Peptide analogues of the VanS catalytic center inhibit VanR binding to its cognate promoter.
The dodecamer peptide SLCHDSVIGWEC, named E12, was selected from a combinatorial peptide library on the basis of its ability to bind to VanR, the two-component signal transduction response regulator which controls expression of vancomycin resistance in Enterococcus faecium. The binding of E12 was localized to the N-terminal, regulatory domain of VanR which contains Asp-55, the residue which accepts the phosphoryl group from His-164 in the activated VanS sensor kinase. E12, along with a related sequence SLAHDSIIGYLS, named E12.1, was found to inhibit the binding of VanR approximately P to a DNA segment which corresponds to its cognate promoter PvanH. With a single gap, both E12 and E12.1 could be aligned with the octadecamer sequence YLAHDIKTPLTSIIGYLS, comprising Tyr-161 through Ser-178, of the catalytic center dimerization domain of VanS, a sequence with which VanR also normally interacts. Alanine substitution analysis of E12.1 identified six amino acids as indispensable for its ability to inhibit VanR approximately P-PvanH DNA complex formation. A similar analysis of the corresponding amino acids in VanS showed a parallel dependence except for the substitutions Leu-162 --> Ala and Gly-175 --> Ala which interfered with the ability of E12.1 to compete with protein-DNA complex formation, but did not inhibit the ability of VanS to bind VanR. Our findings support a model in which E12 mimics the VanS phosphorylatable sequence with which the regulatory domain of VanR interacts, and thus functions as a "minimalist" analogue of VanS. Our results also indicate the usefulness of phage-displayed peptides as a general tool for mimicking the interacting faces of interacting proteins.
Ulijasz
A T
AT
Pharmacology Department, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA.
Kay
B K
BK
Weisblum
B
B
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D017104:Bacteriophage M13 / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D017931:DNA Primers / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D005982:Glutathione Transferase / Q000235:genetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010755:Organophosphates / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry* / Q000494:pharmacology; D019151:Peptide Library; D010750:Phosphoproteins / Q000032:analysis; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D011494:Protein Kinases / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D046911:Macromolecular Substances; D010755:Organophosphates; D010446:Peptide Fragments; D019151:Peptide Library; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C061278:PhoB protein, Bacteria; C073902:VanR protein, bacteria; C073901:VanS protein, Enterococcus; C011632:acetyl phosphate; D005982:Glutathione Transferase; D010770:Phosphotransferases; D011494:Protein Kinases; D000409:Alanine