Retrocyclin-2:  Structural Analysis of a Potent Anti-HIV θ-Defensin^,

Abstract

Retrocyclins are circular mini-defensins with significant potential as agents against human immunodeficiency virus, influenza A, and herpes simplex virus. Retrocyclins bind carbohydrate-containing surface molecules such as gp120 and CD4 with high affinity (Kd, 10-100 nM), promoting their localization on cell membranes. The structural features important for activity have yet to be fully elucidated, but here, we have determined the first three-dimensional structure of a retrocyclin, namely, one of the most potent forms, retrocyclin-2. In the presence of SDS micelles, a well-defined beta-hairpin braced by three disulfide bonds that defines the cystine ladder motif is present. By contrast, a well-defined structure could not be determined in aqueous solution, suggesting that the presence of SDS micelles stabilizes the extended conformation of retrocyclin-2. Translational diffusion measurements indicate that retrocyclin-2 interacts with the SDS micelles, and such a membrane-like interaction may be an important feature in the mechanism of action of these antimicrobial peptides. Analytical ultracentrifugation and the NMR data indicated that retrocyclin-2 self-associates to form a trimer in a concentration-dependent manner. The ability to self-associate may contribute to the high-affinity binding of retrocyclins for glycoproteins by increasing the valency and enhancing the ability of retrocyclins to cross-link cell surface glycoproteins.

Interactions of α-, β-, and θ-Defensins with Influenza A Virus and Surfactant Protein D

“…In the case of RC2 the increased ability to self-aggregate could result in more effective cross-linking of cell membrane receptors through self-association on the cell surface. The increased aggregating activity of RC2 could also reflect a greater ability to form trimers or to self-associate once bound to viral particles (46). The finding of significant differences in antiviral activity between RCs or other defensins based on single amino acid changes is surprising, but not unprecedented (14,44,45).…”

Multivalent Binding of Carbohydrates by the Human α-Defensin, HD5

“…These include four to six arginines plus six cysteines that form a tri-disulfide ladder cross-connecting their antiparallel ␤-strands (23,73). -Defensins kill bacteria (15,17), show broad-spectrum antiviral activity (5,21,74), and inhibit bacterial toxins, including anthrax lethal factor, a nonglycosylated zinc metalloprotease (75).…”

“…In such instances, carbohydrate epitopes of glycan chains afford likely biorecognition sites (19,20). The carbohydrate-binding ability of -defensins has been documented and examined with respect to their antiviral properties (21,22) and also because -defensins are among the smallest known lectins (23)(24)(25). Humans express six different ␣-defensins.…”

The Cyclic Cystine Ladder in θ-Defensins Is Important for Structure and Stability, but Not Antibacterial Activity

“…NMR solution structures have shown that -defensins have a highly constrained symmetrical conformation made up of two antiparallel ␤-strands joined by two turns (Fig. 1b) (9,10). The three disulfide bonds are all on one face of the molecule with the cationic arginine side chains exposed on the other face (Fig.…”