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Systematic structure-activity analysis of microcin J25.

Pavlova O, Mukhopadhyay J, Sineva E, Ebright RH, Severinov K - J. Biol. Chem. (2008)

Bottom Line: The results show that only a small number of residues (three in the cycle and one in the threaded segment of the tail) are important for MccJ25 production.The results further show that only a small number of additional residues (two in the cycle and four in the threaded segment of the tail) are important for inhibition of transcription.The results open the way for design and construction of more potent MccJ25-based inhibitors of bacterial growth.

View Article: PubMed Central - PubMed

Affiliation: Waksman Institute of Microbiology, Piscataway, NJ 08844, USA.

ABSTRACT
Microcin J25 (MccJ25) is a 21-residue plasmid-encoded ribosomally synthesized lariat-protoknot antibacterial peptide that targets bacterial RNA polymerase. MccJ25 consists of an 8-residue cycle followed by a 13-residue tail that loops back and threads through the cycle. We have performed systematic mutational scanning of MccJ25, constructing and analyzing more than 380 singly substituted derivatives of MccJ25. The results define residues important for production of MccJ25 (comprising synthesis of MccJ25 precursor, processing of MccJ25 precursor, export of mature MccJ25, and stability of mature MccJ25), inhibition of RNA polymerase, and inhibition of bacterial growth. The results show that only a small number of residues (three in the cycle and one in the threaded segment of the tail) are important for MccJ25 production. The results further show that only a small number of additional residues (two in the cycle and four in the threaded segment of the tail) are important for inhibition of transcription. The results open the way for design and construction of more potent MccJ25-based inhibitors of bacterial growth.

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Locations of residues important for production/maturation/export/stability of MccJ25 and for inhibition of RNAP by MccJ25. Shown are locations of residues shown here to be important for production/maturation/export/stability of MccJ25 (residues 1, 2, 8, and 20; black) and for inhibition of RNAP by MccJ25 (residues 4, 7, 9, 10, 17, and 19; red) on the three-dimensional structure of MccJ25 (see Ref. 5; see also Refs. 3 and 4). A, stereoviews in solvent-accessible surface representation. B, stereoviews in stick representation.
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fig4: Locations of residues important for production/maturation/export/stability of MccJ25 and for inhibition of RNAP by MccJ25. Shown are locations of residues shown here to be important for production/maturation/export/stability of MccJ25 (residues 1, 2, 8, and 20; black) and for inhibition of RNAP by MccJ25 (residues 4, 7, 9, 10, 17, and 19; red) on the three-dimensional structure of MccJ25 (see Ref. 5; see also Refs. 3 and 4). A, stereoviews in solvent-accessible surface representation. B, stereoviews in stick representation.

Mentions: Of the 381 single-amino acid substitutions analyzed, 242 were shown to be compatible with production of MccJ25 (comprising synthesis of MccJ25 precursor, processing of MccJ25 precursor, export of mature MccJ25, and stability of mature MccJ25) (Fig. 1). Inspection of the lariat-protoknot (threaded lasso) covalent structure of MccJ25 suggests that the residues that form the lactam linkage of the MccJ25 cycle and at least one of the two aromatic residues that lock the threaded MccJ25 tail within the MccJ25 cycle may be critical for production of MccJ25, whereas residues in the loop segment of the MccJ25 tail may be less critical. Our findings support these expectations. The residues that form the lactam linkage (Gly1 and Glu8) and one immediately adjacent residue (Gly2) are the sole residues for which no non-wild-type side chains are tolerated in production of MccJ25, and the distal aromatic residue of the pair of aromatic residues that lock the threaded MccJ25 tail within the MccJ25 cycle (Tyr20) is the sole additional residue for which nearly no non-wild-type side chains are tolerated. In the three-dimensional structure of MccJ25 (5-7), these residues form a discrete, continuous surface determinant on one face of MccJ25 (Fig. 4, black residues). We propose that the MccJ25 maturation machinery (McjB and McjC; see Refs. 9 and 11) recognizes and interacts with this surface determinant during processing of MccJ25 precursor.


