PMC 20201223 pmc.key 4773095 CC BY no 0 0 Structure of the Lantibiotic Resistance Response Regulator 10.1371/journal.pone.0149903 4773095 26930060 PONE-D-15-49972 e0149903 3 This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. surname:Khosa;given-names:Sakshi surname:Hoeppner;given-names:Astrid surname:Gohlke;given-names:Holger surname:Schmitt;given-names:Lutz surname:Smits;given-names:Sander H. J. surname:Cascales;given-names:Eric All relevant data are within the paper and its Supporting Information files. TITLE Data Availability front 11 2016 0 Structure of the Response Regulator NsrR from Streptococcus agalactiae, Which Is Involved in Lantibiotic Resistance 0.9945463 evidence cleaner0 2023-07-27T14:51:22Z DUMMY: Structure 0.998559 protein_type cleaner0 2023-07-27T12:47:37Z MESH: Response Regulator 0.9993967 protein cleaner0 2023-07-27T12:47:43Z PR: NsrR 0.9984838 species cleaner0 2023-07-27T12:47:50Z MESH: Streptococcus agalactiae chemical CHEBI: cleaner0 2023-07-27T12:52:04Z Lantibiotic ABSTRACT abstract 116 Lantibiotics are antimicrobial peptides produced by Gram-positive bacteria. Interestingly, several clinically relevant and human pathogenic strains are inherently resistant towards lantibiotics. The expression of the genes responsible for lantibiotic resistance is regulated by a specific two-component system consisting of a histidine kinase and a response regulator. Here, we focused on a response regulator involved in lantibiotic resistance, NsrR from Streptococcus agalactiae, and determined the crystal structures of its N-terminal receiver domain and C-terminal DNA-binding effector domain. The C-terminal domain exhibits a fold that classifies NsrR as a member of the OmpR/PhoB subfamily of regulators. Amino acids involved in phosphorylation, dimerization, and DNA-binding were identified and demonstrated to be conserved in lantibiotic resistance regulators. Finally, a model of the full-length NsrR in the active and inactive state provides insights into protein dimerization and DNA-binding. 0.99717027 chemical cleaner0 2023-07-27T12:48:04Z CHEBI: Lantibiotics chemical CHEBI: cleaner0 2023-07-27T12:48:28Z antimicrobial peptides 0.97839725 taxonomy_domain cleaner0 2023-07-27T12:48:34Z DUMMY: Gram-positive bacteria 0.9982835 species cleaner0 2023-07-27T12:48:39Z MESH: human 0.99746597 chemical cleaner0 2023-07-27T12:48:05Z CHEBI: lantibiotics chemical CHEBI: cleaner0 2023-07-27T12:52:04Z lantibiotic complex_assembly GO: cleaner0 2023-07-27T12:57:48Z two-component system 0.9987494 protein_type cleaner0 2023-07-27T12:48:45Z MESH: histidine kinase 0.99863535 protein_type cleaner0 2023-07-27T12:47:38Z MESH: response regulator 0.998608 protein_type cleaner0 2023-07-27T12:47:38Z MESH: response regulator chemical CHEBI: cleaner0 2023-07-27T12:52:04Z lantibiotic 0.9994099 protein cleaner0 2023-07-27T12:47:43Z PR: NsrR 0.9986101 species cleaner0 2023-07-27T12:47:51Z MESH: Streptococcus agalactiae 0.99849606 evidence cleaner0 2023-07-27T14:51:26Z DUMMY: crystal structures 0.9992875 structure_element cleaner0 2023-07-27T12:48:55Z SO: receiver domain 0.9989768 structure_element cleaner0 2023-07-27T12:49:01Z SO: DNA-binding effector domain 0.9987072 structure_element cleaner0 2023-07-27T12:49:11Z SO: C-terminal domain 0.9993938 protein cleaner0 2023-07-27T12:47:43Z PR: NsrR protein_type MESH: cleaner0 2023-07-27T12:49:40Z OmpR/PhoB subfamily 0.8666049 ptm cleaner0 2023-07-27T12:50:11Z MESH: phosphorylation chemical CHEBI: cleaner0 2023-07-27T12:49:57Z DNA 0.9957343 protein_state cleaner0 2023-07-27T14:23:03Z DUMMY: conserved 0.99867386 protein_type cleaner0 2023-07-27T12:49:44Z MESH: lantibiotic resistance regulators 0.99908096 protein_state cleaner0 2023-07-27T12:50:18Z DUMMY: full-length 0.9994062 protein cleaner0 2023-07-27T12:47:43Z PR: NsrR 0.99904615 protein_state cleaner0 2023-07-27T12:50:24Z DUMMY: active 0.99918455 protein_state cleaner0 2023-07-27T12:50:30Z DUMMY: inactive chemical CHEBI: cleaner0 2023-07-27T12:49:58Z DNA INTRO title_1 1120 Introduction INTRO paragraph 1133 The dramatic rise in antibiotic resistance has posed a major threat to the treatment of infectious diseases. This has led to the search for novel antibiotics that can be used as pharmaceuticals against human pathogenic bacteria. One of the potential antibiotic alternatives are lantibiotics. Lantibiotics are small antimicrobial peptides (30–50 amino acids in length), which are produced by several Gram-positive bacterial strains. They are post-translationally modified and contain specific lanthionine/methyl-lanthionine rings, which are crucial for their high antimicrobial activity. Lantibiotics are for example highly effective against various Gram-positive, human pathogenic bacteria including Streptococcus pneumoniae and several methicillin-resistant Staphylococcus aureus (MRSA) strains. The high potency of lantibiotics for medical usage has already been noticed, and several lantibiotics are already included in clinical trials. Their high potency is highlighted by the fact that, although being extensively used in food industry, resistance has not been described so far. Nisin is the most prominent member of the lantibiotic family and is able to inhibit cell growth, penetrates the membranes of various Gram-positive bacteria, and is characterized by five specific (methyl-)lanthionine rings, which are crucial for stability and activity in the nanomolar range. Thus, the lantibiotic producer strains have an inbuilt self-protection mechanism (immunity) to prevent cell death caused due to the action of its cognate lantibiotic. This immunity system consists of a membrane–associated lipoprotein (usually referred to as LanI) and/or an ABC transporter (termed as LanFEG and comprising three subunits). Although some lantibiotics such as Pep5, epicidin, epilancin, and lactocin S only require LanI for immunity, other lantibiotics with a dual mode of action involving pore formation and lipid II binding such as nisin, subtilin, epidermin, gallidermin, and lacticin 3147 require additionally the presence of LanFEG. Examples for LanFEG are NisI and NisFEG of the nisin system, SpaI and SpaFEG conferring immunity towards subtilin, and PepI constituting the immunity system of Pep5 producing strains. Structural data are reported for the immunity proteins NisI from Lactococcus lactis, SpaI from Bacillus subtilis and MlbQ from the lantibiotic NAI-107 producer strain Microbispora ATCC PTA-5024. 0.9987451 species cleaner0 2023-07-27T12:48:40Z MESH: human 0.9874534 taxonomy_domain cleaner0 2023-07-27T12:51:04Z DUMMY: bacteria 0.9956435 chemical cleaner0 2023-07-27T12:48:05Z CHEBI: lantibiotics 0.99147624 chemical cleaner0 2023-07-27T12:48:05Z CHEBI: Lantibiotics 0.9319806 chemical cleaner0 2023-07-27T12:48:29Z CHEBI: antimicrobial peptides 0.9792487 taxonomy_domain cleaner0 2023-07-27T14:20:56Z DUMMY: Gram-positive bacterial 0.9981494 chemical cleaner0 2023-07-27T12:52:10Z CHEBI: lanthionine 0.9968582 chemical cleaner0 2023-07-27T12:52:25Z CHEBI: methyl-lanthionine 0.99280035 chemical cleaner0 2023-07-27T12:48:05Z CHEBI: Lantibiotics 0.9802711 taxonomy_domain cleaner0 2023-07-27T14:20:59Z DUMMY: Gram-positive 0.997926 species cleaner0 2023-07-27T12:48:40Z MESH: human 0.6351519 taxonomy_domain cleaner0 2023-07-27T12:51:05Z DUMMY: bacteria 0.9978671 species cleaner0 2023-07-27T14:26:51Z MESH: Streptococcus pneumoniae 0.95391035 species cleaner0 2023-07-27T14:26:59Z MESH: methicillin-resistant Staphylococcus aureus 0.9970284 species cleaner0 2023-07-27T14:27:03Z MESH: MRSA 0.9952632 chemical cleaner0 2023-07-27T12:48:05Z CHEBI: lantibiotics 0.9958437 chemical cleaner0 2023-07-27T12:48:05Z CHEBI: lantibiotics 0.9949949 chemical cleaner0 2023-07-27T12:51:59Z CHEBI: Nisin 0.9946043 chemical cleaner0 2023-07-27T12:52:04Z CHEBI: lantibiotic 0.9794172 taxonomy_domain cleaner0 2023-07-27T12:48:34Z DUMMY: Gram-positive bacteria 0.9782201 chemical cleaner0 2023-07-27T12:52:04Z CHEBI: lantibiotic 0.9982658 chemical cleaner0 2023-07-27T12:52:04Z CHEBI: lantibiotic 0.92543674 protein_type cleaner0 2023-07-27T12:51:28Z MESH: membrane–associated lipoprotein 0.9788287 protein_type cleaner0 2023-07-27T12:54:45Z MESH: LanI 0.9981523 protein_type cleaner0 2023-07-27T12:51:33Z MESH: ABC transporter 0.9421355 protein_type cleaner0 2023-07-27T12:54:56Z MESH: LanFEG 0.9975822 chemical cleaner0 2023-07-27T12:48:05Z CHEBI: lantibiotics 0.9969531 chemical cleaner0 2023-07-27T12:52:55Z CHEBI: Pep5 0.9987821 chemical cleaner0 2023-07-27T12:53:00Z CHEBI: epicidin 0.99894005 chemical cleaner0 2023-07-27T12:53:05Z CHEBI: epilancin 0.9979113 chemical cleaner0 2023-07-27T12:53:10Z CHEBI: lactocin S 0.9869429 protein_type cleaner0 2023-07-27T12:54:46Z MESH: LanI 0.9969144 chemical cleaner0 2023-07-27T12:48:05Z CHEBI: lantibiotics 0.9938975 chemical cleaner0 2023-07-27T12:52:00Z CHEBI: nisin 0.99245435 chemical cleaner0 2023-07-27T12:53:30Z CHEBI: subtilin 0.99520075 chemical cleaner0 2023-07-27T12:53:35Z CHEBI: epidermin 0.9977969 chemical cleaner0 2023-07-27T12:53:40Z CHEBI: gallidermin 0.99786294 chemical cleaner0 2023-07-27T12:53:45Z CHEBI: lacticin 3147 0.9719448 protein_type cleaner0 2023-07-27T12:54:57Z MESH: LanFEG 0.7806 protein_type cleaner0 2023-07-27T12:54:57Z MESH: LanFEG 0.9988275 protein cleaner0 2023-07-27T12:54:12Z PR: NisI 0.9985185 protein cleaner0 2023-07-27T12:54:18Z PR: NisFEG 0.6532558 chemical cleaner0 2023-07-27T12:52:00Z CHEBI: nisin 0.9991843 protein cleaner0 2023-07-27T12:54:25Z PR: SpaI 0.99916637 protein cleaner0 2023-07-27T12:54:30Z PR: SpaFEG 0.99042773 chemical cleaner0 2023-07-27T12:53:31Z CHEBI: subtilin 0.9991824 protein cleaner0 2023-07-27T12:55:05Z PR: PepI 0.9694549 chemical cleaner0 2023-07-27T12:52:56Z CHEBI: Pep5 0.6991327 evidence cleaner0 2023-07-27T14:51:33Z DUMMY: Structural data 0.8691757 protein_type cleaner0 2023-07-27T14:58:00Z MESH: immunity proteins 0.99902976 protein cleaner0 2023-07-27T12:54:13Z PR: NisI 0.9983518 species cleaner0 2023-07-27T12:55:14Z MESH: Lactococcus lactis 0.9991961 protein cleaner0 2023-07-27T12:54:26Z PR: SpaI 0.99803007 species cleaner0 2023-07-27T12:55:19Z MESH: Bacillus subtilis 0.99903035 protein cleaner0 2023-07-27T12:55:24Z PR: MlbQ 0.9953537 chemical cleaner0 2023-07-27T12:52:04Z CHEBI: lantibiotic 0.9741191 chemical cleaner0 2023-07-27T12:55:31Z CHEBI: NAI-107 0.7365977 species cleaner0 2023-07-27T12:55:37Z MESH: Microbispora ATCC PTA-5024 INTRO paragraph 3545 Recently, gene clusters were identified in certain clinically relevant human pathogenic strains such as Streptococcus agalactiae, S. aureus, and others that confer inherent resistance against specific lantibiotics such as nisin and resemble the genetic architecture of the lantibiotic immunity genes found in the producing strains. Within these resistance operons, genes encoding for a membrane-associated protease and an ABC transporter were identified. Expression of these proteins provides resistance against lantibiotics. Recently, the structure of SaNSR from S. agalactiae was solved which provides resistance against nisin by a protease activity. Furthermore, the upregulation of these genes is mediated by a specific two-component system (TCS) similar to the one found in lantibiotic producing strains, consisting of a sensor histidine kinase (HK) and a response regulator (RR), apparently mediate the expression of the resistance proteins: HK senses the external lantibiotic and, upon receiving the stimuli, auto-phosphorylates at a conserved histidine residue within the cytosol; this high-energetic phosphoryl group is then transferred to the associated RR inducing a conformational change there, which activates the RR to evoke the cellular response. Bacteria have the ability to sense and survive various environmental stimuli through adaptive responses, which are regulated by TCSs. These processes include drug resistance, quorum-sensing, phosphate uptake, sporulation, and osmoregulation. The absence of TCSs within mammals makes them unique targets for novel antimicrobial drugs. 