Structural, functional, and bioinformatics studies reveal a new snake venom homologue phospholipase A₂ class.
Phospholipases A₂ (PLA₂s) are enzymes responsible for membrane disruption through Ca(2+) -dependent hydrolysis of phospholipids. Lys49-PLA₂s are well-characterized homologue PLA₂s that do not show catalytic activity but can exert a pronounced local myotoxic effect. These homologue PLA₂s were first believed to present residual catalytic activity but experiments with a recombinant toxin show they are incapable of catalysis. Herein, we present a new homologue Asp49-PLA₂ (BthTX-II) that is also able to exert muscle damage. This toxin was isolated in 1992 and characterized as presenting very low catalytic activity. Interestingly, this myotoxic homologue Asp49-PLA₂ conserves all the residues responsible for Ca(2+) coordination and of the catalytic network, features thought to be fundamental for PLA₂ enzymatic activity. Previous crystallographic studies of apo BthTX-II suggested this toxin could be catalytically inactive since a distortion in the calcium binding loop was observed. In this article, we show BthTX-II is not catalytic based on an in vitro cell viability assay and time-lapse experiments on C2C12 myotube cell cultures, X-ray crystallography and phylogenetic studies. Cell culture experiments show that BthTX-II is devoid of catalytic activity, as already observed for Lys49-PLA₂s. Crystallographic studies of the complex BthTX-II/Ca(2+) show that the distortion of the calcium binding loop is still present and impairs ion coordination even though Ca(2+) are found interacting with other regions of the protein. Phylogenetic studies demonstrate that BthTX-II is more phylogenetically related to Lys49-PLA₂s than to other Asp49-PLA₂s, thus allowing Crotalinae subfamily PLA₂s to be classified into two main branches: a catalytic and a myotoxic one.
dos Santos
Juliana I
JI
Departamento de Física e Biofísica, Instituto de Biociências, UNESP - Univ Estadual Paulista, Botucatu-SP and Instituto Nacional de Ciência e Tecnologia em Toxinas, CNPq, Brazil.
Cintra-Francischinelli
Mariana
M
Borges
Rafael J
RJ
Fernandes
Carlos A H
CA
Pizzo
Paola
P
Cintra
Adélia C O
AC
Braz
Antonio S K
AS
Soares
Andreimar M
AM
Fontes
Marcos R M
MR
D000818:Animals; D017837:Bothrops; D002460:Cell Line; D019295:Computational Biology; D003435:Crotalid Venoms / Q000737:chemistry*; D018360:Crystallography, X-Ray; D003603:Cytotoxins / Q000737:chemistry / Q000235:genetics / Q000633:toxicity; D054501:Group II Phospholipases A2 / Q000737:chemistry*; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000187:drug effects; D054467:Phospholipases A2 / Q000737:chemistry* / Q000235:genetics / Q000633:toxicity*; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D040681:Structural Homology, Protein; D013329:Structure-Activity Relationship
D003435:Crotalid Venoms; D003603:Cytotoxins; D054501:Group II Phospholipases A2; D054467:Phospholipases A2; C072031:bothropstoxin II