BTD-7

Short cationic, amphipathic antimicrobial peptides are multi-functional molecules that have roles in host defense as direct microbicides and modulators of the immune response. While a general mechanism of microbicidal activity involves the selective disruption and permeabilization of cell membranes, the relationships between peptide sequence and membrane activity are still under investigation. Here, we review the diverse functions that AMPs collectively have in host defense, and show that these functions can be multiplexed with a membrane mechanism of activity derived from the generation of negative Gaussian membrane curvature. As AMPs preferentially generate this curvature in model bacterial cell membranes, the selective generation of negative Gaussian curvature provides AMPs with a broad mechanism to target microbial membranes. The amino acid constraints placed on AMPs by the geometric requirement to induce negative Gaussian curvature are consistent with known AMP sequences. This 'saddle-splay curvature selection rule' is not strongly restrictive so AMPs have significant compositional freedom to multiplex membrane activity with other useful functions. The observation that certain proteins involved in cellular processes which require negative Gaussian curvature contain domains with similar motifs as AMPs, suggests this rule may be applicable to other curvature-generating proteins. Since our saddle-splay curvature design rule is based upon both a mechanism of activity and the existing motifs of natural AMPs, we believe it will assist the development of synthetic antimicrobials.

The first cyclic peptide discovered in animals is an antimicrobial octadecapeptide that is expressed in leukocytes of rhesus monkeys. The peptide, termed rhesus Theta-defensin 1 (RTD-1) is the prototype of a new family of antimicrobial peptides, which like the previously characterized alpha- and beta-defensin families, possesses broad spectrum microbicidal activities against bacteria, fungi, and protects mononuclear cells from infection by HIV-1. The cyclic Theta-defensin structure is essential for a number of its antimicrobial properties, as demonstrated by the markedly reduced microbicidal activities of de-cyclized Theta-defensin analogs. Genetic and biochemical experiments disclosed that the biosynthesis of RTD-1 results from the head-to-tail joining of two nine-amino acid peptides, each of which is donated by a separate precursor polypeptide, which are in fact C-terminally truncated pro-alpha-defensins. Alternate combinations of the two nonapeptides generate two additional macaque Theta-defensins, RTD-2 and RTD-3. Humans do not express Theta-defensin peptides, but mRNAs encoding at least two Theta-defensins are expressed in human bone marrow. However, in each case the open reading frame is interrupted by a stop codon in the signal peptide-coding region. The mature Theta-defensin peptide is a two-stranded beta-sheet that, like the alpha- and beta-defensins, is stabilized by three disulfides. However, the parallel orientation of the Theta-defensin disulfide arrangement allows for substantial flexibility around its short axis. Unlike alpha- and beta-defensins, RTD-1 lacks an amphiphilic topology. This may partially explain the unusual interaction between Theta-defensins and phospholipid bilayers.

The conserved tridisulfide array of the α-defensin family imposes a common triple-stranded β-sheet topology on peptides that may have highly diverse primary structures, resulting in differential outcomes after targeted mutagenesis. In mouse cryptdin-4 (Crp4) and rhesus myeloid α-defensin-4 (RMAD4), complete substitutions of Arg with Lys affect bactericidal peptide activity very differently. Lys-for-Arg mutagenesis attenuates Crp4, but RMAD4 activity remains mostly unchanged. Here, we show that the differential biological effect of Lys-for-Arg replacements can be understood by the distinct phase behavior of the experimental peptide-lipid system. In Crp4, small-angle x-ray scattering analyses showed that Arg-to-Lys replacements shifted the induced nanoporous phases to a different range of lipid compositions compared with the Arg-rich native peptide, consistent with the attenuation of bactericidal activity by Lys-for-Arg mutations. In contrast, such phases generated by RMAD4 were largely unchanged. The concordance between small-angle x-ray scattering measurements and biological activity provides evidence that specific types of α-defensin-induced membrane curvature-generating tendencies correspond directly to bactericidal activity via membrane destabilization.