0.99886096 species cleaner0 2023-07-27T12:48:40Z MESH: human 0.9984276 species cleaner0 2023-07-27T12:47:51Z MESH: Streptococcus agalactiae 0.9982524 species cleaner0 2023-07-27T12:55:50Z MESH: S. aureus 0.99864274 chemical cleaner0 2023-07-27T12:48:05Z CHEBI: lantibiotics 0.9991204 chemical cleaner0 2023-07-27T12:52:00Z CHEBI: nisin 0.9943542 protein_type cleaner0 2023-07-27T12:56:19Z MESH: membrane-associated protease 0.99774075 protein_type cleaner0 2023-07-27T12:51:33Z MESH: ABC transporter 0.99697626 chemical cleaner0 2023-07-27T12:48:05Z CHEBI: lantibiotics 0.99493057 evidence cleaner0 2023-07-27T14:51:39Z DUMMY: structure 0.99870455 protein cleaner0 2023-07-27T12:56:31Z PR: SaNSR 0.9984689 species cleaner0 2023-07-27T12:56:38Z MESH: S. agalactiae 0.9989241 chemical cleaner0 2023-07-27T12:52:00Z CHEBI: nisin complex_assembly GO: cleaner0 2023-07-27T12:57:48Z two-component system 0.7977292 complex_assembly cleaner0 2023-07-27T12:57:55Z GO: TCS 0.532767 chemical cleaner0 2023-07-27T12:52:04Z CHEBI: lantibiotic protein_type MESH: cleaner0 2023-07-27T12:48:45Z histidine kinase 0.99778545 protein_type cleaner0 2023-07-27T12:58:38Z MESH: HK 0.9968966 protein_type cleaner0 2023-07-27T12:47:38Z MESH: response regulator 0.9954798 protein_type cleaner0 2023-07-27T12:58:45Z MESH: RR 0.998307 protein_type cleaner0 2023-07-27T12:58:39Z MESH: HK 0.9990539 chemical cleaner0 2023-07-27T12:52:04Z CHEBI: lantibiotic 0.6605025 ptm cleaner0 2023-07-27T12:58:52Z MESH: auto-phosphorylates 0.9977049 protein_state cleaner0 2023-07-27T12:59:07Z DUMMY: conserved 0.99674785 residue_name cleaner0 2023-07-27T14:08:48Z SO: histidine 0.99574405 protein_type cleaner0 2023-07-27T12:58:46Z MESH: RR 0.99509084 protein_type cleaner0 2023-07-27T12:58:46Z MESH: RR 0.99866474 taxonomy_domain cleaner0 2023-07-27T12:51:05Z DUMMY: Bacteria 0.965592 complex_assembly cleaner0 2023-07-27T12:58:13Z GO: TCSs 0.99651206 protein_state cleaner0 2023-07-27T12:59:03Z DUMMY: absence of 0.9872185 complex_assembly cleaner0 2023-07-27T12:58:14Z GO: TCSs 0.9981237 taxonomy_domain cleaner0 2023-07-27T12:58:58Z DUMMY: mammals INTRO paragraph 5141 The expression of the lantibiotic-resistance genes via TCS is generally regulated by microorganism-specific lantibiotics, which act via external stimuli. Some examples of TCS are: BraRS in S. aureus which is induced by bacitracin, nisin and nukacin-ISK-1 resistance, BceRS in Bacillus spp. that is induced by actagardine and mersacidin resistance, LcrRS in Streptococcus mutans induced by nukacin-ISK-1 and lacticin 481 and LisRK of Listeria monocytogenes induced by nisin resistance. Furthermore, multiple lantibiotics can induce the TCS CprRK from Clostridium difficile, leading to the expression of the genes localized on the cpr operon, resulting in resistance against several lantibiotics of which nisin, gallidermin, subtilin, and mutacin 1140 are some examples. Interestingly, the histidine kinase contains two-transmembrane helices but lacks an extracellular sensory domain, and are therefore known as ‘intramembrane-sensing’ histidine kinases. It has been suggested that in addition to conferring general resistance against lantibiotics, the BceAB-type transporters assist in signalling as via the presence of a large extracellular domain within the transmembrane segment indicated by experimental evidence from various systems. 0.668119 chemical cleaner0 2023-07-27T12:52:04Z CHEBI: lantibiotic 0.35961458 complex_assembly cleaner0 2023-07-27T12:57:55Z GO: TCS 0.9985266 chemical cleaner0 2023-07-27T12:48:05Z CHEBI: lantibiotics 0.5892956 complex_assembly cleaner0 2023-07-27T12:57:55Z GO: TCS 0.9971806 protein cleaner0 2023-07-27T12:59:44Z PR: BraRS 0.99837446 species cleaner0 2023-07-27T12:55:51Z MESH: S. aureus 0.99902904 chemical cleaner0 2023-07-27T14:27:20Z CHEBI: bacitracin 0.9989477 chemical cleaner0 2023-07-27T12:52:00Z CHEBI: nisin 0.9878618 chemical cleaner0 2023-07-27T13:01:00Z CHEBI: nukacin-ISK-1 0.9742468 protein cleaner0 2023-07-27T12:59:50Z PR: BceRS taxonomy_domain DUMMY: cleaner0 2023-07-27T13:00:06Z Bacillus spp 0.9970746 chemical cleaner0 2023-07-27T14:27:24Z CHEBI: actagardine 0.99615234 chemical cleaner0 2023-07-27T14:27:27Z CHEBI: mersacidin 0.99828523 protein cleaner0 2023-07-27T13:00:14Z PR: LcrRS 0.9982466 species cleaner0 2023-07-27T14:27:08Z MESH: Streptococcus mutans 0.9896021 chemical cleaner0 2023-07-27T13:01:00Z CHEBI: nukacin-ISK-1 0.9980278 chemical cleaner0 2023-07-27T13:01:06Z CHEBI: lacticin 481 0.998494 protein cleaner0 2023-07-27T13:00:20Z PR: LisRK 0.998415 species cleaner0 2023-07-27T13:00:31Z MESH: Listeria monocytogenes 0.99782085 chemical cleaner0 2023-07-27T12:52:00Z CHEBI: nisin 0.9961661 chemical cleaner0 2023-07-27T12:48:05Z CHEBI: lantibiotics 0.6338489 complex_assembly cleaner0 2023-07-27T12:57:55Z GO: TCS 0.7201387 protein cleaner0 2023-07-27T13:00:25Z PR: CprRK 0.99792933 species cleaner0 2023-07-27T13:00:37Z MESH: Clostridium difficile gene GENE: cleaner0 2023-07-27T14:09:07Z cpr 0.9980063 chemical cleaner0 2023-07-27T12:48:05Z CHEBI: lantibiotics 0.99929786 chemical cleaner0 2023-07-27T12:52:00Z CHEBI: nisin 0.9993292 chemical cleaner0 2023-07-27T12:53:40Z CHEBI: gallidermin 0.99928635 chemical cleaner0 2023-07-27T12:53:31Z CHEBI: subtilin 0.99885315 chemical cleaner0 2023-07-27T13:00:43Z CHEBI: mutacin 1140 0.9988773 protein_type cleaner0 2023-07-27T12:48:45Z MESH: histidine kinase 0.9979497 structure_element cleaner0 2023-07-27T14:46:05Z SO: two-transmembrane helices 0.998926 structure_element cleaner0 2023-07-27T14:46:10Z SO: extracellular sensory domain protein_type MESH: cleaner0 2023-07-27T14:58:28Z ‘intramembrane-sensing’ histidine kinases 0.9964479 chemical cleaner0 2023-07-27T12:48:05Z CHEBI: lantibiotics 0.9987182 protein_type cleaner0 2023-07-27T13:01:28Z MESH: BceAB-type transporters 0.9970465 structure_element cleaner0 2023-07-27T14:46:13Z SO: extracellular domain 0.9991809 structure_element cleaner0 2023-07-27T14:46:16Z SO: transmembrane segment INTRO paragraph 6383 The recently discovered nsr gene cluster of the human pathogen S. agalactiae encodes for the resistance protein NSR and the ABC transporter NsrFP, both conferring resistance against nisin. Homologous operons have been identified in various human pathogenic strains such as Staphylococcus epidermis and Streptococcus ictaluri based on the high sequence identity of NSR and NsrFP. In this gene cluster, the TCS NsrRK is responsible for the expression of the nsr and nsrFP genes. The similarity of the TCS within all the described nisin resistance operons suggests an expression specifically induced by nisin. Thus, NsrRK might be a useful target to combat inherently pathogenic lantibiotic-resistant strains. gene GENE: cleaner0 2023-07-27T13:02:41Z nsr 0.9989058 species cleaner0 2023-07-27T12:48:40Z MESH: human 0.99860716 species cleaner0 2023-07-27T12:56:39Z MESH: S. agalactiae 0.99846685 protein_type cleaner0 2023-07-27T14:58:33Z MESH: resistance protein 0.99936527 protein cleaner0 2023-07-27T15:00:30Z PR: NSR 0.997722 protein_type cleaner0 2023-07-27T12:51:33Z MESH: ABC transporter 0.9993332 protein cleaner0 2023-07-27T15:00:43Z PR: NsrFP 0.99881834 chemical cleaner0 2023-07-27T12:52:00Z CHEBI: nisin 0.9989549 species cleaner0 2023-07-27T12:48:40Z MESH: human 0.99869466 species cleaner0 2023-07-27T13:03:08Z MESH: Staphylococcus epidermis 0.9987157 species cleaner0 2023-07-27T13:03:14Z MESH: Streptococcus ictaluri 0.9989485 protein cleaner0 2023-07-27T15:00:26Z PR: NSR 0.99765635 protein cleaner0 2023-07-27T15:00:35Z PR: NsrFP 0.97538346 complex_assembly cleaner0 2023-07-27T12:57:55Z GO: TCS 0.9808241 protein cleaner0 2023-07-27T14:10:58Z PR: NsrRK 0.9584849 gene cleaner0 2023-07-27T13:02:41Z GENE: nsr 0.902056 gene cleaner0 2023-07-27T13:02:49Z GENE: nsrFP 0.8232556 complex_assembly cleaner0 2023-07-27T12:57:55Z GO: TCS chemical CHEBI: cleaner0 2023-07-27T12:52:00Z nisin 0.9988973 chemical cleaner0 2023-07-27T12:52:00Z CHEBI: nisin 0.9986237 protein cleaner0 2023-07-27T14:10:59Z PR: NsrRK 0.9980398 chemical cleaner0 2023-07-27T12:52:04Z CHEBI: lantibiotic INTRO paragraph 7090 Generally, RRs consist of two distinct structural domains, a receiver domain (RD) and an effector domain (ED), that are separated from each other by a flexible linker. RDs contain a highly conserved aspartate residue, which acts as a phosphoryl acceptor that becomes phosphorylated by the kinase domain of the histidine kinase upon reception of an external signal. The ED is thereby activated and binds to the designated promoters, thus initiating transcription of the target genes. 0.94559 protein_type cleaner0 2023-07-27T13:02:15Z MESH: RRs 0.99930763 structure_element cleaner0 2023-07-27T12:48:55Z SO: receiver domain 0.99952686 structure_element cleaner0 2023-07-27T13:01:57Z SO: RD 0.9992855 structure_element cleaner0 2023-07-27T13:02:02Z SO: effector domain 0.9994966 structure_element cleaner0 2023-07-27T13:02:08Z SO: ED 0.38903397 protein_state cleaner0 2023-07-27T14:46:31Z DUMMY: flexible 0.6832957 structure_element cleaner0 2023-07-27T14:46:36Z SO: linker 0.99942696 structure_element cleaner0 2023-07-27T13:34:52Z SO: RDs 0.9986931 protein_state cleaner0 2023-07-27T14:23:30Z DUMMY: highly conserved 0.9979576 residue_name cleaner0 2023-07-27T13:26:45Z SO: aspartate 0.5846595 protein_state cleaner0 2023-07-27T14:22:49Z DUMMY: phosphorylated 0.9991957 structure_element cleaner0 2023-07-27T14:46:40Z SO: kinase domain 0.99852765 protein_type cleaner0 2023-07-27T12:48:45Z MESH: histidine kinase 0.9995159 structure_element cleaner0 2023-07-27T13:02:08Z SO: ED INTRO paragraph 7573 The RRs are classified into different subfamilies depending on the three-dimensional structure of their EDs. The OmpR/PhoB subfamily is the largest subgroup of RRs and comprises approximately 40% of all response regulators in bacteria. All their members are characterized by a winged helix-turn-helix (wHTH) motif. Although numerous structures of the single domains are known, only a few structures of full-length OmpR/PhoB-type RRs have been determined: RegX3 (PDB code: 2OQR), MtrA (PDB code: 2GWR), PrrA (PDB code: 1YS6) and PhoP (PDB code: 3R0J) from Mycobacterium tuberculosis; DrrB (PDB code: 1P2F) and DrrD (PDB code: 1KGS) from Thermotoga maritima; and KdpE from Escherichia coli (PDB code: 4KNY). The various structures of RRs reveal that in addition to being in either “inactive” or “active” state, the RRs can also exist in two distinct conformations: “open” and “closed”. MtrA and PrrA exhibit a very compact, closed structure with the DNA-binding sequence, called recognition helix, of the ED being inaccessible to DNA. The structures of DrrD and DrrB exist in an open conformation, here the recognition helix is fully exposed, suggesting that RRs are flexible in solution and can adopt multiple conformations. 0.99907553 protein_type cleaner0 2023-07-27T13:02:16Z MESH: RRs 0.9992859 structure_element cleaner0 2023-07-27T14:46:45Z SO: EDs 0.8960476 protein_type cleaner0 2023-07-27T13:03:41Z MESH: OmpR/PhoB subfamily 0.99917954 protein_type cleaner0 2023-07-27T13:02:16Z MESH: RRs 0.99859655 protein_type cleaner0 2023-07-27T13:03:36Z MESH: response regulators 0.9985084 taxonomy_domain cleaner0 2023-07-27T12:51:05Z DUMMY: bacteria 0.99917966 structure_element cleaner0 2023-07-27T13:04:08Z SO: winged helix-turn-helix 0.99807054 structure_element cleaner0 2023-07-27T13:04:13Z SO: wHTH 0.9970993 evidence cleaner0 2023-07-27T14:51:45Z DUMMY: structures 0.9978765 evidence cleaner0 2023-07-27T14:51:53Z DUMMY: structures 0.9991668 protein_state cleaner0 2023-07-27T12:50:19Z DUMMY: full-length 0.9771588 protein_type cleaner0 2023-07-27T13:03:56Z MESH: OmpR/PhoB-type RRs 0.999338 protein cleaner0 2023-07-27T13:04:18Z PR: RegX3 0.99928516 protein cleaner0 2023-07-27T13:04:25Z PR: MtrA 0.99929607 protein cleaner0 2023-07-27T13:04:30Z PR: PrrA 0.99932086 protein cleaner0 2023-07-27T13:04:35Z PR: PhoP 0.99864024 species cleaner0 2023-07-27T13:04:58Z MESH: Mycobacterium tuberculosis 0.99932957 protein cleaner0 2023-07-27T13:04:42Z PR: DrrB 0.99933076 protein cleaner0 2023-07-27T13:04:47Z PR: DrrD 0.99869025 species cleaner0 2023-07-27T13:05:04Z MESH: Thermotoga maritima 0.9992974 protein cleaner0 2023-07-27T13:04:52Z PR: KdpE 0.9986049 species cleaner0 2023-07-27T13:05:09Z MESH: Escherichia coli 0.99784315 evidence cleaner0 2023-07-27T14:51:49Z DUMMY: structures 0.9987684 protein_type cleaner0 2023-07-27T13:02:16Z MESH: RRs 0.99910814 protein_state cleaner0 2023-07-27T12:50:31Z DUMMY: inactive 0.99913764 protein_state cleaner0 2023-07-27T12:50:25Z DUMMY: active 0.9983626 protein_type cleaner0 2023-07-27T13:02:16Z MESH: RRs 0.9992224 protein_state cleaner0 2023-07-27T13:05:18Z DUMMY: open 0.9992557 protein_state cleaner0 2023-07-27T13:05:24Z DUMMY: closed 0.99927944 protein cleaner0 2023-07-27T13:04:25Z PR: MtrA 0.99929 protein cleaner0 2023-07-27T13:04:30Z PR: PrrA 0.7214432 protein_state cleaner0 2023-07-27T14:23:34Z DUMMY: very compact 0.999193 protein_state cleaner0 2023-07-27T13:05:25Z DUMMY: closed 0.99700975 evidence cleaner0 2023-07-27T14:51:57Z DUMMY: structure structure_element SO: cleaner0 2023-07-27T13:06:26Z DNA-binding sequence 0.9984138 structure_element cleaner0 2023-07-27T13:06:31Z SO: recognition helix 0.9992761 structure_element cleaner0 2023-07-27T13:02:08Z SO: ED 0.94537944 chemical cleaner0 2023-07-27T12:49:58Z CHEBI: DNA 0.997687 evidence cleaner0 2023-07-27T14:52:00Z DUMMY: structures 0.9993082 protein cleaner0 2023-07-27T13:04:48Z PR: DrrD 0.999303 protein cleaner0 2023-07-27T13:04:43Z PR: DrrB 0.9992262 protein_state cleaner0 2023-07-27T13:05:19Z DUMMY: open 0.99932104 structure_element cleaner0 2023-07-27T13:06:32Z SO: recognition helix 0.99866134 protein_state cleaner0 2023-07-27T14:23:38Z DUMMY: fully exposed 0.9987412 protein_type