Systematic structure-activity analysis of microcin J25.

Pavlova O, Mukhopadhyay J, Sineva E, Ebright RH, Severinov K - J. Biol. Chem. (2008)

Locations of residues important for production/maturation/export/stability of MccJ25 and for inhibition of RNAP by MccJ25. Shown are locations of residues shown here to be important for production/maturation/export/stability of MccJ25 (residues 1, 2, 8, and 20; black) and for inhibition of RNAP by MccJ25 (residues 4, 7, 9, 10, 17, and 19; red) on the three-dimensional structure of MccJ25 (see Ref. 5; see also Refs. 3 and 4). A, stereoviews in solvent-accessible surface representation. B, stereoviews in stick representation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2533079&req=5

fig4: Locations of residues important for production/maturation/export/stability of MccJ25 and for inhibition of RNAP by MccJ25. Shown are locations of residues shown here to be important for production/maturation/export/stability of MccJ25 (residues 1, 2, 8, and 20; black) and for inhibition of RNAP by MccJ25 (residues 4, 7, 9, 10, 17, and 19; red) on the three-dimensional structure of MccJ25 (see Ref. 5; see also Refs. 3 and 4). A, stereoviews in solvent-accessible surface representation. B, stereoviews in stick representation.
Mentions: Of the 381 single-amino acid substitutions analyzed, 242 were shown to be compatible with production of MccJ25 (comprising synthesis of MccJ25 precursor, processing of MccJ25 precursor, export of mature MccJ25, and stability of mature MccJ25) (Fig. 1). Inspection of the lariat-protoknot (threaded lasso) covalent structure of MccJ25 suggests that the residues that form the lactam linkage of the MccJ25 cycle and at least one of the two aromatic residues that lock the threaded MccJ25 tail within the MccJ25 cycle may be critical for production of MccJ25, whereas residues in the loop segment of the MccJ25 tail may be less critical. Our findings support these expectations. The residues that form the lactam linkage (Gly1 and Glu8) and one immediately adjacent residue (Gly2) are the sole residues for which no non-wild-type side chains are tolerated in production of MccJ25, and the distal aromatic residue of the pair of aromatic residues that lock the threaded MccJ25 tail within the MccJ25 cycle (Tyr20) is the sole additional residue for which nearly no non-wild-type side chains are tolerated. In the three-dimensional structure of MccJ25 (5-7), these residues form a discrete, continuous surface determinant on one face of MccJ25 (Fig. 4, black residues). We propose that the MccJ25 maturation machinery (McjB and McjC; see Refs. 9 and 11) recognizes and interacts with this surface determinant during processing of MccJ25 precursor.

Bottom Line: The results show that only a small number of residues (three in the cycle and one in the threaded segment of the tail) are important for MccJ25 production.The results further show that only a small number of additional residues (two in the cycle and four in the threaded segment of the tail) are important for inhibition of transcription.The results open the way for design and construction of more potent MccJ25-based inhibitors of bacterial growth.

View Article: PubMed Central - PubMed

Affiliation: Waksman Institute of Microbiology, Piscataway, NJ 08844, USA.

ABSTRACT
Microcin J25 (MccJ25) is a 21-residue plasmid-encoded ribosomally synthesized lariat-protoknot antibacterial peptide that targets bacterial RNA polymerase. MccJ25 consists of an 8-residue cycle followed by a 13-residue tail that loops back and threads through the cycle. We have performed systematic mutational scanning of MccJ25, constructing and analyzing more than 380 singly substituted derivatives of MccJ25. The results define residues important for production of MccJ25 (comprising synthesis of MccJ25 precursor, processing of MccJ25 precursor, export of mature MccJ25, and stability of mature MccJ25), inhibition of RNA polymerase, and inhibition of bacterial growth. The results show that only a small number of residues (three in the cycle and one in the threaded segment of the tail) are important for MccJ25 production. The results further show that only a small number of additional residues (two in the cycle and four in the threaded segment of the tail) are important for inhibition of transcription. The results open the way for design and construction of more potent MccJ25-based inhibitors of bacterial growth.

Show MeSH