High resolution structure of cleaved Serpin 42 Da from Drosophila melanogaster

Abstract

BackgroundThe Drosophila melanogaster Serpin 42 Da gene (previously Serpin 4) encodes a serine protease inhibitor that is capable of remarkable functional diversity through the alternative splicing of four different reactive centre loop exons. Eight protein isoforms of Serpin 42 Da have been identified to date, targeting the protease inhibitor to both different proteases and cellular locations. Biochemical and genetic studies suggest that Serpin 42 Da inhibits target proteases through the classical serpin ‘suicide’ inhibition mechanism, however the crystal structure of a representative Serpin 42 Da isoform remains to be determined.ResultsWe report two high-resolution crystal structures of Serpin 42 Da representing the A/B isoforms in the cleaved conformation, belonging to two different space-groups and diffracting to 1.7 Å and 1.8 Å. Structural analysis reveals the archetypal serpin fold, with the major elements of secondary structure displaying significant homology to the vertebrate serpin, neuroserpin. Key residues known to have central roles in the serpin inhibitory mechanism are conserved in both the hinge and shutter regions of Serpin 42 Da. Furthermore, these structures identify important conserved interactions that appear to be of crucial importance in allowing the Serpin 42 Da fold to act as a versatile template for multiple reactive centre loops that have different sequences and protease specificities.ConclusionsIn combination with previous biochemical and genetic studies, these structures confirm for the first time that the Serpin 42 Da isoforms are typical inhibitory serpin family members with the conserved serpin fold and inhibitory mechanism. Additionally, these data reveal the remarkable structural plasticity of serpins, whereby the basic fold is harnessed as a template for inhibition of a large spectrum of proteases by reactive centre loop exon ‘switching’. This is the first structure of a Drosophila serpin reported to date, and will provide a platform for future mutational studies in Drosophila to ascertain the functional role of each of the Serpin 42 Da isoforms.

Serpins in arthropod biology

“…Currently, the structures of seven different arthropod serpins have been solved from M. sexta [122,123], D. melanogaster [124], An. gambiae [125], B. mori [126], I. ricinus [108], and T. molitor [35].…”

“…Two arthropod serpin structures have been determined in their cleaved, inactivated form: D. melanogaster Spn4 (Serpin 42 Da) (pdb codes: 4P0O and 4P0F, [124]) and I. ricinus IRS-2 (pdb code 3NDA, [148]) (Fig. 4E), previously discussed in Sections 2.3 and 4.2, respectively.…”

“…gambiae SRPN2 (blue, pdb code: 3PZF) [125], and M. sexta serpin 1K (teal) demonstrating the partial hinge insertion found in antithrombin, SPN48 and SRPN2. (E) Alignment of the cleaved D. melanogaster Serpin 42 Da (pdb codes: 4P0O) [124] and I. ricinus IRS-2 (pdb code 3NDA) [108] structures indicating complete insertion of the RCL (magenta and purple, respectively), indicated by an arrow. Although neither protein was crystallized in an inhibitory complex with a proteinase, the presumed position of the inactivated proteinase in such a complex is marked with an asterisk.…”

A serine protease inhibitor from the hornfaced bee, Osmia cornifrons, exhibits antimicrobial activities

“…Like many serpins, the C-terminus of OcSPI contains a potential RCL region, including the hinge region and proteinase cleavage site characteristic of active proteinase inhibitors (Osterwalder et al, 2004;Garrett et al, 2009). Additionally, OcSPI contains Phe and Phe residues at the putative P1-P1′ positions, but did not contain a tetrapeptide reminiscent of an ER retention signal, as observed in Drosophila serpin4.1 and vertebrate neuroserpins (Osterwalder et al, 2004;Garrett et al, 2009;Ellisdon et al, 2014). The C-terminal portions of the OcSPI protein (including the conserved hinge region, the reactive site, and the serpin signature region) exhibited 42% protein sequence identity to the Drosophila Serpin27A (Osterwalder et al, 2004).…”

“…In Drosophila, serpin 4 is the closest vertebrate neuroserpin homolog and functions as a neuroserpin-like inhibitor of subtilisin-like proprotein convertases (Osterwalder et al, 2004). A recent study demonstrated that the D. melanogaster Spn42Da (previously known as serpin 4) encodes eight protein isoforms (Ellisdon et al, 2014) that inhibit furin-, subtilase-and chymotrypsin-like serine proteases and papain-like cysteine proteases (Oley et al, 2004;Osterwalder et al, 2004;Richer et al, 2004). From these results, Spn42Da isoforms potentially improve immune defense by inhibiting pathogenic proteolytic enzymes (Brüning et al, 2007).…”