cleaner0 2023-07-27T13:02:16Z MESH: RRs 0.67741996 protein_state cleaner0 2023-07-27T14:23:43Z DUMMY: flexible INTRO paragraph 8813 Here, we describe the crystal structures of the N-terminal RD and the C-terminal ED of the lantibiotic resistance-associated RR NsrR from S. agalactiae. NsrR is part of the nisin resistance operon. The expression of the genes of this operon is induced by a TCS consisting of the HK NsrK and the RR NsrR. Based on the crystal structures of both the domains, modeling was employed to shed light on the putative DNA-bound state of full-length NsrR. 0.9987211 evidence cleaner0 2023-07-27T14:52:03Z DUMMY: crystal structures 0.9995086 structure_element cleaner0 2023-07-27T13:01:57Z SO: RD 0.9994874 structure_element cleaner0 2023-07-27T13:02:09Z SO: ED protein_type MESH: cleaner0 2023-07-27T14:24:11Z lantibiotic resistance-associated RR 0.9993624 protein cleaner0 2023-07-27T12:47:43Z PR: NsrR 0.9983449 species cleaner0 2023-07-27T12:56:39Z MESH: S. agalactiae 0.59359527 protein cleaner0 2023-07-27T12:47:43Z PR: NsrR chemical CHEBI: cleaner0 2023-07-27T12:52:00Z nisin 0.7663454 complex_assembly cleaner0 2023-07-27T12:57:55Z GO: TCS 0.9973092 protein_type cleaner0 2023-07-27T12:58:39Z MESH: HK 0.9976102 protein cleaner0 2023-07-27T15:00:50Z PR: NsrK 0.9794317 protein_type cleaner0 2023-07-27T12:58:46Z MESH: RR 0.9993907 protein cleaner0 2023-07-27T12:47:43Z PR: NsrR 0.998714 evidence cleaner0 2023-07-27T14:52:06Z DUMMY: crystal structures 0.9989874 protein_state cleaner0 2023-07-27T13:06:02Z DUMMY: DNA-bound 0.9991117 protein_state cleaner0 2023-07-27T12:50:19Z DUMMY: full-length 0.99933654 protein cleaner0 2023-07-27T12:47:43Z PR: NsrR METHODS title_1 9259 Materials and Methods METHODS title_2 9281 Cloning, expression and purification NsrR METHODS paragraph 9323 NsrR was constructed, expressed, and purified as described previously. In brief, the nsrR gene (accession no. HG939456.1) from S. agalactiae COH1 was ligated into the expression vector pET24a allowing expression in E. coli with a His6-tag introduced at the C-terminus. The resulting plasmid pET24a-NsrR was transformed into E. coli BL21 (DE3) for expression. A single transformed colony was inoculated into 20 ml LB media containing 30 μg/ml kanamycin. The culture was grown for 14 h at 310 K with shaking at 200 rpm. 4 l LB media with 30 μg/ml kanamycin were inoculated with the overnight culture at an OD600 of 0.05 and grown at 310 K with shaking at 170 rpm until an OD600 of 0.3 was reached. Subsequently, temperature was lowered to 291 K, and cells were further grown until an OD600 of 0.8 was reached before inducing the expression by addition of 1 mM IPTG. Cells were further grown for 15 h and harvested by centrifugation at 8000 rpm for 20 min at 277 K. The harvested cell pellet was re-suspended in 10 ml of buffer A (50 mM Tris pH 8.0, 50 mM NaCl, 2 mM PMSF and 10% (v/v) glycerol) and 10 mg of DNase (Deoxyribonuclease I from bovine pancreas, Sigma Aldrich) was added. Cells were lysed using a cell disruptor (Constant Cell Disruption Systems, United Kingdom) at 2.6 × 105 kPa. The lysate was centrifuged at 42000 rpm for 60 min using a Ti60 rotor to remove non-lysed cells and cell debris. METHODS paragraph 10729 20 mM imidazole was added to the cleared lysate prior to applying it onto a Ni2+ loaded Hi-Trap HP Chelating column (GE Healthcare) pre-equilibrated with buffer B (20 mM Tris pH 8.0, 250 mM NaCl and 20 mM imidazole, 2 mM PMSF). The column was washed with six column volumes of buffer B. Protein was eluted with a linear gradient of imidazole from 20 mM to 400 mM in buffer B. The fractions containing NsrR were pooled and concentrated up to 8 mg/ml in an Amicon centrifugal filter concentrator with a 10 kDa cut-off membrane (Millipore). The concentrated protein was further purified by size exclusion chromatography using a Superdex 200 GL 10/300 column (GE Healthcare), equilibrated with buffer C (25 mM Tris pH 9.0, 50 mM NaCl, 2 mM PMSF). The eluted protein fractions were pooled and concentrated to 11 mg/ml as described above. The purity of the protein was analyzed with 15% SDS-PAGE using colloidal Coomassie blue staining. METHODS title_2 11660 Crystallization of NsrR METHODS paragraph 11684 Crystals were obtained by using 1 μl of protein solution (concentration of 6.0 mg/ml) mixed with 1 μl of reservoir solution using the hanging-drop vapor diffusion method at 285 K. The reservoir solution contained PEG 20000 (11, 13, 15, 17 and 21% (w/v)) and 0.1 M MES pH (6.0, 6.5, 7.0 and 7.5). Crystals were obtained after three weeks and grew to their maximum dimensions within one month. Two different crystal forms, rectangular plate-shaped crystals and thin plates, were observed in the same drop. Both crystals forms were transferred into a buffer containing the reservoir solution plus 30% (v/v) ethylene glycol for 5 min prior to flash cooling using liquid nitrogen. For phasing, 20 mM tetra-chloro platinate IV (Hampton Research) was added to the crystallization drop, and the rectangular plate-shaped crystals were soaked for 30 min. The crystals with no obvious optical damage were harvested and flash-cooled in liquid nitrogen following the procedure above. METHODS title_2 12657 Data collection METHODS paragraph 12673 Initially crystals were screened for quality at beamline P13 (DESY, EMBL Hamburg). All X-ray diffraction data were collected at beamline ID23eh1 of the European Synchrotron Radiation Facility (ESRF), Grenoble. All data sets were processed and scaled using XDS and XSCALE software package. Data sets from both native crystal forms were collected at 100 K. A single-wavelength anomalous dispersion (SAD) dataset from a single heavy-atom derivatized crystal (rectangular plate-shaped crystal) was collected at 1.0714 Å at 100 K. Diffraction data up to 1.7 Å was used for heavy atom localization and subsequent phasing. METHODS title_2 13291 Structure determination of NsrR METHODS paragraph 13323 The structure of the thin plate-shaped crystals was solved by molecular replacement using the structure of the receiver domain of PhoB (PDB entry: 1B00) as a model to phase the native data set at 1.8 Å resolution. The model generated was refined manually in COOT followed by iterative cycles of refinement using the program phenix.refine. Manual adjustments between the refinement cycles were performed with the program COOT and Ramachandran validation was done using MolProbity. METHODS paragraph 13804 The SAD dataset of the rectangular plate-shaped crystal was used for phasing via the Auto-Rickshaw server. The initial model was further built and refined manually using COOT and phenix.refine from the Phenix package with iterative cycles of refinement. This model was used to phase the native data set of the rectangular plate-shaped crystals at a resolution of 1.6 Å. METHODS paragraph 14175 Data collection and refinement statistics are listed in Table 1 and all images of the models were prepared using PyMOL. pone.0149903.t001.xml pone.0149903.t001 TABLE table_title_caption 14295 Data collection, phasing, and refinement statistics for the receiver and effector domains of NsrR. pone.0149903.t001.xml pone.0149903.t001 TABLE table <?xml version="1.0" encoding="UTF-8"?> <table frame="hsides" rules="groups"><colgroup span="1"><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/></colgroup><thead><tr><th align="center" rowspan="1" colspan="1"/><th align="center" rowspan="1" colspan="1">NsrR-RD (native)</th><th align="center" rowspan="1" colspan="1">NsrR-ED (native)</th><th align="center" rowspan="1" colspan="1">NsrR-ED (SAD dataset)</th></tr></thead><tbody><tr><td align="justify" rowspan="1" colspan="1"><bold>Data collection</bold></td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">Space group</td><td align="justify" rowspan="1" colspan="1">P 2₁ 2₁ 2</td><td align="justify" rowspan="1" colspan="1">P 2₁ 2₁ 2</td><td align="justify" rowspan="1" colspan="1">P 2₁ 2₁ 2</td></tr><tr><td align="justify" rowspan="1" colspan="1"><italic>Cell dimensions</italic></td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">    a, b, c (Å)</td><td align="justify" rowspan="1" colspan="1">57.0 107.1 39.4</td><td align="justify" rowspan="1" colspan="1">56.3 60.4 56.8</td><td align="justify" rowspan="1" colspan="1">56.3 60.6 56.7</td></tr><tr><td align="justify" rowspan="1" colspan="1">    α, β, γ (°)</td><td align="justify" rowspan="1" colspan="1">90.0 90.0 90.0</td><td align="justify" rowspan="1" colspan="1">90.0 90.0 90.0</td><td align="justify" rowspan="1" colspan="1">90.0 90.0 90.0</td></tr><tr><td align="justify" rowspan="1" colspan="1">Wavelength (λ)</td><td align="justify" rowspan="1" colspan="1">1.0688</td><td align="justify" rowspan="1" colspan="1">0.9677</td><td align="justify" rowspan="1" colspan="1">1.0714</td></tr><tr><td align="justify" rowspan="1" colspan="1">Resolution (Å)</td><td align="justify" rowspan="1" colspan="1">39.48–1.80 (1.86–1.80)</td><td align="justify" rowspan="1" colspan="1">56.85–1.60 (1.65–1.60)</td><td align="justify" rowspan="1" colspan="1">100.00–1.70 (1.75–1.70)</td></tr><tr><td align="justify" rowspan="1" colspan="1">R_merge <xref ref-type="table-fn" rid="t001fn002">^a</xref></td><td align="justify" rowspan="1" colspan="1">3.4 (33.3)</td><td align="justify" rowspan="1" colspan="1">4.8 (30.5)</td><td align="justify" rowspan="1" colspan="1">6.8 (97.0)</td></tr><tr><td align="justify" rowspan="1" colspan="1">I /σ(I)</td><td align="justify" rowspan="1" colspan="1">26.2 (5.1)</td><td align="justify" rowspan="1" colspan="1">18.2 (4.6)</td><td align="justify" rowspan="1" colspan="1">21.6 (1.7)</td></tr><tr><td align="justify" rowspan="1" colspan="1">Completeness (%)</td><td align="justify" rowspan="1" colspan="1">98.8 (98.8)</td><td align="justify" rowspan="1" colspan="1">99.5 (99.7)</td><td align="justify" rowspan="1" colspan="1">98.8 (90.2)</td></tr><tr><td align="justify" rowspan="1" colspan="1">Redundancy</td><td align="justify" rowspan="1" colspan="1">4.8 (4.8)</td><td align="justify" rowspan="1" colspan="1">4.8 (4.9)</td><td align="justify" rowspan="1" colspan="1">11.7 (6.5)</td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>Structure Refinement</bold></td><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">Resolution (Å)</td><td align="justify" rowspan="1" colspan="1">39.48–1.80 (1.86–1.80)</td><td align="justify" rowspan="1" colspan="1">56.85–1.60 (1.65–1.60)</td><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">No. of reflections</td><td align="justify" rowspan="1" colspan="1">109201 (10602)</td><td align="justify" rowspan="1" colspan="1">124810 (12438)</td><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">CC1/2</td><td align="left" rowspan="1" colspan="1">0.999 (0.924)</td><td align="left" rowspan="1" colspan="1">0.999 (0.923)</td><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">R_work ^b / R_free <xref ref-type="table-fn" rid="t001fn003">^b</xref></td><td align="justify" rowspan="1" colspan="1">0.17 (0.20)/ 0.22 (0.27)</td><td align="justify" rowspan="1" colspan="1">0.18 (0.22)/ 0.22 (0.27)</td><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1"><italic>No</italic>. <italic>of atoms</italic></td><td align="justify" rowspan="1" colspan="1">2027</td><td align="justify" rowspan="1" colspan="1">1843</td><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">    Macromolecules</td><td align="justify" rowspan="1" colspan="1">1894</td><td align="justify" rowspan="1" colspan="1">1580</td><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">    Ligand/ion</td><td align="justify" rowspan="1" colspan="1">20</td><td align="justify" rowspan="1" colspan="1">8</td><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">    Water</td><td align="justify" rowspan="1" colspan="1">113</td><td align="justify" rowspan="1" colspan="1">255</td><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1"><italic>B-factors (Å</italic>^{<italic>2</italic>}<italic>)</italic></td><td align="justify" rowspan="1" colspan="1">28.3</td><td align="justify" rowspan="1" colspan="1">21.7</td><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">    Macromolecules</td><td align="justify" rowspan="1" colspan="1">27.7</td><td align="justify" rowspan="1" colspan="1">20.2</td><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">    Ligand/ion</td><td align="justify" rowspan="1" colspan="1">34.0</td><td align="justify" rowspan="1" colspan="1">38.6</td><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">    Solvent</td><td align="justify" rowspan="1" colspan="1">36.9</td><td align="justify" rowspan="1" colspan="1">30.4</td><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1"><italic>R</italic>.<italic>m</italic>.