins

The θ-defensins are, to date, the only known ribosomally synthesized cyclic peptides in mammals, and they have promising antimicrobial bioactivities. The characteristic structural motif of the θ-defensins is the cyclic cystine ladder, comprising a cyclic peptide backbone and three parallel disulfide bonds. In contrast to the cyclic cystine knot, which characterizes the plant cyclotides, the cyclic cystine ladder has not been as well described as a structural motif. Here we report the solution structures and nuclear magnetic resonance relaxation properties in aqueous solution of three representative θ-defensins from different species. Our data suggest that the θ-defensins are more rigid and structurally defined than previously thought. In addition, all three θ-defensins were found to self-associate in aqueous solution in a concentration-dependent and reversible manner, a property that might have a role in their mechanism of action. The structural definition of the θ-defensins and the cyclic cystine ladder will help to guide exploitation of these molecules as structural frameworks for the design of peptide drugs

Insights into the Molecular Flexibility of θ‑Defensins by NMR Relaxation Analysis

θ-Defensins are mammalian cyclic peptides that have antimicrobial activity and show potential as stable scaffolds for peptide-based drug design. The cyclic cystine ladder structural motif of θ-defensins has been characterized using NMR spectroscopy and is important for their structure and stability. However, the effect of the pronounced elongated topology of θ-defensins on their molecular motion is not yet understood. Studies of molecular motion by NMR relaxation measurements have been facilitated by the recent development of a semirecombinant method for producing cyclic peptides that allows for isotopic labeling. Here we have undertaken a multifield ¹⁵N NMR relaxation analysis of the anti-HIV θ-defensin, HTD-2, and interpreted the experimental data using various models of overall and internal molecular motion. We found that it was necessary to apply a model that includes internal motion to account for the variations in the experimental T₁ and NOE data at different backbone amide sites in the peptide. Although an isotropic model with internal motion was the simplest model that provided a satisfactory fit with the experimental data, we cannot exclude the possibility that overall motion is anisotropic, especially considering the strikingly elongated topology of θ-defensins. The presence of flexible side chains, self-association, interactions with solvent, and internal motions are all potential contributors to the observed relaxation data. Internal motion consistent with the constraints imposed by the cyclic cystine ladder was observed in that the order parameters, <i>S</i>², show that residues in the turns are more flexible than those in the β-sheet. This study provides insights into the dynamics of θ-defensins and information that might be useful in their application as scaffolds in drug design