Structural and functional characterization of a highly specific serpin in the insect innate immunity.
The Toll signaling pathway, an essential innate immune response in invertebrates, is mediated via the serine protease cascade. Once activated, the serine proteases are irreversibly inactivated by serine protease inhibitors (serpins). Recently, we identified three serpin-serine protease pairs that are directly involved in the regulation of Toll signaling cascade in a large beetle, Tenebrio molitor. Of these, the serpin SPN48 was cleaved by its target serine protease, Spätzle-processing enzyme, at a noncanonical P1 residue of the serpin's reactive center loop. To address this unique cleavage, we report the crystal structure of SPN48, revealing that SPN48 exhibits a native conformation of human antithrombin, where the reactive center loop is partially inserted into the center of the largest β-sheet of SPN48. The crystal structure also shows that SPN48 has a putative heparin-binding site that is distinct from those of the mammalian serpins. Ensuing biochemical studies demonstrate that heparin accelerates the inhibition of Spätzle-processing enzyme by a proximity effect in targeting the SPN48. Our finding provides the molecular mechanism of how serpins tightly regulate innate immune responses in invertebrates.
College of Pharmacy and Research Institute for Drug Development, Pusan National University, Jangjeon-dong, Geumjeong-gu, Busan 609-735, Republic of Korea.
Sun Hee
SH
Park
Rui
R
Jiang
Shunfu
S
Piao
Bing
B
Zhang
Eun-Hye
EH
Kim
Hyun-Mi
HM
Kwon
Xiao Ling
XL
Jin
Bok Luel
BL
Lee
Nam-Chul
NC
Ha
D000595:Amino Acid Sequence; D000818:Animals; D000991:Antithrombins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics / Q000276:immunology; D002118:Calcium / Q000378:metabolism; D003461:Crystallography; D004789:Enzyme Activation / Q000276:immunology; D006493:Heparin / Q000378:metabolism; D006801:Humans; D007113:Immunity, Innate / Q000502:physiology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D015843:Serpins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D015398:Signal Transduction / Q000276:immunology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013712:Tenebrio / Q000201:enzymology* / Q000276:immunology*; D051193:Toll-Like Receptors / Q000276:immunology / Q000378:metabolism
D000991:Antithrombins; D015843:Serpins; D051193:Toll-Like Receptors; D006493:Heparin; D002118:Calcium