<italic>s</italic>. <italic>deviations</italic></td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">    Bond lengths (Å)</td><td align="justify" rowspan="1" colspan="1">0.007</td><td align="justify" rowspan="1" colspan="1">0.008</td><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">    Bond angles (°)</td><td align="justify" rowspan="1" colspan="1">1.11</td><td align="justify" rowspan="1" colspan="1">1.18</td><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1"><italic>Ramachandran plot (%)</italic></td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">    Favored</td><td align="justify" rowspan="1" colspan="1">99.0</td><td align="justify" rowspan="1" colspan="1">97.0</td><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">    Allowed</td><td align="justify" rowspan="1" colspan="1">1.0</td><td align="justify" rowspan="1" colspan="1">2.48</td><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">    Outliers</td><td align="justify" rowspan="1" colspan="1">0.0</td><td align="justify" rowspan="1" colspan="1">0.52</td><td align="justify" rowspan="1" colspan="1"/></tr></tbody></table> 14394 NsrR-RD (native) NsrR-ED (native) NsrR-ED (SAD dataset) Data collection Space group P 21 21 2 P 21 21 2 P 21 21 2 Cell dimensions     a, b, c (Å) 57.0 107.1 39.4 56.3 60.4 56.8 56.3 60.6 56.7     α, β, γ (°) 90.0 90.0 90.0 90.0 90.0 90.0 90.0 90.0 90.0 Wavelength (λ) 1.0688 0.9677 1.0714 Resolution (Å) 39.48–1.80 (1.86–1.80) 56.85–1.60 (1.65–1.60) 100.00–1.70 (1.75–1.70) Rmergea 3.4 (33.3) 4.8 (30.5) 6.8 (97.0) I /σ(I) 26.2 (5.1) 18.2 (4.6) 21.6 (1.7) Completeness (%) 98.8 (98.8) 99.5 (99.7) 98.8 (90.2) Redundancy 4.8 (4.8) 4.8 (4.9) 11.7 (6.5) Structure Refinement Resolution (Å) 39.48–1.80 (1.86–1.80) 56.85–1.60 (1.65–1.60) No. of reflections 109201 (10602) 124810 (12438) CC1/2 0.999 (0.924) 0.999 (0.923) Rworkb / Rfreeb 0.17 (0.20)/ 0.22 (0.27) 0.18 (0.22)/ 0.22 (0.27) No. of atoms 2027 1843     Macromolecules 1894 1580     Ligand/ion 20 8     Water 113 255 B-factors (Å2) 28.3 21.7     Macromolecules 27.7 20.2     Ligand/ion 34.0 38.6     Solvent 36.9 30.4 R.m.s. deviations     Bond lengths (Å) 0.007 0.008     Bond angles (°) 1.11 1.18 Ramachandran plot (%)     Favored 99.0 97.0     Allowed 1.0 2.48     Outliers 0.0 0.52 pone.0149903.t001.xml pone.0149903.t001 TABLE table_footnote 15717 Values in parentheses are for the highest resolution shell. pone.0149903.t001.xml pone.0149903.t001 TABLE table_footnote 15777 a Rmerge is defined as Rsym = ∑hkl∑i|Ii(hkl) − ⟨I(hkl)⟩|/∑hkl∑iIi(hkl) and pone.0149903.t001.xml pone.0149903.t001 TABLE table_footnote 15866 b RF as Rf = ∑hkl‖Fobs|−|Fcalc‖/∑hkl|Fobs| METHODS title_2 15919 Accession numbers METHODS paragraph 15937 Coordinates and structure factors have been deposited in the PDB with accession numbers 5DCL (NsrR-RD) and 5DCM (NsrR-ED). RESULTS title_1 16060 Results and Discussion RESULTS paragraph 16083 NsrR was expressed and purified as described, resulting in a homogenous protein as observed by size exclusion chromatography (Fig 1A), with a yield of 2 mg per liter of cell culture. By calibrating the column with proteins of known molecular weight the NsrR full length protein elutes as a dimer. The purified NsrR protein has a theoretical molecular mass of 27.7 kDa and was >98% pure as assessed by SDS-PAGE (Fig 1B, indicated by *). Surprisingly, over time NsrR degraded into two distinct fragments as visible on SDS-PAGE analysis using the same purified protein sample after one week (Fig 1C, indicated by ** and ***, respectively). This was also observed by size exclusion chromatography where a peak at an elution time of 18 min appeared (Fig 1A). Both bands were subjected to mass spectrometry analysis. The analysis revealed that the larger fragment (**) represents the N-terminal receiver domain (residues 1–119; referred to as NsrR-RD) whereas the smaller fragment (***) contained the C-terminal DNA-binding effector domain of NsrR (residues 129–243 including 21 amino acids derived from the expression tag; referred to as NsrR-ED) (Fig 1C). Residues 120–128 form the linker connecting the RD and ED. Such a cleavage of the full-length RR into two specific domains is not unusual and has been previously reported for other RRs as well. Mass spectrometry analysis did not reveal the presence of any specific protease in the purified NsrR sample. Furthermore, addition of a protease inhibitor, such as PMSF (Phenylmethylsulfonyl fluoride) and AEBSF {4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride}, even at high concentrations, did not inhibit proteolysis (data not shown). 0.99927205 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.98291045 experimental_method cleaner0 2023-07-27T14:35:16Z MESH: expressed and purified 0.99884963 experimental_method cleaner0 2023-07-27T14:35:20Z MESH: size exclusion chromatography 0.99928325 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.9991232 protein_state cleaner0 2023-07-27T14:28:34Z DUMMY: full length 0.9988662 oligomeric_state cleaner0 2023-07-27T13:37:59Z DUMMY: dimer 0.99927896 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR evidence DUMMY: cleaner0 2023-07-27T13:07:27Z molecular mass 0.9988087 experimental_method cleaner0 2023-07-27T14:36:27Z MESH: SDS-PAGE 0.99922633 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.99886465 experimental_method cleaner0 2023-07-27T14:35:24Z MESH: SDS-PAGE 0.9988444 experimental_method cleaner0 2023-07-27T14:35:28Z MESH: size exclusion chromatography experimental_method MESH: cleaner0 2023-07-27T14:35:48Z mass spectrometry analysis 0.9993094 structure_element cleaner0 2023-07-27T12:48:55Z SO: receiver domain 0.99736327 residue_range cleaner0 2023-07-27T14:08:24Z DUMMY: 1–119 0.9771807 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.9815507 structure_element cleaner0 2023-07-27T13:01:57Z SO: RD 0.9990264 structure_element cleaner0 2023-07-27T12:49:02Z SO: DNA-binding effector domain 0.99936086 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.99765706 residue_range cleaner0 2023-07-27T14:08:27Z DUMMY: 129–243 0.9743844 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.7150254 structure_element cleaner0 2023-07-27T13:02:09Z SO: ED 0.9978265 residue_range cleaner0 2023-07-27T14:08:31Z DUMMY: 120–128 0.9986193 structure_element cleaner0 2023-07-27T14:46:51Z SO: linker 0.9995346 structure_element cleaner0 2023-07-27T13:01:57Z SO: RD 0.9995073 structure_element cleaner0 2023-07-27T13:02:09Z SO: ED 0.9991009 protein_state cleaner0 2023-07-27T12:50:19Z DUMMY: full-length 0.98067904 protein_type cleaner0 2023-07-27T12:58:46Z MESH: RR 0.99906784 protein_type cleaner0 2023-07-27T13:02:16Z MESH: RRs experimental_method MESH: cleaner0 2023-07-27T14:36:21Z Mass spectrometry analysis 0.99928313 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.99845946 chemical cleaner0 2023-07-27T13:08:05Z CHEBI: PMSF 0.9973788 chemical cleaner0 2023-07-27T13:08:10Z CHEBI: Phenylmethylsulfonyl fluoride 0.99913484 chemical cleaner0 2023-07-27T13:08:14Z CHEBI: AEBSF 0.9984695 chemical cleaner0 2023-07-27T13:08:19Z CHEBI: 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride pone.0149903.g001.jpg pone.0149903.g001 FIG fig_title_caption 17780 Purification of NsrR and SDS PAGE analysis of purified NsrR directly and one week after purification. experimental_method MESH: cleaner0 2023-07-27T14:36:54Z Purification 0.99923396 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.9983078 experimental_method cleaner0 2023-07-27T14:36:42Z MESH: SDS PAGE 0.9992512 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR pone.0149903.g001.jpg pone.0149903.g001 FIG fig_caption 17882 (a) Elution profile of size-exclusion chromatography step of NsrR. The y-axis represents the UV absorption of the protein at 280 nm, while the x-axis represents the elution volume. a, b, c refer to the protein standards dextran blue (2,000 kDa), BSA (67 kDa), and lysozyme (14.3 kDa), respectively. The bold line represents the chromatogram of freshly purified NsrR while the dashed line shows the chromatogram of the same NsrR protein after one week. (b) Freshly purified NsrR protein, and (c) NsrR protein after one week. Lanes: M represents the PAGE Ruler Unstained Ladder; 1: NsrR after a two-step purification; 2: NsrR one week after purification. * corresponds to full-length NsrR protein at 27 kDa, while ** and *** correspond to the NsrR-RD and NsrR-ED domain at around 13 kDa, respectively. 0.624071 evidence cleaner0 2023-07-27T13:09:02Z DUMMY: Elution profile 0.9987503 experimental_method cleaner0 2023-07-27T14:36:58Z MESH: size-exclusion chromatography 0.99920565 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.9852395 evidence cleaner0 2023-07-27T13:08:56Z DUMMY: chromatogram 0.99915934 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.9861688 evidence cleaner0 2023-07-27T13:08:55Z DUMMY: chromatogram 0.9992047 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.99925953 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.99921334 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.99917966 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.99925095 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.9990738 protein_state cleaner0 2023-07-27T12:50:19Z DUMMY: full-length 0.99925727 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.99833435 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.99890995 structure_element cleaner0 2023-07-27T13:01:57Z SO: RD 0.99834394 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.9976876 structure_element cleaner0 2023-07-27T13:02:09Z SO: ED RESULTS paragraph 18682 Since formation of the crystals took around one month, it is not surprising that this cleavage also occurred in the crystallization drop. NsrR was crystallized yielding two crystal forms, which were distinguishable by visual inspection. Initially, we tried to solve the structure of NsrR by molecular replacement, which was not successful. Therefore, we tried heavy atom phasing using a platinum compound. This succeeded for the rectangular plate-shaped crystals. After the structure was solved, it became evident that these crystals contained two monomers of the ED of NsrR in the asymmetric unit. 0.96927536 evidence cleaner0 2023-07-27T14:52:11Z DUMMY: crystals 0.9993742 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.99806994 experimental_method cleaner0 2023-07-27T14:37:27Z MESH: crystallized 0.98949033 evidence cleaner0 2023-07-27T14:52:14Z DUMMY: structure 0.99938893 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.998575 experimental_method cleaner0 2023-07-27T14:37:31Z MESH: molecular replacement 0.99857587 experimental_method cleaner0 2023-07-27T14:37:34Z MESH: heavy atom phasing 0.8848682 chemical cleaner0 2023-07-27T14:27:32Z CHEBI: platinum 0.86987334 evidence cleaner0 2023-07-27T14:52:17Z DUMMY: crystals 0.9626586 evidence cleaner0 2023-07-27T14:52:19Z DUMMY: structure 0.9928011 evidence cleaner0 2023-07-27T14:52:22Z DUMMY: crystals 0.99839395 oligomeric_state cleaner0 2023-07-27T13:18:37Z DUMMY: monomers 0.99861085 structure_element cleaner0 2023-07-27T13:02:09Z SO: ED 0.99940157 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR RESULTS paragraph 19281 We also tried to solve the structure of the thin plate-shaped crystals with this template, but the resulting model generated was not sufficient. Therefore, we thought that these crystals contained the N-terminal domain of NsrR and successfully phased this dataset using molecular replacement with the N-terminal domain of PhoB (PDB code: 1B00; as a template. This approach revealed that this crystal form indeed contained two monomers of the RD of NsrR in the asymmetric unit. Since both crystals forms were obtained in the same drop it is not surprising that, when dissolving several crystals and performing subsequent mass-spectrometry to identify the protein in the crystals, it yielded peptide fragments throughout the NsrR sequence. 0.9663912 evidence cleaner0 2023-07-27T14:52:26Z DUMMY: structure 0.98445845 evidence cleaner0 2023-07-27T14:52:29Z DUMMY: crystals 0.9924428 evidence cleaner0 2023-07-27T14:52:32Z DUMMY: crystals 0.99763584 structure_element cleaner0 2023-07-27T14:47:24Z SO: N-terminal domain 0.9993974 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.9987139 experimental_method cleaner0 2023-07-27T14:37:48Z MESH: molecular replacement 0.9982074 structure_element cleaner0 2023-07-27T14:47:26Z SO: N-terminal domain 0.9993304 protein cleaner0 2023-07-27T13:28:31Z PR: PhoB 0.99808645 oligomeric_state cleaner0 2023-07-27T13:18:37Z DUMMY: monomers 0.99933535 structure_element cleaner0 2023-07-27T13:01:57Z SO: RD 0.99941313 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.56550413 evidence cleaner0 2023-07-27T14:52:35Z DUMMY: crystals 0.99880904 experimental_method cleaner0 2023-07-27T14:38:11Z MESH: mass-spectrometry 0.7979644 evidence cleaner0 2023-07-27T14:52:38Z DUMMY: crystals 0.9993994 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR RESULTS paragraph 20019 In summary, the two crystal forms contained one of the two domains, respectively, such that both domains were successfully crystallized. We determined the crystal structures of NsrR-RD and NsrR-ED separately. However, a part of the linker region (residues 120–128; 120RRSQQFIQQ128; underlined are the amino acid residues not visible in either domain) could not be traced in the electron density. 