The C-terminal mutation beyond the catalytic site of brown spider phospholipase D significantly impacts its biological activities

• Published in: Biochimie Vol. 211; pp. 122 - 130
• Main Authors: Cunha, Laís Cardoso, Barreto, Lucas Passos, Valadares, Veronica Silva, Oliveira, Camila Franco Batista, Vuitika, Larissa, Vilela, Maura Páscoa, Cino, Elio A., Silva, Adolfo Henrique de Moraes, Nagem, Ronaldo A.P., Chávez-Olórtegui, Carlos, Dias-Lopes, Camila, Molina, Franck, Felicori, Liza
• Format: Journal Article
• Language: English
• Published: France Elsevier B.V 01-08-2023; Elsevier
• Subjects: Animals; brown spider; Catalytic Domain; Circular dichroism; Dermonecrosis; Life Sciences; Mutation; Phospholipase D - chemistry; Phospholipase D - genetics; Phospholipase D - metabolism; Phospholipases D; Phosphoric Diester Hydrolases - genetics; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Site-directed mutagenesis; Sphingomyelin Phosphodiesterase; Spider Venoms - chemistry; Spider Venoms - genetics; Spiders - genetics; Phospholipases D; Dermonecrosis; brown spider; Site-directed mutagenesis; Circular dichroism
• ISSN: 0300-9084; 1638-6183
• DOI: 10.1016/j.biochi.2023.03.010

Loxosceles spider envenomation results in dermonecrosis, principally due to phospholipases D (PLDs) present in the venom. These enzymes have a strongly conserved sequence, 273ATXXDNPW280, in the C-terminal region (SMD-tail) that make contact with β-sheets of the TIM barrel, in which the amino acids Asp277 and Trp280 establish the energetically strongest contacts. The SMD-tail is conserved in PLDs from different species but absent in the non-toxic PLD ancestral glycerophosphodiester phosphodiesterases (GDPDs). This work aims to understand the role of the C-terminal region in the structural stability and/or function of phospholipases D. Through site-directed mutagenesis of the rLiD1 protein (recombinant Loxosceles intermedia dermonecrotic protein 1), we produced two mutants: rLiD1D277A and rLiD1W280A (both with sphingomyelinase activity), in which Asp277 and Trp280 were replaced by alanine. rLiD1D277A showed similar sphingomyelinase activity but at least 2 times more dermonecrotic activity than rLiD1 (wild-type protein). Conversely, while the rLiD1W280A displayed a slight increase in sphingomyelinase activity, its biological activity was similar or lower compared to rLiD1, potentially due to its decreased thermostability and formation of amyloid aggregates. In conclusion, these new findings provide evidence that SMD-tail mutants impact the structure and function of these proteins and point out that residues outside the active site can even increase the function of these enzymes.
•Spider PLD's C-terminal mutation, Asp277 to Ala, increases dermonecrosis in vivo.•The Asp277 to Ala mutation does not affect enzymatic activity in vitro.•Spider PLD's C-terminal mutation, Trp280 to Ala, lowers enzyme thermostability.•Trp280 to Ala mutation slightly increases enzyme activity in vitro.•Mutations in PLD's conserved C-terminal amino acids affect its structure and function.