Functional and structural characterization of 2B viroporin membranolytic domains.
Nonstructural 2B viroporin is an intracellularly produced pore-forming protein required for effective enteroviral and rhinoviral replication. The sequence of 2B displays two putative interconnected transmembrane domains, which are predicted to insert into the negatively charged membranes of target organelles forming an integral hairpin. The use of an overlapping peptide library that spanned the complete 2B sequence has recently allowed the mapping of the cell plasma membrane porating activity to the partially amphipathic, amino-terminal transmembrane domain (TM1, residues 35-55). We describe here that although the TM1 peptide was effective in permeabilizing uncharged membranes, it induced marginal lysis of anionic bilayers. In fact, only the peptide representing the highly conserved carboxy-terminal transmembrane domain (TM2, residues 59-82) reproduced the capacity of the full 2B protein to efficiently permeabilize bilayers made of anionic phospholipids. Insertion into lipid monolayers and circular dichroism determinations were, however, consistent with penetration of the TM1 helix into both anionic and zwitterionic membranes, while TM2 interacting with membranes assumed a mixture of conformations. Moreover, addition of TM1 strongly stimulated TM2-induced permeabilization of the anionic membranes. In combination, TM1 and TM2 formed a complex that had structural properties, including a high proportion of extended nonhelical secondary structure, that were distinct from those of the individual peptides. Finally, a comparison of antimicrobial and hemolytic activities further underscored the TM1 domain's cytolytic character. Overall, our data support the idea that the cytolytic activity of TM1 in the negatively charged cell endomembranes targeted by 2B viroporin requires the cooperation of both transmembrane domains.
Sánchez-Martínez
Silvia
S
Unidad de Biofísica (CSIC-UPV/EHU) and Departamento de Bioquímica, Universidad del País Vasco, Aptdo. 644, 48080 Bilbao, Spain.
Huarte
Nerea
N
Maeso
Rubén
R
Madan
Vanessa
V
Carrasco
Luis
L
Nieva
José L
JL
D002942:Circular Dichroism; D008081:Liposomes / Q000737:chemistry*; D010713:Phosphatidylcholines / Q000737:chemistry; D010716:Phosphatidylinositols / Q000737:chemistry; D017434:Protein Structure, Tertiary; D017361:Viral Nonstructural Proteins / Q000737:chemistry*
D008081:Liposomes; D010713:Phosphatidylcholines; D010716:Phosphatidylinositols; D017361:Viral Nonstructural Proteins