0.97675586 evidence cleaner0 2023-07-27T14:52:41Z DUMMY: crystal forms 0.9501939 experimental_method cleaner0 2023-07-27T14:38:26Z MESH: crystallized 0.99843526 evidence cleaner0 2023-07-27T14:52:44Z DUMMY: crystal structures protein PR: cleaner0 2023-07-27T12:47:44Z NsrR structure_element SO: cleaner0 2023-07-27T13:01:57Z RD protein PR: cleaner0 2023-07-27T12:47:44Z NsrR structure_element SO: cleaner0 2023-07-27T13:02:09Z ED 0.9992579 structure_element cleaner0 2023-07-27T13:52:42Z SO: linker region 0.9973113 residue_range cleaner0 2023-07-27T14:08:37Z DUMMY: 120–128 0.86698794 structure_element cleaner0 2023-07-27T14:08:40Z SO: 120RRSQQFIQQ128 0.9987925 evidence cleaner0 2023-07-27T14:52:46Z DUMMY: electron density RESULTS title_2 20417 Overall structure of the N-terminal NsrR receiver domain (NsrR-RD) 0.99032 evidence cleaner0 2023-07-27T14:52:52Z DUMMY: structure 0.9990251 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.99905497 structure_element cleaner0 2023-07-27T12:48:55Z SO: receiver domain protein PR: cleaner0 2023-07-27T12:47:44Z NsrR structure_element SO: cleaner0 2023-07-27T13:01:57Z RD RESULTS paragraph 20484 The structure of the NsrR-RD was determined at a resolution of 1.8 Å (Table 1). The Rwork and Rfree values after refinement were 0.17 and 0.22, respectively. Ramachandran validation revealed that all residues (100%, 236 amino acids) were in the preferred or allowed regions. The structure contained many ethylene glycol molecules arising from the cryo-protecting procedure. Data collection and refinement statistics are listed in Table 1. 0.99849343 evidence cleaner0 2023-07-27T14:52:56Z DUMMY: structure protein PR: cleaner0 2023-07-27T12:47:44Z NsrR structure_element SO: cleaner0 2023-07-27T13:01:57Z RD 0.9982931 evidence cleaner0 2023-07-27T14:52:59Z DUMMY: Rwork 0.99802566 evidence cleaner0 2023-07-27T14:53:03Z DUMMY: Rfree 0.97388566 evidence cleaner0 2023-07-27T13:41:47Z DUMMY: Ramachandran validation 0.99851376 evidence cleaner0 2023-07-27T14:53:06Z DUMMY: structure 0.6619525 chemical cleaner0 2023-07-27T14:27:38Z CHEBI: ethylene glycol RESULTS paragraph 20924 The asymmetric unit contains two copies of NsrR-RD. Although the entire N-terminal receiver domain is composed of residues Met1-Leu119, only residues Asn4 to Arg121 of chain A (including residues Arg120 and Arg121 of the linker) and Gln5 to Ser122 of chain B (including residues Arg120 until Ser122 of the linker) could be traced in the electron density of NsrR-RD. For Asn85, Asp86, and Glu87 of chain A, poor electron density was observed for the side chains and, thus, these side chains were deleted during refinement and are not present in the final structure. Since the two monomers of NsrR-RD were virtually identical (rmsd of 0.6 Å over 116 Cα atoms for the two monomers). Therefore, the overall structure is described for monomer A only. protein PR: cleaner0 2023-07-27T12:47:44Z NsrR structure_element SO: cleaner0 2023-07-27T13:01:57Z RD 0.9993507 structure_element cleaner0 2023-07-27T12:48:55Z SO: receiver domain 0.99704796 residue_range cleaner0 2023-07-27T13:18:20Z DUMMY: Met1-Leu119 0.9967788 residue_range cleaner0 2023-07-27T13:18:24Z DUMMY: Asn4 to Arg121 0.9990872 structure_element cleaner0 2023-07-27T13:43:00Z SO: chain A 0.9973508 residue_name_number cleaner0 2023-07-27T13:19:04Z DUMMY: Arg120 0.9985317 residue_name_number cleaner0 2023-07-27T13:19:09Z DUMMY: Arg121 0.9991097 structure_element cleaner0 2023-07-27T14:47:31Z SO: linker 0.99671906 residue_range cleaner0 2023-07-27T13:18:27Z DUMMY: Gln5 to Ser122 0.99902666 structure_element cleaner0 2023-07-27T13:43:05Z SO: chain B residue_range DUMMY: cleaner0 2023-07-27T13:18:09Z Arg120 until Ser122 0.9988379 structure_element cleaner0 2023-07-27T14:47:36Z SO: linker 0.9987049 evidence cleaner0 2023-07-27T14:53:10Z DUMMY: electron density protein PR: cleaner0 2023-07-27T12:47:44Z NsrR structure_element SO: cleaner0 2023-07-27T13:01:57Z RD 0.99953294 residue_name_number cleaner0 2023-07-27T13:19:25Z DUMMY: Asn85 0.9995608 residue_name_number cleaner0 2023-07-27T13:19:29Z DUMMY: Asp86 0.9995659 residue_name_number cleaner0 2023-07-27T13:19:33Z DUMMY: Glu87 0.99893194 structure_element cleaner0 2023-07-27T13:43:00Z SO: chain A 0.99816 evidence cleaner0 2023-07-27T14:53:12Z DUMMY: electron density 0.997624 evidence cleaner0 2023-07-27T14:53:16Z DUMMY: structure 0.9985448 oligomeric_state cleaner0 2023-07-27T13:18:36Z DUMMY: monomers protein PR: cleaner0 2023-07-27T12:47:44Z NsrR structure_element SO: cleaner0 2023-07-27T13:01:58Z RD 0.99848664 evidence cleaner0 2023-07-27T13:18:48Z DUMMY: rmsd 0.9985921 oligomeric_state cleaner0 2023-07-27T13:18:37Z DUMMY: monomers 0.99662185 evidence cleaner0 2023-07-27T14:53:18Z DUMMY: structure 0.99799687 oligomeric_state cleaner0 2023-07-27T13:18:42Z DUMMY: monomer 0.9981139 structure_element cleaner0 2023-07-27T14:47:41Z SO: A RESULTS paragraph 21674 NsrR-RD structurally adopts a αβ doubly-wound fold previously observed in OmpR/PhoB type regulators. Five β-strands (β1-β5) are arranged in a parallel fashion constituting the central core of the structure, which is surrounded by two α-helices (α1 and α5) on one and three helices (α2, α3, α4) on the other side (Fig 2). The NsrR-RD structure shows a β1-α1-β2-α2-β3-α3-β4-α4-β5-α5 topology as also observed for other RRs. protein PR: cleaner0 2023-07-27T12:47:44Z NsrR structure_element SO: cleaner0 2023-07-27T13:01:58Z RD 0.9978269 structure_element cleaner0 2023-07-27T14:47:46Z SO: αβ doubly-wound fold protein_type MESH: cleaner0 2023-07-27T13:19:55Z OmpR/PhoB type regulators 0.92422724 structure_element cleaner0 2023-07-27T13:44:03Z SO: β-strands 0.99796885 structure_element cleaner0 2023-07-27T13:23:38Z SO: β1-β5 0.9941896 evidence cleaner0 2023-07-27T14:53:21Z DUMMY: structure 0.9989569 structure_element cleaner0 2023-07-27T13:43:54Z SO: α-helices 0.9994605 structure_element cleaner0 2023-07-27T13:23:14Z SO: α1 0.9994593 structure_element cleaner0 2023-07-27T13:23:19Z SO: α5 0.9925608 structure_element cleaner0 2023-07-27T14:48:06Z SO: helices 0.9994474 structure_element cleaner0 2023-07-27T13:23:25Z SO: α2 0.9994404 structure_element cleaner0 2023-07-27T13:23:30Z SO: α3 0.99948514 structure_element cleaner0 2023-07-27T13:20:47Z SO: α4 protein PR: cleaner0 2023-07-27T12:47:44Z NsrR structure_element SO: cleaner0 2023-07-27T13:01:58Z RD 0.9972779 evidence cleaner0 2023-07-27T14:53:24Z DUMMY: structure structure_element SO: cleaner0 2023-07-27T13:23:09Z β1-α1-β2-α2-β3-α3-β4-α4-β5-α5 0.9992448 protein_type cleaner0 2023-07-27T13:02:16Z MESH: RRs pone.0149903.g002.jpg pone.0149903.g002 FIG fig_title_caption 22171 Structure of NsrR-RD. 0.9948383 evidence cleaner0 2023-07-27T14:53:30Z DUMMY: Structure protein PR: cleaner0 2023-07-27T12:47:44Z NsrR structure_element SO: cleaner0 2023-07-27T13:01:58Z RD pone.0149903.g002.jpg pone.0149903.g002 FIG fig_caption 22193 Cartoon representation of the helices (α1 – α5) and β-sheets (β1 - β5). Structural areas with the highest variations to the receiver domains of DrrB (pink, 1P2F), MtrA (grey, 2GWR), and PhoB (blue, 1B00) are marked in separate boxes. 0.9325321 structure_element cleaner0 2023-07-27T14:48:12Z SO: helices 0.99635005 structure_element cleaner0 2023-07-27T13:24:02Z SO: α1 – α5 0.9991474 structure_element cleaner0 2023-07-27T13:43:49Z SO: β-sheets 0.9984813 structure_element cleaner0 2023-07-27T14:48:22Z SO: β1 - β5 0.99939233 structure_element cleaner0 2023-07-27T14:48:25Z SO: receiver domains 0.99928397 protein cleaner0 2023-07-27T13:04:43Z PR: DrrB 0.99928206 protein cleaner0 2023-07-27T13:04:25Z PR: MtrA 0.99928015 protein cleaner0 2023-07-27T13:28:31Z PR: PhoB RESULTS title_2 22444 Comparison with structures of other receiver domains experimental_method MESH: cleaner0 2023-07-27T14:53:43Z Comparison 0.9972778 evidence cleaner0 2023-07-27T14:53:33Z DUMMY: structures 0.99821997 structure_element cleaner0 2023-07-27T14:48:30Z SO: receiver domains RESULTS paragraph 22497 NsrR belongs to the OmpR/PhoB family of RRs. The receiver domain of NsrR was superimposed with other structurally characterized receiver domains from the OmpR/PhoB family, such as DrrB, KdpE, MtrA, and the crystal structure of only the receiver domain of PhoB. The rmsd of the overlays and the corresponding PDB codes used are highlighted in Table 2. Superimposition of the structures revealed that helix α4 is slightly rotated outward in NsrR-RD (Fig 2). In receiver domains of response regulators, helix α4 has been shown to be a crucial part of the dimerization interface. Furthermore, helix α4 in NsrR is shorter than in other RRs. The first helical turn is unwound and adopts an unstructured region (see Fig 2). A slightly outward rotation or unwinding of helix α4 has been observed in the structures of other RD of regulators. For example, the structure of BaeR and RegX3 displayed a completely unwound helix α4. In the structure of DrrD, helix α4 is only partially displaced. In the receiver domain of NsrR, helix α4 is also partially displaced but in a different direction (S1 Fig). Inspection of the crystal contacts revealed no major interactions in this region that could have influenced the orientation of helix α4. Furthermore, NsrR is crystallized as a monomer, and investigation of the symmetry-related molecules did not reveal a functional dimer within the crystal. This could explain the flexibility and thereby the different orientation of helix α4 in NsrR. 0.9992686 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.92631733 protein_type cleaner0 2023-07-27T14:29:15Z MESH: OmpR/PhoB family 0.9988243 protein_type cleaner0 2023-07-27T13:02:16Z MESH: RRs 0.9993143 structure_element cleaner0 2023-07-27T12:48:55Z SO: receiver domain 0.99924767 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.99867636 experimental_method cleaner0 2023-07-27T14:38:37Z MESH: superimposed 0.9992309 structure_element cleaner0 2023-07-27T14:48:34Z SO: receiver domains 0.9120915 protein_type cleaner0 2023-07-27T14:29:23Z MESH: OmpR/PhoB family 0.9993724 protein cleaner0 2023-07-27T13:04:43Z PR: DrrB 0.9992994 protein cleaner0 2023-07-27T13:04:53Z PR: KdpE 0.9993486 protein cleaner0 2023-07-27T13:04:25Z PR: MtrA 0.9981092 evidence cleaner0 2023-07-27T14:53:48Z DUMMY: crystal structure 0.99913347 structure_element cleaner0 2023-07-27T12:48:55Z SO: receiver domain 0.9990823 protein cleaner0 2023-07-27T13:28:31Z PR: PhoB 0.9985311 evidence cleaner0 2023-07-27T13:18:49Z DUMMY: rmsd 0.99806637 experimental_method cleaner0 2023-07-27T14:38:41Z MESH: overlays 0.99843746 experimental_method cleaner0 2023-07-27T14:38:45Z MESH: Superimposition 0.9913635 evidence cleaner0 2023-07-27T14:53:51Z DUMMY: structures 0.99868447 structure_element cleaner0 2023-07-27T13:24:13Z SO: helix 0.99937207 structure_element cleaner0 2023-07-27T13:20:47Z SO: α4 0.51685774 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR structure_element SO: cleaner0 2023-07-27T13:01:58Z RD 0.99866635 structure_element cleaner0 2023-07-27T14:48:38Z SO: receiver domains 0.99828744 protein_type cleaner0 2023-07-27T13:03:37Z MESH: response regulators 0.99895096 structure_element cleaner0 2023-07-27T13:24:12Z SO: helix 0.99940157 structure_element cleaner0 2023-07-27T13:20:47Z SO: α4 0.99892545 site cleaner0 2023-07-27T14:33:02Z SO: dimerization interface 0.9989354 structure_element cleaner0 2023-07-27T13:24:13Z SO: helix 0.99936026 structure_element cleaner0 2023-07-27T13:20:46Z SO: α4 0.9992988 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.99918586 protein_type cleaner0 2023-07-27T13:02:16Z MESH: RRs 0.9848738 structure_element cleaner0 2023-07-27T14:48:48Z SO: first helical turn protein_state DUMMY: cleaner0 2023-07-27T14:28:59Z unwound 0.9173802 protein_state cleaner0 2023-07-27T14:28:41Z DUMMY: unstructured 0.998276 structure_element cleaner0 2023-07-27T13:24:13Z SO: helix 0.9991788 structure_element cleaner0 2023-07-27T13:20:47Z SO: α4 0.99831796 evidence cleaner0 2023-07-27T14:53:56Z DUMMY: structures 0.99908435 structure_element cleaner0 2023-07-27T13:01:58Z SO: RD 0.9976528 evidence cleaner0 2023-07-27T14:54:01Z DUMMY: structure 0.99934536 protein cleaner0 2023-07-27T15:00:57Z PR: BaeR 0.9994049 protein cleaner0 2023-07-27T13:04:20Z PR: RegX3 0.75553304 protein_state cleaner0 2023-07-27T14:29:02Z DUMMY: unwound 0.9991953 structure_element cleaner0 2023-07-27T13:24:13Z SO: helix 0.9988997 structure_element cleaner0 2023-07-27T13:20:47Z SO: α4 0.99758387 evidence cleaner0 2023-07-27T14:53:59Z DUMMY: structure 0.99940825 protein cleaner0 2023-07-27T13:04:48Z PR: DrrD 0.99679846 structure_element cleaner0 2023-07-27T13:24:13Z SO: helix 0.99936146 structure_element cleaner0 2023-07-27T13:20:47Z SO: α4 0.9991323 structure_element cleaner0 2023-07-27T12:48:55Z SO: receiver domain 0.99933666 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.9980818 structure_element cleaner0 2023-07-27T13:24:13Z SO: helix 0.9993893 structure_element cleaner0 2023-07-27T13:20:47Z SO: α4 0.99822336 structure_element cleaner0 2023-07-27T13:24:13Z SO: helix 0.9993075 structure_element cleaner0 2023-07-27T13:20:47Z SO: α4 0.99936146 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.9984592 experimental_method cleaner0 2023-07-27T14:39:05Z MESH: crystallized 0.9988293 oligomeric_state cleaner0 2023-07-27T13:18:43Z DUMMY: monomer 0.998806 oligomeric_state cleaner0 2023-07-27T13:38:00Z DUMMY: dimer 0.85652447 evidence cleaner0 2023-07-27T14:54:04Z DUMMY: crystal 0.9982503 structure_element cleaner0 2023-07-27T13:24:13Z SO: helix 0.9992778 structure_element cleaner0 2023-07-27T13:20:47Z SO: α4 0.99934965 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR pone.0149903.t002.xml pone.0149903.t002 TABLE table_title_caption 24009 The structures of the RD and ED domains of NsrR aligned to other response regulators. 