Structure and Lytic Activity of a Bacillus anthracis Prophage Endolysin
We report a structural and functional analysis of the λ 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 the of We report a structural and functional analysis of the λ 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 the of are that the cell wall the lytic of the of an N-terminal catalytic domain and a C-terminal domain that the enzyme to the cell species and specificity the and but are in the of the cell wall-binding domain analysis of Bacillus anthracis a a endolysin the of the λ Ba02 which we PlyL a of in its catalytic domain an endolysin from the which and B. anthracis and related species when to is a and we a structural and functional analysis of We show that the N-terminal domain is an amidase lytic activity the cell wall of several Bacillus to we that the C-terminal domain a not a cell wall targeting domain but an of catalytic activity in the of the and of and C-terminal PlyL by from the Bacillus anthracis by of using the and We the the and using the from to of and and to to of to PlyL by of wall-binding domain. and to the cell by and by a an of and by the of of in a of applied to the to the the full-length PlyL, more than by and to to further the and of by using an and its N-terminal domain and cell the and of and similar to the but to the N-terminal by and of the PlyL N-terminal catalytic domain by of the full-length PlyL using a of a to the catalytic domain. to and analysis that a and the by by using a of of of and of to in cell a To data the in a of to a of data C-terminal and to that of the of the C-terminal domain by and in by crystal to a of in to using a and cell a of the PlyL data the using a and using the and of a zinc in the crystal by a the zinc using and to a of by further using and the data a using the of the structure the to a of are in the structure of and a of the two in the and are the and its are than the other two are in and structure have the the and in the of the in to the is the of of the is the data using of data from of of from is the the is the data using of data from in a of activity of PlyL and when applied to of B. anthracis Bacillus cereus Bacillus Bacillus and tested. to and and in of cell upon of endolysin of the in from B. PlyL in of PlyL a and and the of using a in of and of in and the in the in the by a a from to in in and detected of the by a using the of using the and by and and the of the C-terminal domain of PlyL, which using and the that the of the full-length domain and a a and and using and the binding by to and to a analysis the and and the of to the and of using the the two an prophage of B. λ and λ Ba02 are and in the of B. anthracis from other Bacillus species and detected in than catalytic are in PlyL is related to in the and C-terminal is similar but the C-terminal and a B. anthracis of the full-length using to a N-terminal the the catalytic and cell wall-binding more than the full-length and We the C-terminal domain; we have not its the of that to that is an autonomously folded of PlyL in N-terminal the activity of PlyL, from B. full-length PlyL and the N-terminal in from that the enzyme is a a of the the by the of which that the enzyme is an N-acetylmuramoyl-l-alanine amidase, the and the N-terminal that comprises a catalytic domain. N-terminal domain more than the full-length in the that the C-terminal domain is of the PlyL N-terminal the structure of the PlyL catalytic domain using from a fold is similar to of the T7 B. and the which we have the and of T7 fold of a by the and the and a of and T7 lysozyme, an N-terminal an of the zinc to the of the the of the by from and by two and of is a from the structure of PlyL catalytic domain and related of the of PlyL, T7 and zinc is a the structure T7 and are and and are and of PlyL, and are the of the of PlyL and site of PlyL and are of these are except which in the is by the but is by a to the in of of the site of PlyL T7 and site is and in a the the to a and more the in PlyL than in T7 lysozyme, that the T7 is not site that of T7 and are conserved PlyL and T7 is an in PlyL is to of T7 lysozyme, which to by the the in the a in T7 and in are and are to the to the the of the in PlyL to in the crystal structure the of a and the of a the to the T7 To demonstrate a catalytic in PlyL, we to and the amidase is folded by its to the not are the N-terminal of PlyL and that three-dimensional These are the three-dimensional of PlyL of the are the of the and of are from the site and a binding that the two catalytic have similar specificity and catalytic of PlyL and the lytic activity of PlyL of several by and by We that the full-length PlyL B. cereus an that B. anthracis and of B. cereus in a lytic activity of PlyL B. and but activity B. and B. and cell wall binding activity of PlyL and of of Bacillus species by full-length PlyL and its N-terminal catalytic domain except B. anthracis the the full-length and catalytic domain of PlyL to the by the from of Bacillus species by the B. cereus and its N-terminal domain. are the in of the binding to the cell wall of B. cereus, the other Bacillus species the not unexpectedly, that the N-terminal catalytic domain of PlyL is more than the full-length in B. B. and B. anthracis B. and the of the C-terminal domain a the lytic activity B. the of we the lytic activity of the from the B. cereus phage, is PlyL in its catalytic domain, its C-terminal domain to B. cereus when B. are We specificity the full-length but