PEPSTATIN-A - POLYMERIZATION OF AN OLIGOPEPTIDE

Pepstatin A, a pentapeptide with the molecular weight of 686, is a nat urally occurring inhibitor of aspartyl proteases secreted by Streptomy ces species. Above a critical concentration of 0.1 mM at low ionic str ength and neutral pH, it can polymerize into filaments which may exten d over several micrometers. After negative staining, these filaments s how a helical substructure with characteristic diameters ranging from 6 to 12 nm. Selected images at higher magnification suggest the filame nts are composed of two intertwined 6 nm strands. This is in agreement with the optical diffraction analysis which additionally established a periodic pitch of 25 nm for the helical intertwining. Rotary shadowi ng of the pepstatin A filaments clearly demonstrated the right-handedn ess of the helical twist. In physiological salt solution or at higher concentrations of pepstatin A, a variety of higher order structures we re observed, including ribbons, sheets and cylinders with both regular and twisted or irregular geometries. Pepstatin A can interact with in termediate filament subunit proteins. These proteins possess a long, a lpha-helical rod domain that forms coiled-coil dimers, which through b oth hydrophobic and ionic interactions form tetramers which, in turn: in the presence of physiological salt concentrations, polymerize into the 10 nm intermediate filaments. In the absence of salt, pepstatin A and intermediate filament proteins polymerize into long filaments with a rough surface and a diameter of 15-17 nm. This polymerization appea rs to be primarily driven by nonionic interactions between pepstatin A and polymerization-competent forms of intermediate filament proteins, resulting in a composite filament. Polymerization-incompetent proteol ytic fragments of vimentin, lacking portions of the head and/or tail d omain, failed to copolymerize with pepstatin A into long filaments und er these conditions. These peptides, as well as bovine serum albumin, were found to stick to the surface of pepstatin A filaments, ribbons a nd sheets. Independent evidence for direct association of pepstatin A with intermediate filament subunit proteins was provided not only by e lectron microscopy but also by UV difference spectra. Pepstatin A lose s its ability to inhibit the aspartyl protease of the human immunodefi ciency virus type 1 following polymerization into the higher order str uctures described here. The amazing fact that pepstatin A can spontane ously self-associate to form very large polymers seems to be a more ra re event for such small peptides. The other examples of synthetic or n aturally occurring oligopeptides discussed in this review which are ab le to polymerize into higher order structures possess a common propert y, their hydrophobicity, often manifested by clusters of valine or iso leucine residues. The mechanism of homo- or heteropolymerization may b e of interest not only for model studies on the polymerization of smal l peptides, but also for practical development of soluble protease inh ibitors and for the elucidation of the origin of the amyloid plaques o f Alzheimer's disease and Down's syndrome, to name a few examples.