0.9971283 evidence cleaner0 2023-07-27T14:54:10Z DUMMY: structures 0.9994692 structure_element cleaner0 2023-07-27T13:01:58Z SO: RD 0.9994423 structure_element cleaner0 2023-07-27T13:02:09Z SO: ED 0.99930155 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.6662647 experimental_method cleaner0 2023-07-27T14:39:08Z MESH: aligned 0.9969348 protein_type cleaner0 2023-07-27T13:03:37Z MESH: response regulators pone.0149903.t002.xml pone.0149903.t002 TABLE table_caption 24095 The rmsd values of the superimpositions of the structures of NsrR-RD and NsrR-ED with the available structures of members of the OmpR/PhoB subfamily are highlighted. *Seq ID (%) corresponds to the full-length protein sequence. 0.9984945 evidence cleaner0 2023-07-27T13:18:49Z DUMMY: rmsd 0.9987993 experimental_method cleaner0 2023-07-27T14:39:12Z MESH: superimpositions 0.9979717 evidence cleaner0 2023-07-27T14:54:13Z DUMMY: structures protein PR: cleaner0 2023-07-27T12:47:44Z NsrR structure_element SO: cleaner0 2023-07-27T13:01:58Z RD protein PR: cleaner0 2023-07-27T12:47:44Z NsrR structure_element SO: cleaner0 2023-07-27T13:02:09Z ED 0.9972584 evidence cleaner0 2023-07-27T14:54:16Z DUMMY: structures protein_type MESH: cleaner0 2023-07-27T13:03:42Z OmpR/PhoB subfamily protein_state DUMMY: cleaner0 2023-07-27T12:50:19Z full-length pone.0149903.t002.xml pone.0149903.t002 TABLE table <?xml version="1.0" encoding="UTF-8"?> <table frame="hsides" rules="groups"><colgroup span="1"><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/></colgroup><thead><tr><th align="left" rowspan="1" colspan="1"><italic>Protein</italic></th><th align="left" rowspan="1" colspan="1"><italic>PDB</italic></th><th align="left" rowspan="1" colspan="1"><italic>Z-score</italic></th><th align="left" rowspan="1" colspan="1"><italic>RMSD (Å)</italic></th><th align="left" rowspan="1" colspan="1"><italic>Number of residues (total number of residues)</italic></th><th align="left" rowspan="1" colspan="1"><italic>Seq</italic>. <italic>ID (%)*</italic></th><th align="left" rowspan="1" colspan="1"><italic>Reference</italic></th></tr></thead><tbody><tr><td align="center" colspan="7" rowspan="1"><bold>Receiver domain</bold></td></tr><tr><td align="justify" rowspan="1" colspan="1">KdpE</td><td align="justify" rowspan="1" colspan="1">4KNY</td><td align="justify" rowspan="1" colspan="1">18.8</td><td align="justify" rowspan="1" colspan="1">1.9</td><td align="justify" rowspan="1" colspan="1">117 (222)</td><td align="left" rowspan="1" colspan="1">28</td><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref042" ref-type="bibr">42</xref></td></tr><tr><td align="justify" rowspan="1" colspan="1">YycF</td><td align="justify" rowspan="1" colspan="1">2ZWM</td><td align="justify" rowspan="1" colspan="1">18.3</td><td align="justify" rowspan="1" colspan="1">1.7</td><td align="justify" rowspan="1" colspan="1">115 (120)</td><td align="left" rowspan="1" colspan="1">35</td><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref053" ref-type="bibr">53</xref></td></tr><tr><td align="justify" rowspan="1" colspan="1">YycF</td><td align="justify" rowspan="1" colspan="1">3F6P</td><td align="justify" rowspan="1" colspan="1">18.1</td><td align="justify" rowspan="1" colspan="1">1.7</td><td align="justify" rowspan="1" colspan="1">114 (120)</td><td align="left" rowspan="1" colspan="1">35</td><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref081" ref-type="bibr">81</xref></td></tr><tr><td align="justify" rowspan="1" colspan="1">DivK</td><td align="justify" rowspan="1" colspan="1">1M5T</td><td align="justify" rowspan="1" colspan="1">18.1</td><td align="justify" rowspan="1" colspan="1">1.9</td><td align="justify" rowspan="1" colspan="1">116 (123)</td><td align="left" rowspan="1" colspan="1">27</td><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref065" ref-type="bibr">65</xref></td></tr><tr><td align="justify" rowspan="1" colspan="1">KdpE</td><td align="justify" rowspan="1" colspan="1">1ZH2</td><td align="justify" rowspan="1" colspan="1">18.0</td><td align="justify" rowspan="1" colspan="1">1.9</td><td align="justify" rowspan="1" colspan="1">115 (120)</td><td align="left" rowspan="1" colspan="1">28</td><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref074" ref-type="bibr">74</xref></td></tr><tr><td align="justify" rowspan="1" colspan="1">PhoB</td><td align="justify" rowspan="1" colspan="1">1B00</td><td align="justify" rowspan="1" colspan="1">17.0</td><td align="justify" rowspan="1" colspan="1">1.9</td><td align="justify" rowspan="1" colspan="1">113 (122)</td><td align="left" rowspan="1" colspan="1">30</td><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref047" ref-type="bibr">47</xref></td></tr><tr><td align="center" colspan="7" rowspan="1"><bold>Effector domain</bold></td></tr><tr><td align="justify" rowspan="1" colspan="1">PhoB</td><td align="justify" rowspan="1" colspan="1">1GXQ</td><td align="justify" rowspan="1" colspan="1">13.7</td><td align="justify" rowspan="1" colspan="1">1.7</td><td align="justify" rowspan="1" colspan="1">92 (105)</td><td align="left" rowspan="1" colspan="1">30</td><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref054" ref-type="bibr">54</xref></td></tr><tr><td align="justify" rowspan="1" colspan="1">PhoP</td><td align="justify" rowspan="1" colspan="1">2PMU</td><td align="justify" rowspan="1" colspan="1">13.4</td><td align="justify" rowspan="1" colspan="1">1.7</td><td align="justify" rowspan="1" colspan="1">87 (93)</td><td align="left" rowspan="1" colspan="1">30</td><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref082" ref-type="bibr">82</xref></td></tr><tr><td align="justify" rowspan="1" colspan="1">PhoB</td><td align="justify" rowspan="1" colspan="1">2Z33</td><td align="justify" rowspan="1" colspan="1">13.3</td><td align="justify" rowspan="1" colspan="1">1.8</td><td align="justify" rowspan="1" colspan="1">92 (104)</td><td align="left" rowspan="1" colspan="1">30</td><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref083" ref-type="bibr">83</xref></td></tr><tr><td align="left" rowspan="1" colspan="1">PhoB (DNA bound)</td><td align="justify" rowspan="1" colspan="1">1GXP</td><td align="justify" rowspan="1" colspan="1">13.3</td><td align="justify" rowspan="1" colspan="1">2.0</td><td align="justify" rowspan="1" colspan="1">92 (101)</td><td align="left" rowspan="1" colspan="1">30</td><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref054" ref-type="bibr">54</xref></td></tr><tr><td align="justify" rowspan="1" colspan="1">SaeR</td><td align="justify" rowspan="1" colspan="1">4IXA</td><td align="justify" rowspan="1" colspan="1">13.0</td><td align="justify" rowspan="1" colspan="1">2.1</td><td align="justify" rowspan="1" colspan="1">94 (102)</td><td align="left" rowspan="1" colspan="1">29</td><td align="justify" rowspan="1" colspan="1">Not available</td></tr><tr><td align="justify" rowspan="1" colspan="1">RstA</td><td align="justify" rowspan="1" colspan="1">4NHJ</td><td align="justify" rowspan="1" colspan="1">11.8</td><td align="justify" rowspan="1" colspan="1">1.9</td><td align="justify" rowspan="1" colspan="1">85 (101)</td><td align="left" rowspan="1" colspan="1">29</td><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref084" ref-type="bibr">84</xref></td></tr><tr><td align="justify" rowspan="1" colspan="1">KdpE</td><td align="justify" rowspan="1" colspan="1">4KNY</td><td align="justify" rowspan="1" colspan="1">11.5</td><td align="justify" rowspan="1" colspan="1">2.6</td><td align="justify" rowspan="1" colspan="1">86 (222)</td><td align="left" rowspan="1" colspan="1">28</td><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref042" ref-type="bibr">42</xref></td></tr><tr><td align="center" colspan="7" rowspan="1"><bold>Full-length Response Regulators</bold></td></tr><tr><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"><italic>PDB code</italic></td><td align="center" rowspan="1" colspan="1"><italic>N-terminal rmsd (Å)</italic></td><td align="center" rowspan="1" colspan="1"><italic>C-terminal rmsd (Å)</italic></td><td align="center" rowspan="1" colspan="1"><italic>DNA bound</italic></td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"><italic>Reference</italic></td></tr><tr><td align="justify" rowspan="1" colspan="1">DrrB</td><td align="justify" rowspan="1" colspan="1">1P2F</td><td align="justify" rowspan="1" colspan="1">2.1</td><td align="justify" rowspan="1" colspan="1">2.3</td><td align="justify" rowspan="1" colspan="1">No</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref040" ref-type="bibr">40</xref></td></tr><tr><td align="justify" rowspan="1" colspan="1">DrrD</td><td align="justify" rowspan="1" colspan="1">1KGS</td><td align="justify" rowspan="1" colspan="1">2.1</td><td align="justify" rowspan="1" colspan="1">1.9</td><td align="justify" rowspan="1" colspan="1">No</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref041" ref-type="bibr">41</xref></td></tr><tr><td align="justify" rowspan="1" colspan="1">KdpE</td><td align="justify" rowspan="1" colspan="1">4KNY</td><td align="justify" rowspan="1" colspan="1">1.9</td><td align="justify" rowspan="1" colspan="1">2.6</td><td align="justify" rowspan="1" colspan="1">Yes</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref042" ref-type="bibr">42</xref></td></tr><tr><td align="justify" rowspan="1" colspan="1">MtrA</td><td align="justify" rowspan="1" colspan="1">2GWR</td><td align="justify" rowspan="1" colspan="1">2.1</td><td align="justify" rowspan="1" colspan="1">2.0</td><td align="justify" rowspan="1" colspan="1">No</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref037" ref-type="bibr">37</xref></td></tr><tr><td align="justify" rowspan="1" colspan="1">PrrA</td><td align="justify" rowspan="1" colspan="1">1YS6</td><td align="justify" rowspan="1" colspan="1">2.0</td><td align="justify" rowspan="1" colspan="1">2.2</td><td align="justify" rowspan="1" colspan="1">No</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref038" ref-type="bibr">38</xref></td></tr><tr><td align="justify" rowspan="1" colspan="1">RegX3</td><td align="justify" rowspan="1" colspan="1">2OQR</td><td align="justify" rowspan="1" colspan="1">2.3</td><td align="justify" rowspan="1" colspan="1">2.1</td><td align="justify" rowspan="1" colspan="1">No</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref036" ref-type="bibr">36</xref></td></tr><tr><td align="justify" rowspan="1" colspan="1">BaeR</td><td align="justify" rowspan="1" colspan="1">4B09</td><td align="justify" rowspan="1" colspan="1">2.1</td><td align="justify" rowspan="1" colspan="1">2.1</td><td align="justify" rowspan="1" colspan="1">No</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref057" ref-type="bibr">57</xref></td></tr><tr><td align="justify" rowspan="1" colspan="1">VraR</td><td align="justify" rowspan="1" colspan="1">4GVP</td><td align="justify" rowspan="1" colspan="1">2.3</td><td align="justify" rowspan="1" colspan="1">2.6</td><td align="justify" rowspan="1" colspan="1">No</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref085" ref-type="bibr">85</xref></td></tr></tbody></table> 24322 Protein PDB Z-score RMSD (Å) Number of residues (total number of residues) Seq. ID (%)* Reference Receiver domain KdpE 4KNY 18.8 1.9 117 (222) 28 YycF 2ZWM 18.3 1.7 115 (120) 35 YycF 3F6P 18.1 1.7 114 (120) 35 DivK 1M5T 18.1 1.9 116 (123) 27 KdpE 1ZH2 18.0 1.9 115 (120) 28 PhoB 1B00 17.0 1.9 113 (122) 30 Effector domain PhoB 1GXQ 13.7 1.7 92 (105) 30 PhoP 2PMU 13.4 1.7 87 (93) 30 PhoB 2Z33 13.3 1.8 92 (104) 30 PhoB (DNA bound) 1GXP 13.3 2.0 92 (101) 30 SaeR 4IXA 13.0 2.1 94 (102) 29 Not available RstA 4NHJ 11.8 1.9 85 (101) 29 KdpE 4KNY 11.5 2.6 86 (222) 28 Full-length Response Regulators PDB code N-terminal rmsd (Å) C-terminal rmsd (Å) DNA bound Reference DrrB 1P2F 2.1 2.3 No DrrD 1KGS 2.1 1.9 No KdpE 4KNY 1.9 2.6 Yes MtrA 2GWR 2.1 2.0 No PrrA 1YS6 2.0 2.2 No RegX3 2OQR 2.3 2.1 No BaeR 4B09 2.1 2.1 No VraR 4GVP 2.3 2.6 No RESULTS paragraph 25243 Based on the Dali server, the NsrR-RD domain is structurally closely related to KdpE (PDB code: 4KNY) from E. coli, displaying a sequence identity of 28%. This structural homology is also reflected by the low rmsd of 1.9 Å over 117 Cα atoms after superimposition of the receiver domains of NsrR and KdpE (Table 2). Furthermore, the orientation of the helix α4 in NsrR is close to that present in KdpE (S1 Fig). 