Structure and lytic activity of a Bacillus anthracis prophage endolysin.
We report a structural and functional analysis of the lambda prophage Ba02 endolysin (PlyL) encoded by the Bacillus anthracis genome. We show that PlyL comprises two autonomously folded domains, an N-terminal catalytic domain and a C-terminal cell wall-binding domain. We determined the crystal structure of the catalytic domain; its three-dimensional fold is related to that of the cell wall amidase, T7 lysozyme, and contains a conserved zinc coordination site and other components of the catalytic machinery. We demonstrate that PlyL is an N-acetylmuramoyl-L-alanine amidase that cleaves the cell wall of several Bacillus species when applied exogenously. We show, unexpectedly, that the catalytic domain of PlyL cleaves more efficiently than the full-length protein, except in the case of Bacillus cereus, and using GFP-tagged cell wall-binding domain, we detected strong binding of the cell wall-binding domain to B. cereus but not to other species tested. We further show that a related endolysin (Ply21) from the B. cereus phage, TP21, shows a similar pattern of behavior. To explain these data, and the species specificity of PlyL, we propose that the C-terminal domain inhibits the activity of the catalytic domain through intramolecular interactions that are relieved upon binding of the C-terminal domain to the cell wall. Furthermore, our data show that (when applied exogenously) targeting of the enzyme to the cell wall is not a prerequisite of its lytic activity, which is inherently high. These results may have broad implications for the design of endolysins as therapeutic agents.
Low
Lieh Yoon
LY
Infectious and Inflammatory Disease Center, The Burnham Institute, La Jolla, California 92037, USA.
Yang
Chen
C
Perego
Marta
M
Osterman
Andrei
A
Liddington
Robert C
RC
D000581:Amidohydrolases / Q000737:chemistry / Q000502:physiology*; D000595:Amino Acid Sequence; D001408:Bacillus anthracis / Q000378:metabolism*; D001426:Bacterial Proteins; D001665:Binding Sites; D020134:Catalytic Domain; D002473:Cell Wall / Q000378:metabolism; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000737:chemistry*; D016680:Genome, Bacterial; D008954:Models, Biological; D008958:Models, Molecular; D015233:Models, Statistical; D008969:Molecular Sequence Data; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000737:chemistry; D010457:Peptidoglycan / Q000737:chemistry; D039002:Prophages / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013997:Time Factors; D015032:Zinc / Q000737:chemistry
D001426:Bacterial Proteins; D010457:Peptidoglycan; D010450:Endopeptidases; C001143:endolysin; D000581:Amidohydrolases; C506058:PlyL protein, Bacillus anthracis; D009238:N-Acetylmuramoyl-L-alanine Amidase; C019665:amidase; D015032:Zinc