a in lytic activity B. by the N-terminal domain. of B. cereus by the N-terminal domain the full-length enzyme further the of the C-terminal domain of PlyL, we cell binding using a C-terminal domain to B. cereus and a a the binding B. B. anthracis not have that the endolysin from the B. anthracis λ prophage PlyL, is a cell wall lytic amidase a an N-terminal catalytic domain and a C-terminal cell wall-binding domain. We determined the three-dimensional structure of the catalytic domain and that the fold and site are similar to but from that of T7 and other and are the Bacillus in are the other and of not from T7 lysozyme, but its a similar to the in T7 lysozyme, and we a in by results that of the in have an N-acetylmuramoyl-l-alanine amidase activity and a similar catalytic the and we that its N-terminal domain a similar lytic specificity Bacillus species that the catalytic domain of the B. have a similar the that the PlyL and catalytic not to the that the lytic of the full-length are by the C-terminal domain, which is that the C-terminal domain is a cell wall-binding domain and that B. cereus We further that the of the the full-length PlyL an the lytic activity of the catalytic domain when the of B. to a B. and B. the of the a the activity of PlyL B. We that is not a of PlyL, we a similar pattern of activity a endolysin that is B. cereus, the domain that of the PlyL and a similar catalytic domain but in the C-terminal cell wall-binding domain. that in the cell wall-binding domain an of by the catalytic domain and the catalytic domain to in the of a cell propose the the of PlyL and have the of cell the an similar to a in the structure of the catalytic domain, is that the is the of the domain from a that is to the in cell wall binding which the the and the catalytic domain the the in the activity of the full-length PlyL and the N-terminal domain B. by binding of the to the B. cell the cell wall is to the amidase the case of B. cereus the full-length and have an activity, we propose that strong binding of the to the cell wall the the catalytic domain. results that of the domain to the cell the of a of of in full-length PlyL, the C-terminal domain to and the catalytic activity of the N-terminal domain binding of the C-terminal domain to a by of a B. the the catalytic domain, to an the of an in the case of B. B. a in the full-length PlyL in the a of the C-terminal domain the enzyme in a are to a species of by of that cell wall that the catalytic domain of PlyL strong lytic activity a of Bacillus species and that activity not is the that the PlyL of are that the of are when the is applied than We that are have to lytic activity when the is These may have implications the of are that the cell wall the lytic of the of an N-terminal catalytic domain and a C-terminal domain that the enzyme to the cell species and specificity the and but are in the of the cell wall-binding domain analysis of Bacillus anthracis a a endolysin the of the λ Ba02 which we PlyL a of in its catalytic domain an endolysin from the which and B. anthracis and related species when to is a and we a structural and functional analysis of We show that the N-terminal domain is an amidase lytic activity the cell wall of several Bacillus to we that the C-terminal domain a not a cell wall targeting domain but an of catalytic activity in the of the and of and C-terminal PlyL by from the Bacillus anthracis by of using the and We the the and using the from to of and and to to of to PlyL by of wall-binding domain. and to the cell by and by a an of and by the of of in a of applied to the to the the full-length PlyL, more than by and to to further the and of by using an and its N-terminal domain and cell the and of and similar to the but to the N-terminal by and of the PlyL N-terminal catalytic domain by of the full-length PlyL using a of a to the catalytic domain. to and analysis that a and the by by using a of of of and of to in cell a To data the in a of to a of data C-terminal and to that of the of the C-terminal domain by and in by crystal to a of in to using a and cell a of the PlyL data the using a and using the and of a zinc in the crystal by a the zinc using and to a of by further using and the data a using the of the structure the to a of are in the structure of and a of the two in the and are the and its are than the other two are in and structure have the the and in the of the in to the is the of of the is the data using of data from of of from is the the is the data using of data from in a of activity of PlyL and when applied to of B. anthracis Bacillus cereus Bacillus Bacillus and tested. to and and in of cell upon of endolysin of the in from B. PlyL in of PlyL a and and the of using a in of and of in and the in the in the by a a from to in in and detected of the by a using the of using the and by and and the of the C-terminal domain of PlyL, which using and the that the of the full-length domain and a a and and using and the binding by to and to a analysis the and and the of to the and of and C-terminal PlyL by from the Bacillus anthracis by of using the and We the the and using the from to of and and to to of to PlyL by of wall-binding domain. and to the cell by and by a an of and by the of of in a of applied to the to the the full-length PlyL, more than by and to to further the cell wall-binding domain. and of by using an and its N-terminal domain and cell the and of and similar to the but to the N-terminal by and of the PlyL N-terminal catalytic domain