Subtleties among subtilases. The structural biology of Kex2 and furin-related prohormone convertases.

Prohormone processing was discovered 35 years ago with the observation that proinsulin is the precursor of both the A and the B chains of insulin (Steiner, 1969). During the next 15 years, many candidate proteases were proposed and rejected before genetic and biochemical evidence identified specific processing proteases in Saccharomyces cerevisiae that are involved in α-factor biosynthesis ( Julius et al., 1983, 1984). The initial cleavages of the precursor—endoproteolytic scissions carboxyl to Lys-Arg sites—are due to the action of Kex2 protease. Mammalian Kex2 homologues, known as furin, PC1/3, PC2, PC4, PACE4, PC5/6 and PC7/LPC were cloned throughout the 1990s. They were shown to process hormone and neuropeptide precursors, growth factors, receptors, metalloand aspartyl proteases, envelope glycoproteins of many viruses including HIV, and bacterial toxins such as anthrax-protective antigen (Zhou et al., 1999; Thomas, 2002). These Kex2-homologous prohormone convertases (PCs) are unrelated to the tryptic enzymes of the blood coagulation cascade that hydrolyse precursors carboxyl to basic amino acids. By contrast, PCs are related to degradative enzymes, such as subtilisin and proteinase K (Fuller et al., 1989). Thus, accounting for the refined functions and specificity of PCs has been a major research problem. Molecular-genetic studies have established a ‘P-domain’ carboxyl to the subtilisin domain that is essential for the biosynthesis and/or activity of PCs. Biochemical analyses have further indicated that PCs are calcium-dependent and contain several specificity sites that confer substrate discrimination. The recent publication of the crystal structures of the secreted, soluble forms of yeast Kex2 (Holyoak et al., 2003) and murine furin (Henrich et al., 2003), each bound to specific peptidyl inhibitors, have brought these features into focus and accounted for the subtleties of these refined subtilases. Moreover, the structural coordinates may accelerate the development of inhibitors to target the bacteria and viruses that rely on processing by these proteases for infectivity. PCs are multidomain proteins that are targeted to late secretory compartments. Carboxyl to the signal sequence is an 80–90 aminoacid prodomain that is autocatalytically removed during maturation (Germain et al., 1992) in a process that involves acid-induced dissociation and cleavage at a second, internal pro-domain site (Anderson et al., 2002). From residue 144 to 438 (numbering according to Kex2), PCs contain a catalytic domain ~30% identical to subtilisin. Carboxyl to the subtilisin domain is the essential P-domain (Gluschankof & Fuller, 1994), followed by isozymespecific features that are primarily involved in localization (Zhou et al., 1999). The experiments that identified and delimited the P-domain (Gluschankof & Fuller, 1994) could not determine whether it forms part of the active site or has some other essential role in biosynthesis. The new crystal structures physically establish the P-domain as a true polypeptide domain (Holyoak et al., 2003; Henrich et al., 2003). As shown in Fig. 1, the P-domain is a closed ‘thicket’ of β-strands that contributes no amino acid to the active site or specificity pockets. Although the P-domain is located on the far side of the molecule relative to the amino terminus, the prodomain,