β-Trefoil inhibitors – from the work of Kunitz onward

Abstract

Protein protease inhibitors are the tools of nature in controlling proteolytic enzymes. They come in different shapes and sizes. The β -trefoil protease inhibitors that come from plants, fi rst discovered by Kunitz, were later complemented with representatives from higher fungi. They inhibit serine (families S1 and S8) and cysteine proteases (families C1 and C13) as well as other hydrolases. Their versatility is the result of the plasticity of the loops coming out of the stable β -trefoil scaffold. For this reason, they display several different mechanisms of inhibition involving different positions of the loops and their combinations. Natural diversity, as well as the initial successes in de novo protein engineering, makes the β -trefoil proteins a promising starting point for the generation of strong, specifi c, multitarget inhibitors capable of inhibiting multiple types of hydrolytic enzymes and simultaneously interacting with different protein, carbohydrate, or DNA molecules. This pool of knowledge opens up new possibilities for the exploration of their naturally occurring as well as modifi ed properties for applications in many fi elds of medicine, biotechnology, and agriculture.

Crystal Structures of a Plant Trypsin Inhibitor from Enterolobium contortisiliquum (EcTI) and of Its Complex with Bovine Trypsin

“…As found in most known Kunitz-type inhibitors [2], EcTI has a single reactive site located on loop L5, between β-strands 4 and 5. An exception is the recently published complex structure of a double-headed serine arrowhead protease inhibitor, API-A complexed with two molecules of trypsin, in which two reactive sites are located in loops L6 and L10 [21].…”

“…Members of the superfamily characterized by the β-trefoil fold [1] are structurally similar although their biological functions may be widely different [2]. Such functions may include chlorophyll binding [3], taste modification (miraculin [4]), binding to cytokine receptors (IL-1β [5]), tight binding to ribosomes (ricin [6]) or carbohydrate binding, exemplified by the Clitocybe nebularis lectin, CNL [7].…”

Structures of a bi-functional Kunitz-type STI family inhibitor of serine and aspartic proteases: Could the aspartic protease inhibition have evolved from a canonical serine protease-binding loop?

“…These inhibitors have a b-trefoil fold, composed of 12 b-strands arranged in threefold pseudo-symmetry units or subdomains with the presence of helices in some cases (Broom et al, 2012;McLachlan, 1979;Murzin et al, 1992;Renko et al, 2012). The pseudo-symmetry units are composed of four b-strands, two of them forming a sixstranded b-barrel with the other subdomains, and the other two of each unit forming a b-hairpin acting like the cap for the bbarrel.…”

“…The structural regions reported to be involved in protease-inhibitor interactions are loops connecting the b-strand elements (i.e. loop L4 between b-strands 4 and 5 in STI), which can be highly variable in sequence and length (Renko et al, 2012). The most accepted model for the b-trefoil fold evolution proposes that the fold arose from a duplication and fusion of the monomeric unit from an ancestral trimeric protein (Broom et al, 2012;Ponting and Russell, 2000).…”

Crystal Structure of Crataeva tapia Bark Protein (CrataBL) and Its Effect in Human Prostate Cancer Cell Lines

“…Members of this superfamily share structural features but not necessarily other properties and their biological role may be widely different [2]. Prominent among them are plant protease inhibitors of the Kunitz type, called Kunitz-P or Kunitz-STI inhibitors [2], [3]. These proteins primarily inhibit serine proteases, although some also inhibit cysteine and aspartic proteases [4].…”