by of the full-length PlyL using a of a to the catalytic domain. to and analysis that a and the by by using a of of of and of to in cell a To data the in a of to a of data PlyL C-terminal and to that of the of the C-terminal domain by and in by crystal to a of in to using a and cell a of the PlyL data the using a and using the and of a zinc in the crystal by a the zinc using and to a of by further using and the data a using the of the structure the to a of are in the structure of and a of the two in the and are the and its are than the other two are in and structure have the the and in the of the of activity of PlyL and when applied to of B. anthracis Bacillus cereus Bacillus Bacillus and tested. to and and in of cell upon of endolysin of the in from B. PlyL in of PlyL a and and the of using a in of and of in and the in the in the by a a from to in in and detected of the by a using the C-terminal of using the and by and and the of the C-terminal domain of PlyL, which using and the that the of the full-length domain and a a and and using and the binding by to and to a analysis the and and the of to the and of using the the two an prophage of B. λ and λ Ba02 are and in the of B. anthracis from other Bacillus species and detected in than catalytic are in PlyL is related to in the and C-terminal is similar but the C-terminal and a B. anthracis of the full-length using to a N-terminal the the catalytic and cell wall-binding more than the full-length and We the C-terminal domain; we have not its the of that to that is an autonomously folded of PlyL in N-terminal the activity of PlyL, from B. full-length PlyL and the N-terminal in from that the enzyme is a a of the the by the of which that the enzyme is an N-acetylmuramoyl-l-alanine amidase, the and the N-terminal that comprises a catalytic domain. N-terminal domain more than the full-length in the that the C-terminal domain is of the PlyL N-terminal the structure of the PlyL catalytic domain using from a fold is similar to of the T7 B. and the which we have the and of T7 fold of a by the and the and a of and T7 lysozyme, an N-terminal an of the zinc to the of the the of the by from and by two and of is a from the of PlyL and site of PlyL and are of these are except which in the is by the but is by a to the in of of the site of PlyL T7 and site is and in a the the to a and more the in PlyL than in T7 lysozyme, that the T7 is not site that of T7 and are conserved PlyL and T7 is an in PlyL is to of T7 lysozyme, which to by the the in the a in T7 and in are and are to the to the the of the in PlyL to in the crystal structure the of a and the of a the to the T7 To demonstrate a catalytic in PlyL, we to and the amidase is folded by its to the not are the N-terminal of PlyL and that three-dimensional These are the three-dimensional of PlyL of the are the of the and of are from the site and a binding that the two catalytic have similar specificity and catalytic of PlyL and the lytic activity of PlyL of several by and by We that the full-length PlyL B. cereus an that B. anthracis and of B. cereus in a lytic activity of PlyL B. and but activity B. and B. and cell wall binding activity of PlyL and of of Bacillus species by full-length PlyL and its N-terminal catalytic domain except B. anthracis the the full-length and catalytic domain of PlyL to the by the from of Bacillus species by the B. cereus and its N-terminal domain. are the in of the binding to the cell wall of B. cereus, the other Bacillus species the not unexpectedly, that the N-terminal catalytic domain of PlyL is more than the full-length in B. B. and B. anthracis B. and the of the C-terminal domain a the lytic activity B. the of we the lytic activity of the from the B. cereus phage, is PlyL in its catalytic domain, its C-terminal domain to B. cereus when B. are We specificity the full-length but a in lytic activity B. by the N-terminal domain. of B. cereus by the N-terminal domain the full-length enzyme further the of the C-terminal domain of PlyL, we cell binding using a C-terminal domain to B. cereus and a a the binding B. B. anthracis not and of using the the two an prophage of B. λ and λ Ba02 are and in the of B. anthracis from other Bacillus species and detected in than catalytic are in PlyL is related to in the and C-terminal is similar but the C-terminal domain. We and a B. anthracis of the full-length using to a N-terminal the the catalytic and cell wall-binding more than the full-length and We the C-terminal domain; we have not its the of that to that is an autonomously folded domain. of PlyL in N-terminal the activity of PlyL, from B. full-length PlyL and the N-terminal in from that the enzyme is a a of the the by the of which that the enzyme is an N-acetylmuramoyl-l-alanine amidase, the and the N-terminal that comprises a catalytic domain. N-terminal domain more than the full-length in the that the C-terminal domain is of the PlyL N-terminal the structure of the PlyL catalytic domain using from a fold is similar to of the T7 B. and the which we have the and of T7 fold of a by the and the and a of and T7 lysozyme, an N-terminal an of the zinc to the of the the of the by from and by two and of is a from the site is and in a the the to a and more the in PlyL than in T7 lysozyme, that the T7 is not site that of T7 and are conserved PlyL and T7 is an in PlyL is to of T7 lysozyme, which to by the the in the a in T7 and in are and are to the to the the of the in PlyL to in the crystal structure the of a and the of a