ABSTRACT
Two-component signaling systems involving receptor-histidine kinases are ubiquitous in bacteria and have been found in yeast and plants. These systems provide the major means by which bacteria communicate with each other and the outside world. Remarkably, very little is known concerning the extracellular ligands that presumably bind to receptor-histidine kinases to initiate signaling. The two-component agr signaling circuit in Staphylococcus aureus is one system where the ligands are known in chemical detail, thus opening the door for detailed structure-activity relationship studies. These ligands are short (8- to 9-aa) peptides containing a thiolactone structure, in which the alpha-carboxyl group of the C-terminal amino acid is linked to the sulfhydryl group of a cysteine, which is always the fifth amino acid from the C terminus of the peptide. One unique aspect of the agr system is that peptides that activate virulence expression in one group of S. aureus strains also inhibit virulence expression in other groups of S. aureus strains. Herein, it is demonstrated by switching the receptor-histidine kinase, AgrC, between strains of different agr specificity types, that intragroup activation and intergroup inhibition are both mediated by the same group-specific receptors. These results have facilitated the development of a global inhibitor of virulence in S. aureus, which consists of a truncated version of one of the naturally occurring thiolactone peptides.
Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Oligopeptides/pharmacology , Protein Kinase Inhibitors , Staphylococcus aureus/pathogenicity , Amino Acid Sequence , Binding Sites , Drug Design , Escherichia coli/genetics , Histidine Kinase , Kinetics , Lactones , Ligands , Oligopeptides/chemistry , Plasmids , Protein Kinases/genetics , Signal Transduction , Staphylococcus aureus/drug effects , Staphylococcus aureus/growth & development , Structure-Activity Relationship , VirulenceABSTRACT
The crystal structure of the triple-helical peptide, (Pro-Hyp-Gly)(4)-Glu-Lys-Gly-(Pro-Hyp-Gly)(5) has been determined to 1.75 A resolution. This peptide was designed to examine the effect of a pair of adjacent, oppositely charged residues on collagen triple-helical conformation and intermolecular interactions. The molecular conformation (a 7(5) triple helix) and hydrogen bonding schemes are similar to those previously reported for collagen triple helices and provides a second instance of water mediated N--H . . . O==C interchain hydrogen bonds for the amide group of the residue following Gly. Although stereochemically capable of forming intramolecular or intermolecular ion pairs, the lysine and glutamic acid side-chains instead display direct interactions with carbonyl groups and hydroxyproline hydroxyl groups or interactions mediated by water molecules. Solution studies on the EKG peptide indicate stabilization at neutral pH values, where both Glu and Lys are ionized, but suggest that this occurs because of the effects of ionization on the individual residues, rather than ion pair formation. The EKG structure suggests a molecular mechanism for such stabilization through indirect hydrogen bonding. The molecular packing in the crystal includes an axial stagger between molecules, reminiscent of that observed in D-periodic collagen fibrils. The presence of a Glu-Lys-Gly triplet in the middle of the sequence appears to mediate this staggered molecular packing through its indirect water-mediated interactions with backbone C==O groups and side chains.
Subject(s)
Collagen/chemistry , Peptides/chemistry , Peptides/metabolism , Amino Acid Sequence , Biopolymers/chemistry , Biopolymers/metabolism , Crystallography, X-Ray , Glutamic Acid/metabolism , Hydrogen Bonding , Hydrogen-Ion Concentration , Hydroxyproline/metabolism , Lysine/metabolism , Models, Molecular , Molecular Sequence Data , Protein Structure, Quaternary , Static Electricity , Water/metabolismABSTRACT
The 2 A crystal structure reported here of the collagen-like model peptide, T3-785, provides the first visualization of how the sequence of collagen defines distinctive local conformational variations in triple-helical structure.
Subject(s)
Collagen/chemistry , Peptide Fragments/chemistry , Amino Acid Sequence , Collagen/metabolism , Crystallization , Crystallography, X-Ray , Humans , Hydrogen Bonding , Models, Molecular , Molecular Sequence Data , Peptide Fragments/metabolism , Protein Conformation , Protein Structure, Secondary , Sequence Alignment , Water/chemistry , Water/metabolismABSTRACT
The synthesis of virulence factors and other extracellular proteins responsible for pathogenicity in Staphylococcus aureus is under the control of the agr locus. A secreted agr-encoded peptide, AgrD, processed from the AgrD gene product, is known to be an effector of self-strain activation and cross-strain inhibition of the agr response. Biochemical analysis of AgrD peptides isolated from culture supernatants has suggested that they contain an unusual thiol ester-linked cyclic structure. In the present work, chemical synthesis is used to confirm that the mature AgrD peptides contain a thiolactone structure and that this feature is absolutely necessary for full biological activity. The AgrD synthetic thiolactone peptides exhibited biological activity in vivo in a mouse protection test. Structure-activity studies have allowed key aspects of the peptide structure involved in the differential activation and inhibition functions to be identified. Accordingly, we propose a model for activation and inhibition of the agr response in which the former, but not the latter, involves specific acylation of the agr transmembrane receptor, AgrC.
