High resolution structure of cleaved Serpin 42 Da from Drosophila melanogaster.
BACKGROUND
The 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.


RESULTS
We 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.


CONCLUSIONS
In 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.
Andrew M
AM
Ellisdon
Qingwei
Q
Zhang
Michelle A
MA
Henstridge
Travis K
TK
Johnson
Coral G
CG
Warr
Ruby Hp
RH
Law
Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC 3800, Australia. james.whisstock@monash.edu.
James C
JC
Whisstock
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D018360:Crystallography, X-Ray; D029721:Drosophila Proteins / Q000737:chemistry* / Q000378:metabolism; D004331:Drosophila melanogaster / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000378:metabolism; D015843:Serpins / Q000737:chemistry* / Q000378:metabolism
D029721:Drosophila Proteins; D020033:Protein Isoforms; D015842:Serine Proteinase Inhibitors; D015843:Serpins; C488721:Spn42Da protein, Drosophila