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Multidrug Recognition PNAS PLUS in a Minimal Bacterial Multidrug Resistance System

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Multidrug Recognition PNAS PLUS in a Minimal Bacterial Multidrug Resistance System Structural basis and dynamics of multidrug recognition PNAS PLUS in a minimal bacterial multidrug resistance system Judith Habazettla, Martin Allana,1, Pernille Rose Jensena,2, Hans-Jürgen Sassa, Charles J. Thompsonb, and Stephan Grzesieka,3 aFocal Area Structural Biology and Biophysics, Biozentrum, University of Basel, CH-4056 Basel, Switzerland; and bDepartment of Microbiology and Immunology, Life Sciences Center, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada Edited by Adriaan Bax, National Institutes of Health, Bethesda, MD, and approved November 11, 2014 (received for review June 27, 2014) TipA is a transcriptional regulator found in diverse bacteria. It con- induces folding of a larger, intrinsically unstructured part of stitutes a minimal autoregulated multidrug resistance system the protein (24). By this mechanism, TipA recognizes a variety of against numerous thiopeptide antibiotics. Here we report the thiopeptide antibiotics such as thiostrepton, nosiheptide, or structures of its drug-binding domain TipAS in complexes with promothiocin A, and confers resistance via up-regulation of the promothiocin A and nosiheptide, and a model of the thiostrepton tipA gene and possibly other MDR systems (23–25). The thio- complex. Drug binding induces a large transition from a partially peptide antibiotics induce expression of the tipA gene as two unfolded to a globin-like structure. The structures rationalize the alternate in-frame translation products: a long protein, TipAL mechanism of promiscuous, yet specific, drug recognition: (i)a (253 aa), and a short protein, TipAS (144 aa), which also con- four-ring motif present in all known TipA-inducing antibiotics is stitutes the C-terminal part of TipAL (24, 26). TipAL belongs to recognized specifically by conserved TipAS amino acids; and (ii) the MerR family of stress response regulators, characterized by the variable part of the antibiotic is accommodated within a flex- their homologous N-terminal DNA-recognition domains; how- ible cleft that rigidifies upon drug binding. Remarkably, the iden- ever, its mechanism of promoter activation has distinctive char- tified four-ring motif is also the major interacting part of the acteristics (27). Their highly diverse C-terminal domains (19, 20, antibiotic with the ribosome. Hence the TipA multidrug resistance 28) recognize a wide variety of ligands ranging from divalent mechanism is directed against the same chemical motif that inhibits metal ions to large antibiotics (29). The C-terminal TipAS do- protein synthesis. The observed identity of chemical motifs respon- main of the TipA proteins recognizes thiopeptides and defines BIOCHEMISTRY sible for antibiotic function and resistance may be a general princi- a large subfamily within the MerR proteins (24). The tipA gene is ple and could help to better define new leads for antibiotics. often found in Streptomyces strains that do not produce thio- peptides, whereas thiopeptide-producing strains rather carry the thiopeptides | antibiotic recognition | transcriptional regulation | thiostrepton-resistance gene tsr (30), which provides stronger protein dynamics | solution NMR thiostrepton protection via ribosome methylation (31). Cur- rently, the TipAS subfamily in the Pfam database (32) includes ultidrug resistance (MDR) systems that respond to and 1,938 proteins widely distributed over many common pathogenic Minactivate cytotoxic compounds with diverse structures and and environmental bacteria (Fig. S1A). A phylogenetic tree of targets are found in most forms of life (1, 2). These mechanisms these bacteria reveals homologs in Firmicutes (1,321 proteins are usually active against a large variety of chemically and struc- in 969 species), Actinobacteria (252 proteins in 205 species), turally diverse compounds while still being selective for certain substance classes, such as heterocyclic, hydrophobic compounds Significance with specific hydrogen bond acceptor motifs (3). Structural in- formation, which would provide a rationale for this polyspecific Multidrug recognition is an important phenomenon that is not recognition is scarce, as few transporter structures with bound well understood. TipA, a bacterial transcriptional regulator, substrates have been solved with limited resolution (4, 5). constitutes a minimal multidrug resistance system against nu- The most well-known MDR mechanism is active transport by merous thiopeptide antibiotics. We show that motions in the efflux pumps, like ABC (6–11), RND, SMR, and MFS (4) efflux millisecond to microsecond time range form the basis of the pumps, which confer resistance to numerous anticancer (5) and TipA multidrug recognition mechanism. This may be common antimicrobial drugs (12). However, cytoplasmic proteins can also to many multidrug recognition systems. The discovery that the sequester and thereby inactivate toxic compounds such as bleo- structural antibiotic motifs essential for binding to TipA and to mycin, β-lactams, and fusidic acid (13–16) or drugs such as cis- the ribosome are identical makes the multidrug recognition platinum, melphalan, and chlorambucil (17). The expression of mechanism of TipA a useful model for ribosomal thiopeptide MDR transporters is often regulated by transcription factors, binding and current antibiotic drug development. which sense the presence of the substrates by direct interaction and have a similar spectrum of multidrug recognition (18). This over- Author contributions: J.H., C.J.T., and S.G. designed research; J.H., M.A., and P.R.J. per- lap of recognition specificity by regulatory and effector gene part- formed research; H.-J.S. contributed new reagents/analytic tools; J.H., M.A., P.R.J., and S.G. analyzed data; and J.H. and S.G. wrote the paper. ners also favors the evolution of MDR systems. High-resolution structures of such transcription factors as complexes with various The authors declare no conflict of interest. substrates (19–22) reveal structural motifs, which are assumed to This article is a PNAS Direct Submission. be similar to those found in MDR transporters (4, 10). Multidrug Data deposition: The NMR chemical shifts have been deposited in the BioMagResBank, www.bmrb.wisc.edu [accession nos. 19421 (TipAS·promothiocin A) and 19422 (TipAS· recognition is often achieved in a large hydrophobic pocket with nosiheptide)]. The atomic coordinates and structure factors have been deposited in the multiple binding possibilities, but two-site binding is also observed, Protein Data Bank, www.pdb.org [PDB ID codes 2MBZ (TipAS·promothiocin A) and whereby specific recognition of a part of the ligand occurs in a 2MC0 (TipAS·nosiheptide)]. rigid slot connected to a larger vestibule that accommodates the 1Present address: Harvard Medical School, Boston, MA 02115. variable ligand remainder (10). 2Present address: Albeda Research, 1799 Copenhagen, Denmark. The bacterial transcriptional activator TipA (thiostrepton 3To whom correspondence should be addressed. Email: [email protected]. Streptomyces lividans induced protein A) of (23) exhibits a nov- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. el multidrug recognition mechanism, in which drug binding 1073/pnas.1412070111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1412070111 PNAS Early Edition | 1of10 Downloaded by guest on October 8, 2021 Proteobacteria (176 proteins in 145 species), and other phyla and dynamics of TipAS complexes with the antibiotics promo- (Chloroflexi, Bacteroidetes, Cyanobacteria, Acidobacteria, and thiocin A and nosiheptide as well as a high-resolution model of Thermotogae). the TipAS·thiostrepton complex. These data give unique insights Antibiotics belonging to the thiostrepton class react with into multidrug recognition by a complete MDR system: (i) TipAS or TipAL to form a covalent bond between a dehy- specific recognition of a conserved antibiotic chemical motif is droalanine residue of the antibiotic and cysteine-214 of the achieved by conserved TipAS amino acids and induces folding of protein (33). Antibiotic binding to TipAL increases the affinity the unstructured apo N terminus; and (ii) the variable part of the to its operator site and stabilizes binding of the RNA polymerase antibiotic is accommodated in the large, flexible hydrophobic to the ptipA promoter (27). TipAL binds DNA as a dimer and cleft, which rigidifies upon binding. This mechanism allows presumably, by analogy to MerR (28, 29, 34), might activate promiscuous, yet specific recognition of the entire class of TipA- transcription of tipA by twisting the DNA. In S. lividans cultures inducing thiostrepton antibiotics. The identified recognition induced with thiostrepton, TipAS is expressed in large molar motif is also the major interacting part of thiostrepton and excess (>20:1) over TipAL (26). The sequestration of the drug by nosiheptide in complexes with the D. radiodurans ribosome (38). TipAS constitutes a resistance mechanism (25), and the drug- Hence, the tipA multidrug resistance mechanism is directed induced TipAL activation provides a feedback loop for tipA against the same motif, which is responsible for the antibiotic expression. Thus, TipAL/TipAS represents a minimal autoregu- inhibition of protein synthesis. This structural determinant might lated MDR system (Fig. 1) that responds to and inactivates mul- serve as a scaffold for the development of new antibiotics. tiple thiopeptides. The wide distribution of TipA homologs suggests that the TipAS family provides similar MDR
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