0.9987018 experimental_method cleaner0 2023-07-27T14:39:20Z MESH: Dali server 0.99931526 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.86894065 structure_element cleaner0 2023-07-27T13:01:58Z SO: RD 0.9992918 protein cleaner0 2023-07-27T13:04:53Z PR: KdpE 0.99862313 species cleaner0 2023-07-27T13:21:52Z MESH: E. coli 0.9976701 evidence cleaner0 2023-07-27T13:18:49Z DUMMY: rmsd 0.99874544 experimental_method cleaner0 2023-07-27T14:39:23Z MESH: superimposition 0.99932015 structure_element cleaner0 2023-07-27T14:48:54Z SO: receiver domains 0.9993705 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.99928564 protein cleaner0 2023-07-27T13:04:53Z PR: KdpE 0.99937433 structure_element cleaner0 2023-07-27T13:24:13Z SO: helix 0.99948055 structure_element cleaner0 2023-07-27T13:20:47Z SO: α4 0.99938893 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.99926966 protein cleaner0 2023-07-27T13:04:53Z PR: KdpE RESULTS title_2 25662 Active site residues and dimerization site SO: cleaner0 2023-07-27T13:26:23Z Active site RESULTS paragraph 25700 All RRs contain a highly conserved aspartate residue in the active site (Fig 3; shown in red). Phosphorylation of this aspartate residue induces a conformational change leading to the activation of the effector domain that binds DNA and regulates the transcription of target genes. This site of phosphorylation is conserved throughout the family of response regulators, including the lantibiotic resistance-associated RRs such as BraR from L. monocytogenes, BceR from Bacillus subtilis, CprR from C. difficile, GraR from S. aureus, LcrR from S. mutans, LisR, and VirR from L. monocytogenes (Fig 3). 0.9992574 protein_type cleaner0 2023-07-27T13:02:16Z MESH: RRs 0.99878275 protein_state cleaner0 2023-07-27T13:26:48Z DUMMY: highly conserved 0.99776244 residue_name cleaner0 2023-07-27T13:26:44Z SO: aspartate 0.99911153 site cleaner0 2023-07-27T13:26:22Z SO: active site 0.9962476 ptm cleaner0 2023-07-27T12:50:12Z MESH: Phosphorylation 0.99746764 residue_name cleaner0 2023-07-27T13:26:45Z SO: aspartate 0.998433 structure_element cleaner0 2023-07-27T13:02:03Z SO: effector domain chemical CHEBI: cleaner0 2023-07-27T12:49:58Z DNA 0.9949208 ptm cleaner0 2023-07-27T12:50:12Z MESH: phosphorylation 0.99892527 protein_state cleaner0 2023-07-27T13:26:50Z DUMMY: conserved 0.99866056 protein_type cleaner0 2023-07-27T13:03:37Z MESH: response regulators protein_type MESH: cleaner0 2023-07-27T13:26:11Z lantibiotic resistance-associated RRs 0.9993311 protein cleaner0 2023-07-27T15:01:01Z PR: BraR 0.9983806 species cleaner0 2023-07-27T13:24:30Z MESH: L. monocytogenes 0.9992518 protein cleaner0 2023-07-27T13:25:32Z PR: BceR 0.9978348 species cleaner0 2023-07-27T12:55:20Z MESH: Bacillus subtilis 0.999303 protein cleaner0 2023-07-27T13:25:27Z PR: CprR 0.99798745 species cleaner0 2023-07-27T13:24:37Z MESH: C. difficile 0.9993249 protein cleaner0 2023-07-27T13:25:21Z PR: GraR 0.9983581 species cleaner0 2023-07-27T12:55:51Z MESH: S. aureus 0.9993357 protein cleaner0 2023-07-27T13:25:16Z PR: LcrR 0.9983671 species cleaner0 2023-07-27T13:24:43Z MESH: S. mutans 0.999337 protein cleaner0 2023-07-27T13:25:01Z PR: LisR 0.9993486 protein cleaner0 2023-07-27T13:24:55Z PR: VirR 0.9984111 species cleaner0 2023-07-27T13:24:31Z MESH: L. monocytogenes pone.0149903.g003.jpg pone.0149903.g003 FIG fig_title_caption 26299 Sequence alignment of NsrR protein with other response regulators. 0.9984927 experimental_method cleaner0 2023-07-27T14:39:33Z MESH: Sequence alignment 0.99662125 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.9976491 protein_type cleaner0 2023-07-27T13:03:37Z MESH: response regulators pone.0149903.g003.jpg pone.0149903.g003 FIG fig_caption 26366 A sequence alignment of NsrR with RRs belonging to the OmpR/PhoB subfamily (marked in grey) and RRs involved in lantibiotic resistance (black) is shown. The active site aspartate residue (highlighted in red), the residues forming the acidic pocket surrounding it (highlighted in pink), the switch residues (highlighted in blue), the conserved lysine residue (highlighted in green), the highly conserved residues of the linker region (colored in purple), the residues involved in dimer interface of receiver domain (highlighted in yellow), residues involved in interdomain interactions (shown in orange boxes and in cyan) and the residues involved in interaction with DNA (colored in blue) are shown. The linker region of the known structures is underlined within the sequence. 0.99870265 experimental_method cleaner0 2023-07-27T14:39:36Z MESH: sequence alignment 0.9993049 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.99912876 protein_type cleaner0 2023-07-27T13:02:16Z MESH: RRs protein_type MESH: cleaner0 2023-07-27T13:03:42Z OmpR/PhoB subfamily 0.99905664 protein_type cleaner0 2023-07-27T13:02:16Z MESH: RRs chemical CHEBI: cleaner0 2023-07-27T12:52:04Z lantibiotic 0.9988451 site cleaner0 2023-07-27T13:26:23Z SO: active site 0.9969194 residue_name cleaner0 2023-07-27T13:26:45Z SO: aspartate 0.99909055 site cleaner0 2023-07-27T14:33:08Z SO: acidic pocket 0.99875534 site cleaner0 2023-07-27T13:29:10Z SO: switch residues 0.99890363 protein_state cleaner0 2023-07-27T13:27:17Z DUMMY: conserved 0.99776745 residue_name cleaner0 2023-07-27T13:27:21Z SO: lysine 0.9988321 protein_state cleaner0 2023-07-27T13:27:15Z DUMMY: highly conserved 0.9983248 structure_element cleaner0 2023-07-27T13:52:42Z SO: linker region 0.9989053 site cleaner0 2023-07-27T13:52:10Z SO: dimer interface 0.99926007 structure_element cleaner0 2023-07-27T12:48:56Z SO: receiver domain 0.99575883 chemical cleaner0 2023-07-27T12:49:58Z CHEBI: DNA 0.9976224 structure_element cleaner0 2023-07-27T13:52:42Z SO: linker region 0.6837149 evidence cleaner0 2023-07-27T14:54:20Z DUMMY: structures RESULTS paragraph 27143 The putative phosphorylation site of NsrR is Asp55, which is localized at the end of strand β3 (Fig 3, shown in red; Fig 4) and lies within an acidic environment composed of the side chains of Glu12 and Asp13 (Fig 3, highlighted in pink). This pocket is similar to the acidic active site observed within most structures of RRs such as PhoB from E. coli, PhoP from M. tuberculosis, and DivK from Caulobacter crescentus. In NsrR, Glu12, Asp13, and Asp55 are in close proximity of a highly conserved Lys104 residue (highlighted in green in Fig 3). 0.9982375 site cleaner0 2023-07-27T13:27:36Z SO: phosphorylation site 0.9994178 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.9995726 residue_name_number cleaner0 2023-07-27T13:27:43Z DUMMY: Asp55 0.9946991 structure_element cleaner0 2023-07-27T14:49:00Z SO: strand 0.99752575 structure_element cleaner0 2023-07-27T13:27:50Z SO: β3 0.999574 residue_name_number cleaner0 2023-07-27T13:28:01Z DUMMY: Glu12 0.99957496 residue_name_number cleaner0 2023-07-27T13:28:07Z DUMMY: Asp13 0.99850416 site cleaner0 2023-07-27T14:33:14Z SO: pocket 0.645693 protein_state cleaner0 2023-07-27T14:30:06Z DUMMY: acidic 0.9391881 site cleaner0 2023-07-27T13:26:23Z SO: active site 0.9979899 evidence cleaner0 2023-07-27T14:54:24Z DUMMY: structures 0.9990188 protein_type cleaner0 2023-07-27T13:02:16Z MESH: RRs 0.99903286 protein cleaner0 2023-07-27T13:28:31Z PR: PhoB 0.9978824 species cleaner0 2023-07-27T13:21:53Z MESH: E. coli 0.9987301 protein cleaner0 2023-07-27T13:04:36Z PR: PhoP 0.99839306 species cleaner0 2023-07-27T13:28:24Z MESH: M. tuberculosis 0.9990707 protein cleaner0 2023-07-27T13:28:37Z PR: DivK 0.9986853 species cleaner0 2023-07-27T13:28:43Z MESH: Caulobacter crescentus 0.99941945 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.9995726 residue_name_number cleaner0 2023-07-27T13:28:02Z DUMMY: Glu12 0.999577 residue_name_number cleaner0 2023-07-27T13:28:07Z DUMMY: Asp13 0.9995803 residue_name_number cleaner0 2023-07-27T13:27:44Z DUMMY: Asp55 0.99885833 protein_state cleaner0 2023-07-27T13:28:51Z DUMMY: highly conserved 0.9995635 residue_name_number cleaner0 2023-07-27T13:28:55Z DUMMY: Lys104 pone.0149903.g004.jpg pone.0149903.g004 FIG fig_title_caption 27691 Location of the highly conserved Asp55 and inactive state conformation of the key switch residues, Ser82 and Phe101 in NsrR-RD. 0.99882954 protein_state cleaner0 2023-07-27T13:29:02Z DUMMY: highly conserved 0.999592 residue_name_number cleaner0 2023-07-27T13:27:44Z DUMMY: Asp55 0.9993012 protein_state cleaner0 2023-07-27T12:50:31Z DUMMY: inactive 0.9986496 site cleaner0 2023-07-27T13:29:09Z SO: switch residues 0.9995976 residue_name_number cleaner0 2023-07-27T13:29:15Z DUMMY: Ser82 0.9996113 residue_name_number cleaner0 2023-07-27T13:29:19Z DUMMY: Phe101 0.49365017 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR structure_element SO: cleaner0 2023-07-27T13:01:58Z RD pone.0149903.g004.jpg pone.0149903.g004 FIG fig_caption 27819 NsrR (represented in yellow) displays a geometry representing the inactive state as deduced from the inactive state structure of PhoB (shown in brown, PDB code 1B00) (a). The inactive conformation of NsrR differs from the active state structure of PhoB (light blue, PDB code 1ZES) (b) in the orientation of the corresponding switch residues, Ser82 and Phe101, which adopt a conformation pointing away from the active site (Asp55 in NsrR). 0.9993332 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.99925977 protein_state cleaner0 2023-07-27T12:50:31Z DUMMY: inactive 0.999292 protein_state cleaner0 2023-07-27T12:50:31Z DUMMY: inactive 0.9965479 evidence cleaner0 2023-07-27T14:54:29Z DUMMY: structure 0.999335 protein cleaner0 2023-07-27T13:28:32Z PR: PhoB 0.99931276 protein_state cleaner0 2023-07-27T12:50:31Z DUMMY: inactive 0.99935955 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR 0.9992592 protein_state cleaner0 2023-07-27T12:50:25Z DUMMY: active 0.9966697 evidence cleaner0 2023-07-27T14:54:31Z DUMMY: structure 0.99936277 protein cleaner0 2023-07-27T13:28:32Z PR: PhoB 0.9986987 site cleaner0 2023-07-27T13:29:10Z SO: switch residues 0.9996013 residue_name_number cleaner0 2023-07-27T13:29:15Z DUMMY: Ser82 0.9996087 residue_name_number cleaner0 2023-07-27T13:29:21Z DUMMY: Phe101 0.99907035 site cleaner0 2023-07-27T13:26:23Z SO: active site 0.9995894 residue_name_number cleaner0 2023-07-27T13:27:44Z DUMMY: Asp55 0.9993617 protein cleaner0 2023-07-27T12:47:44Z PR: NsrR RESULTS paragraph 28258 A divalent metal ion is usually bound in this acidic environment and is essential for phosphorylation and de-phosphorylation of RRs. In some RRs like CheY, Mg2+ is observed in the structure, bound near the phosphorylation site. In the KdpE regulator from E. coli that is involved in osmoregulation, a divalent calcium ion is present. However, the structure of NsrR-RD did not contain any divalent ion. Instead, a water molecule is present, which interacts with Glu12 of the acidic pocket, Lys104, and another water molecule in the vicinity. 0.9805444 ptm cleaner0 2023-07-27T12:50:12Z MESH: phosphorylation 0.8213034 ptm cleaner0 2023-07-27T13:30:09Z MESH: de-phosphorylation 0.99926823 protein_type cleaner0 2023-07-27T13:02:16Z MESH: RRs 0.99929166 protein_type cleaner0 2023-07-27T13:02:16Z MESH: RRs 0.998963 protein cleaner0 2023-07-27T13:30:25Z PR: CheY 0.9990341 chemical cleaner0 2023-07-27T13:30:17Z CHEBI: Mg2+ 0.9984463 evidence cleaner0 2023-07-27T14:54:35Z DUMMY: structure 0.9743113 protein_state cleaner0 2023-07-27T14:30:17Z DUMMY: bound 0.9988899 site cleaner0 2023-07-27T13:27:37Z SO: phosphorylation site protein PR: cleaner0 2023-07-27T13:04:53Z KdpE protein_type MESH: cleaner0 2023-07-27T13:30:53Z regulator 0.9986133 species cleaner0 2023-07-27T13:21:53Z MESH: E. coli 0.99884295 chemical cleaner0 2023-07-27T14:28:05Z CHEBI: calcium 0.9983334 evidence cleaner0 2023-07-27T14:54:38Z DUMMY: structure protein PR: cleaner0 2023-07-27T12:47:45Z NsrR structure_element SO: cleaner0 2023-07-27T13:01:58Z RD 0.9990127 chemical cleaner0 2023-07-27T14:28:09Z CHEBI: water 0.99956554 residue_name_number cleaner0 2023-07-27T13:28:02Z DUMMY: Glu12 0.9985113 site cleaner0 2023-07-27T14:33:19Z SO: acidic pocket 0.9995339 residue_name_number cleaner0 2023-07-27T13:28:56Z DUMMY: Lys104 0.99894077 chemical cleaner0 2023-07-27T14:28:14Z CHEBI: water RESULTS paragraph 28799 Within the β4-α4 loop and in β5 of the RD of RRs, specific amino acids are crucial for signal transduction from the RD to the ED via conformational changes that are a consequence of phosphorylation of the RD. These amino acids are Ser/Thr and Phe/Tyr located at the end of β4 and before β5, respectively, and designated as “signature switch residues”. As seen in the alignment (Fig 3, highlighted in blue), these signature residues (Ser/Thr and Phe/Tyr) are highly conserved in the lantibiotic resistance-associated RRs. The orientation of the side chains of these residues determines whether the RD is in an active or inactive state. In the inactive state, the phenylalanine or tyrosine residue faces away from the active site, and the corresponding serine or threonine residue adopts an outward-facing conformation as well (Fig 4A). In contrast, the switch residues face towards the active site in the active state conformation (Fig 4B). 