Molecular Interactions between Thiostrepton and the TipAS Protein from Streptomyces lividans

Abstract

In Streptomyces lividans, the expression of several proteins is stimulated by the thiopeptide antibiotic thiostrepton. Two of these, TipAL and TipAS, autoregulate their expression after covalently binding to thiostrepton; this irreversibly sequesters the antibiotic and desensitizes the organism to its effects. In this work, additional molecular recognition interactions involved in this critical event were explored by utilizing various thiostrepton analogues and several site-directed mutants of the TipAS antibiotic binding protein. Dissociation constants for several thiostrepton analogues ranged from 0.19 to 12.95 μM, depending on the analogue. The contributions of specific structural elements of the thiostrepton molecule to this interaction have been discerned, and an unusual covalent modification between the antibiotic and a new residue in a TipAS mutant has been detected.

YcaO-Dependent Posttranslational Amide Activation: Biosynthesis, Structure, and Function

“…346 Upon binding, dehydroamino acids often present in thiopeptides can undergo a Michael-like addition with Cys residues of TipA present within the flexible binding pocket. 346, 495–497 However, thiopeptides with a 29-membered macrocycles lack the conserved four-ring motif. Fittingly, they also do not induce or bind to TipA.…”

Functional roles inS‐adenosyl‐l‐methionine binding and catalysis for active site residues of the thiostrepton resistance methyltransferase

“…Such enzymes are in various instances responsible for rRNA methylations that bring about bacterial resistance to certain ribosome‐targeting antibiotics, noteworthy examples of which can be found in clinically relevant classes such as aminoglycosides and macrolides[2]. This resistance mechanism also accounts for the majority of natural bacterial immunity to thiostrepton, the prototype of the ribosome‐targeting thiopeptide antibiotics that have attracted renewed attention as a potential source for antimicrobial lead compounds[3–6].…”

Antibiotic binding releases autoinhibition of the TipA multidrug-resistance transcriptional regulator

“…This family constitutes a sensitive autoregulated MDR system in that the MDR functional elements as drug sensor, transcriptional activators and drug neutralizers are all integrated within a single tipA gene and executed by its two translation products (14). While the molecular mechanisms of DNA binding of its MerR-like domain and antibiotic recognition of the TipAS domain have been independently well elaborated (14,18,20,24,26), how drug binding promotes its transcriptional activation remains largely unclear ( Figure 1A). The α6-α9 region involved in antibiotic recognition was revealed to be flexible loops in apo-state TipAS (14,18), but it may result from a lack of the N-terminal part, especially a coiled-coil α5 helix that contributes to both dimerization and stabilization.…”

“…Investigation of Streptomyces lividans had revealed that expression of the tipA gene with two alternative start codons produces two protein isoforms: a full-length transcriptional regulatory TipAL protein and a short TipAS protein consisting only of the C-terminal domain of TipAL (17)(18)(19). The TipAS protein is the predominant form that is capable of recognizing thiopeptide compounds via covalent binding by an active cysteine (13,20), which then induces structural reordering to form a large hydrophobic cleft for permanent sequestration and neutralization of the antibiotics (14,18). However, upon thiopeptide binding into the C-terminal TipAS domain, the Nterminus of TipAL (TipAN) is activated to bind promotors via the HTH domain and promote transcriptional activation of multiple genes, including tipA for antibiotic resistance (14,21).…”