the to the T7 To demonstrate a catalytic in PlyL, we to and the amidase is folded by its to the not are the N-terminal of PlyL and that three-dimensional These are the three-dimensional of PlyL of the are the of the and of are from the site and a binding that the two catalytic have similar specificity and catalytic of PlyL and the lytic activity of PlyL of several by and by We that the full-length PlyL B. cereus an that B. anthracis and of B. cereus in a lytic activity of PlyL B. and but activity B. and B. We unexpectedly, that the N-terminal catalytic domain of PlyL is more than the full-length in B. B. and B. anthracis B. and the of the C-terminal domain a the lytic activity B. To the of we the lytic activity of the from the B. cereus phage, is PlyL in its catalytic domain, its C-terminal domain to B. cereus when B. are We specificity the full-length but a in lytic activity B. by the N-terminal domain. of B. cereus by the N-terminal domain the full-length enzyme To further the of the C-terminal domain of PlyL, we cell binding using a C-terminal domain to B. cereus and a a the binding B. B. anthracis not have that the endolysin from the B. anthracis λ prophage PlyL, is a cell wall lytic amidase a an N-terminal catalytic domain and a C-terminal cell wall-binding domain. We determined the three-dimensional structure of the catalytic domain and that the fold and site are similar to but from that of T7 and other and are the Bacillus in are the other and of not from T7 lysozyme, but its a similar to the in T7 lysozyme, and we a in by results that of the in have an N-acetylmuramoyl-l-alanine amidase activity and a similar catalytic the and we that its N-terminal domain a similar lytic specificity Bacillus species that the catalytic domain of the B. have a similar the that the PlyL and catalytic not to the that the lytic of the full-length are by the C-terminal domain, which is that the C-terminal domain is a cell wall-binding domain and that B. cereus We further that the of the the full-length PlyL an the lytic activity of the catalytic domain when the of B. to a B. and B. the of the a the activity of PlyL B. We that is not a of PlyL, we a similar pattern of activity a endolysin that is B. cereus, the domain that of the PlyL and a similar catalytic domain but in the C-terminal cell wall-binding domain. that in the cell wall-binding domain an of by the catalytic domain and the catalytic domain to in the of a cell propose the the of PlyL and have the of cell the an similar to a in the structure of the catalytic domain, is that the is the of the domain from a that is to the in cell wall binding which the the and the catalytic domain the the in the activity of the full-length PlyL and the N-terminal domain B. by binding of the to the B. cell the cell wall is to the amidase the case of B. cereus the full-length and have an activity, we propose that strong binding of the to the cell wall the the catalytic domain. results that of the domain to the cell the of a are to a species of by of that cell wall that the catalytic domain of PlyL strong lytic activity a of Bacillus species and that activity not is the that the PlyL of are that the of are when the is applied than We that are have to lytic activity when the is These may have implications the of We have that the endolysin from the B. anthracis λ prophage PlyL, is a cell wall lytic amidase a an N-terminal catalytic domain and a C-terminal cell wall-binding domain. We determined the three-dimensional structure of the catalytic domain and that the fold and site are similar to but from that of T7 and other and are the Bacillus in are the other and of not from T7 lysozyme, but its a similar to the in T7 lysozyme, and we a in by results that of the in have an N-acetylmuramoyl-l-alanine amidase activity and a similar catalytic the and we that its N-terminal domain a similar lytic specificity Bacillus species that the catalytic domain of the B. have a similar the that the PlyL and catalytic not to the that the lytic of the full-length are by the C-terminal domain, which is We that the C-terminal domain is a cell wall-binding domain and that B. cereus We further that the of the the full-length PlyL an the lytic activity of the catalytic domain when the of B. to a B. and B. the of the a the activity of PlyL B. We that is not a of PlyL, we a similar pattern of activity a endolysin that is B. cereus, the domain that of the PlyL and a similar catalytic domain but in the C-terminal cell wall-binding domain. that in the cell wall-binding domain an of by the catalytic domain and the catalytic domain to in the of a cell wall. We propose the the of PlyL and have the of cell the an similar to a in the structure of the catalytic domain, is that the is the of the domain from a that is to the in cell wall binding which the the and the catalytic domain the the in the activity of the full-length PlyL and the N-terminal domain B. by binding of the to the B. cell the cell wall is to the amidase the case of B. cereus the full-length and have an activity, we propose that strong binding of the to the cell wall the the catalytic domain. results that of the domain to the cell the of a are to a species of by of that cell wall that the catalytic domain of PlyL strong lytic activity a of Bacillus species and that activity not is the that the PlyL of are that the of are when the is applied than We that are have to lytic activity when the is These may have implications the of We of the of the and of the cell the