Structural insights into catalytic and substrate binding mechanisms of the strategic EndA nuclease from Streptococcus pneumoniae.
EndA is a sequence non-specific endonuclease that serves as a virulence factor during Streptococcus pneumoniae infection. Expression of EndA provides a strategy for evasion of the host's neutrophil extracellular traps, digesting the DNA scaffold structure and allowing further invasion by S. pneumoniae. To define mechanisms of catalysis and substrate binding, we solved the structure of EndA at 1.75 Å resolution. The EndA structure reveals a DRGH (Asp-Arg-Gly-His) motif-containing ββα-metal finger catalytic core augmented by an interesting 'finger-loop' interruption of the active site α-helix. Subsequently, we delineated DNA binding versus catalytic functionality using structure-based alanine substitution mutagenesis. Three mutants, H154A, Q186A and Q192A, exhibited decreased nuclease activity that appears to be independent of substrate binding. Glu205 was found to be crucial for catalysis, while residues Arg127/Lys128 and Arg209/Lys210 contribute to substrate binding. The results presented here provide the molecular foundation for development of specific antibiotic inhibitors for EndA.
Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Andrea F
AF
Moon
Marika
M
Midon
Gregor
G
Meiss
Alfred
A
Pingoud
Robert E
RE
London
Lars C
LC
Pedersen
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D002413:Cations, Divalent / Q000737:chemistry; D004247:DNA / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007093:Imidazoles / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011485:Protein Binding; D013296:Streptococcus pneumoniae / Q000201:enzymology*
D001426:Bacterial Proteins; D002413:Cations, Divalent; D007093:Imidazoles; D008565:Membrane Proteins; D004247:DNA; D004706:Endodeoxyribonucleases; C064668:endA protein, bacteria; D000409:Alanine