0.9991443 structure_element cleaner0 2023-07-27T13:31:13Z SO: β4-α4 loop 0.99936444 structure_element cleaner0 2023-07-27T13:31:29Z SO: β5 0.99957186 structure_element cleaner0 2023-07-27T13:01:58Z SO: RD 0.9990752 protein_type cleaner0 2023-07-27T13:02:16Z MESH: RRs 0.99949634 structure_element cleaner0 2023-07-27T13:01:58Z SO: RD 0.9982326 structure_element cleaner0 2023-07-27T13:02:09Z SO: ED 0.9962458 ptm cleaner0 2023-07-27T12:50:12Z MESH: phosphorylation 0.99957305 structure_element cleaner0 2023-07-27T13:01:58Z SO: RD 0.9958106 residue_name cleaner0 2023-07-27T13:31:39Z SO: Ser 0.99469405 residue_name cleaner0 2023-07-27T13:31:44Z SO: Thr 0.9963182 residue_name cleaner0 2023-07-27T13:31:48Z SO: Phe 0.9953441 residue_name cleaner0 2023-07-27T13:31:53Z SO: Tyr 0.9993119 structure_element cleaner0 2023-07-27T13:31:33Z SO: β4 0.99923587 structure_element cleaner0 2023-07-27T13:31:28Z SO: β5 0.9972958 site cleaner0 2023-07-27T13:32:48Z SO: signature switch residues 0.99501884 experimental_method cleaner0 2023-07-27T14:39:45Z MESH: alignment 0.99686414 residue_name cleaner0 2023-07-27T13:31:40Z SO: Ser 0.9956923 residue_name cleaner0 2023-07-27T13:31:45Z SO: Thr 0.9970174 residue_name cleaner0 2023-07-27T13:31:49Z SO: Phe 0.99610436 residue_name cleaner0 2023-07-27T13:31:53Z SO: Tyr 0.99895 protein_state cleaner0 2023-07-27T14:30:22Z DUMMY: highly conserved 0.99675304 protein_type cleaner0 2023-07-27T13:32:10Z MESH: lantibiotic resistance-associated RRs 0.99955064 structure_element cleaner0 2023-07-27T13:01:58Z SO: RD 0.99911636 protein_state cleaner0 2023-07-27T12:50:25Z DUMMY: active 0.99923444 protein_state cleaner0 2023-07-27T12:50:31Z DUMMY: inactive 0.99929166 protein_state cleaner0 2023-07-27T12:50:31Z DUMMY: inactive 0.9975184 residue_name cleaner0 2023-07-27T13:32:23Z SO: phenylalanine 0.9971637 residue_name cleaner0 2023-07-27T13:32:32Z SO: tyrosine 0.99902064 site cleaner0 2023-07-27T13:26:23Z SO: active site 0.9974087 residue_name cleaner0 2023-07-27T13:32:18Z SO: serine 0.9970175 residue_name cleaner0 2023-07-27T13:32:27Z SO: threonine 0.99904776 protein_state cleaner0 2023-07-27T14:30:26Z DUMMY: outward-facing 0.9982003 site cleaner0 2023-07-27T13:29:10Z SO: switch residues 0.99894565 site cleaner0 2023-07-27T13:26:23Z SO: active site 0.99922574 protein_state cleaner0 2023-07-27T12:50:25Z DUMMY: active RESULTS paragraph 29755 By sequence alignment with other lantibiotic resistance-associated RRs, these “signature switch residues” are identified as Ser82 and Phe101 in NsrR (see above). Although some RRs such as KdpE, BraR, BceR, GraR, and VirR contain a serine residue as the first switch residue, the others possess a threonine instead. Furthermore, the second switch residue is mostly a tyrosine, with NsrR, BraR, and BceR being the only exceptions containing a phenylalanine at that position. A comparison of the NsrR-RD structure with the available structures of PhoB (Fig 4) in the active (PDB code: 1ZES) and inactive (PDB code: 1B00) states demonstrates that Ser82 (NsrR-RD) is oriented away from the active site Asp55, and that Phe101 is also in an outward conformation suggesting an inactive state of the NsrR-RD (Fig 4A). 0.9987155 experimental_method cleaner0 2023-07-27T14:39:54Z MESH: sequence alignment 0.9867547 protein_type cleaner0 2023-07-27T13:32:11Z MESH: lantibiotic resistance-associated RRs 0.9982168 site cleaner0 2023-07-27T13:32:49Z SO: signature switch residues 0.9995956 residue_name_number cleaner0 2023-07-27T13:29:15Z DUMMY: Ser82 0.9995952 residue_name_number cleaner0 2023-07-27T13:29:21Z DUMMY: Phe101 0.9993794 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR 0.9990345 protein_type cleaner0 2023-07-27T13:02:16Z MESH: RRs 0.9992231 protein cleaner0 2023-07-27T13:04:53Z PR: KdpE 0.99924433 protein cleaner0 2023-07-27T15:01:06Z PR: BraR 0.99924636 protein cleaner0 2023-07-27T13:25:33Z PR: BceR 0.999281 protein cleaner0 2023-07-27T13:25:22Z PR: GraR 0.9993087 protein cleaner0 2023-07-27T13:24:56Z PR: VirR 0.99800855 residue_name cleaner0 2023-07-27T13:32:19Z SO: serine 0.9939422 site cleaner0 2023-07-27T13:33:54Z SO: first switch residue 0.9978789 residue_name cleaner0 2023-07-27T13:32:28Z SO: threonine 0.9968428 site cleaner0 2023-07-27T13:33:59Z SO: second switch residue 0.99760485 residue_name cleaner0 2023-07-27T13:32:33Z SO: tyrosine 0.9993279 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR 0.99930656 protein cleaner0 2023-07-27T15:01:10Z PR: BraR 0.99928623 protein cleaner0 2023-07-27T13:25:33Z PR: BceR 0.99770194 residue_name cleaner0 2023-07-27T13:32:23Z SO: phenylalanine protein PR: cleaner0 2023-07-27T12:47:45Z NsrR structure_element SO: cleaner0 2023-07-27T13:01:58Z RD 0.9980069 evidence cleaner0 2023-07-27T14:54:45Z DUMMY: structure 0.9976042 evidence cleaner0 2023-07-27T14:54:50Z DUMMY: structures 0.9993369 protein cleaner0 2023-07-27T13:28:32Z PR: PhoB 0.9991748 protein_state cleaner0 2023-07-27T12:50:25Z DUMMY: active 0.99925095 protein_state cleaner0 2023-07-27T12:50:31Z DUMMY: inactive 0.9996037 residue_name_number cleaner0 2023-07-27T13:29:15Z DUMMY: Ser82 0.99407935 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR structure_element SO: cleaner0 2023-07-27T13:01:58Z RD 0.998924 site cleaner0 2023-07-27T13:26:23Z SO: active site 0.9995777 residue_name_number cleaner0 2023-07-27T13:27:44Z DUMMY: Asp55 0.99959034 residue_name_number cleaner0 2023-07-27T13:29:21Z DUMMY: Phe101 0.99907637 protein_state cleaner0 2023-07-27T14:30:33Z DUMMY: outward 0.999186 protein_state cleaner0 2023-07-27T12:50:31Z DUMMY: inactive 0.9952152 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR structure_element SO: cleaner0 2023-07-27T13:01:58Z RD RESULTS paragraph 30568 As mentioned above, RRs contain a phosphorylation-activated switch and normally exist in equilibrium between the active and inactive conformations. Phosphorylation shifts the equilibrium towards the active conformation and induces the formation of rotationally symmetric dimers on the α4-β5-α5 interface of RDs. It has been suggested that dimerization is crucial for DNA-binding of RRs of the OmpR/PhoB subfamily. 0.9986951 protein_type cleaner0 2023-07-27T13:02:16Z MESH: RRs 0.90515757 protein_state cleaner0 2023-07-27T13:34:19Z DUMMY: phosphorylation-activated 0.9873713 site cleaner0 2023-07-27T14:33:36Z SO: switch 0.9990515 protein_state cleaner0 2023-07-27T12:50:25Z DUMMY: active 0.9990963 protein_state cleaner0 2023-07-27T12:50:31Z DUMMY: inactive 0.99587303 ptm cleaner0 2023-07-27T12:50:12Z MESH: Phosphorylation 0.99908197 protein_state cleaner0 2023-07-27T12:50:25Z DUMMY: active 0.9985526 oligomeric_state cleaner0 2023-07-27T13:37:55Z DUMMY: dimers 0.9987912 site cleaner0 2023-07-27T13:34:35Z SO: α4-β5-α5 interface 0.6789144 structure_element cleaner0 2023-07-27T13:34:51Z SO: RDs chemical CHEBI: cleaner0 2023-07-27T12:49:58Z DNA 0.99902534 protein_type cleaner0 2023-07-27T13:02:16Z MESH: RRs protein_type MESH: cleaner0 2023-07-27T13:03:42Z OmpR/PhoB subfamily RESULTS paragraph 30993 The RD domain of NsrR was crystallized with two separate monomers in the asymmetric unit. Therefore, we performed a DALI search and focused on RD domains that were structurally determined as functional dimers. In this context, the dimer of full-length KdpE from E. coli (Z-score 18.8, rmsd 1.9 Å over 117 Cα atoms) (PDB code: 4KNY) and the structure of the functional dimer of the RD of KdpE from E. coli (PDB code: 1ZH2) represent the most structurally related structures. 0.9994293 structure_element cleaner0 2023-07-27T13:01:58Z SO: RD 0.99937123 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR 0.99852055 experimental_method cleaner0 2023-07-27T14:40:01Z MESH: crystallized 0.998896 oligomeric_state cleaner0 2023-07-27T13:18:37Z DUMMY: monomers 0.998592 experimental_method cleaner0 2023-07-27T14:40:04Z MESH: DALI search 0.9994253 structure_element cleaner0 2023-07-27T13:01:58Z SO: RD 0.57029694 protein_state cleaner0 2023-07-27T14:30:38Z DUMMY: functional 0.99879634 oligomeric_state cleaner0 2023-07-27T13:37:55Z DUMMY: dimers 0.9987651 oligomeric_state cleaner0 2023-07-27T13:38:00Z DUMMY: dimer 0.9991476 protein_state cleaner0 2023-07-27T12:50:19Z DUMMY: full-length 0.9984451 protein cleaner0 2023-07-27T13:04:53Z PR: KdpE 0.9985321 species cleaner0 2023-07-27T13:21:53Z MESH: E. coli 0.99824125 evidence cleaner0 2023-07-27T13:34:58Z DUMMY: Z-score 0.9985576 evidence cleaner0 2023-07-27T13:18:49Z DUMMY: rmsd 0.99799037 evidence cleaner0 2023-07-27T14:54:55Z DUMMY: structure 0.9678832 protein_state cleaner0 2023-07-27T14:30:42Z DUMMY: functional 0.99880075 oligomeric_state cleaner0 2023-07-27T13:38:00Z DUMMY: dimer 0.9994287 structure_element cleaner0 2023-07-27T13:01:58Z SO: RD 0.9983266 protein cleaner0 2023-07-27T13:04:53Z PR: KdpE 0.99853164 species cleaner0 2023-07-27T13:21:53Z MESH: E. coli 0.58302724 evidence cleaner0 2023-07-27T14:54:57Z DUMMY: structures RESULTS paragraph 31471 We aligned NsrR-RD on both monomers of the RD of KdpE. Since helix α4 of NsrR-RD is orientated slightly different when compared with other structures of RDs (Fig 2), helix α4 and the N-terminal loop of one monomer were clashing with the second monomer (S2A Fig). Therefore, helix α4 and the N-terminal loop were shifted to the position of KdpE by primarily modifying backbone torsion angles in the region immediately C-terminal to helix α4. Afterwards, helix α4 and the adjacent loops were energy minimized with the MAB force field as implemented in the program Moloc; all other atoms of NsrR-RD were kept fixed. The result is highlighted in S2B Fig. The energy minimized structure of NsrR-RD was then superimposed on the dimeric structure of KdpE. 0.99855644 experimental_method cleaner0 2023-07-27T14:40:15Z MESH: aligned protein PR: cleaner0 2023-07-27T12:47:45Z NsrR structure_element SO: cleaner0 2023-07-27T13:01:58Z RD 0.99868745 oligomeric_state cleaner0 2023-07-27T13:18:37Z DUMMY: monomers 0.9988589 structure_element cleaner0 2023-07-27T13:01:58Z SO: RD 0.9914846 protein cleaner0 2023-07-27T13:04:53Z PR: KdpE 0.9993125 structure_element cleaner0 2023-07-27T13:24:13Z SO: helix 0.9994419 structure_element cleaner0 2023-07-27T13:20:47Z SO: α4 protein PR: cleaner0 2023-07-27T12:47:45Z NsrR structure_element SO: cleaner0 2023-07-27T13:01:58Z RD 0.9982222 evidence cleaner0 2023-07-27T14:55:02Z DUMMY: structures 0.97735256 structure_element cleaner0 2023-07-27T13:34:52Z SO: RDs 0.99917585 structure_element cleaner0 2023-07-27T13:24:13Z SO: helix 0.99945635 structure_element cleaner0 2023-07-27T13:20:47Z SO: α4 0.9992138 structure_element cleaner0 2023-07-27T14:49:05Z SO: loop 0.99885714 oligomeric_state cleaner0 2023-07-27T13:18:43Z DUMMY: monomer 0.99884015 oligomeric_state cleaner0 2023-07-27T13:18:43Z DUMMY: monomer 0.9991743 structure_element cleaner0 2023-07-27T13:24:13Z SO: helix 0.9994629 structure_element cleaner0 2023-07-27T13:20:47Z SO: α4 0.9989548 structure_element cleaner0 2023-07-27T14:49:09Z SO: loop 0.9748784 protein cleaner0 2023-07-27T13:04:53Z PR: KdpE 0.9989729 structure_element cleaner0 2023-07-27T13:24:13Z SO: helix 0.9994205 structure_element cleaner0 2023-07-27T13:20:47Z SO: α4 0.9989839 structure_element cleaner0 2023-07-27T13:24:13Z SO: helix 0.9994455 structure_element cleaner0 2023-07-27T13:20:47Z SO: α4 0.99017185 structure_element cleaner0 2023-07-27T14:49:12Z SO: loops experimental_method MESH: cleaner0 2023-07-27T14:40:43Z energy minimized with the MAB force field protein PR: cleaner0 2023-07-27T12:47:45Z NsrR structure_element SO: cleaner0 2023-07-27T13:01:58Z RD 0.90363157 protein_state cleaner0 2023-07-27T14:41:01Z DUMMY: energy minimized 0.9972216 evidence cleaner0 2023-07-27T14:55:06Z DUMMY: structure protein PR: cleaner0 2023-07-27T12:47:45Z NsrR structure_element SO: cleaner0 2023-07-27T13:01:58Z RD 0.99862397 experimental_method cleaner0 2023-07-27T14:41:04Z MESH: superimposed 0.9988412 oligomeric_state cleaner0 2023-07-27T13:37:49Z DUMMY: dimeric 0.99755234 evidence cleaner0 2023-07-27T14:55:09Z DUMMY: structure 0.9974003 protein cleaner0 2023-07-27T13:04:53Z PR: KdpE RESULTS paragraph 32240 The putative functional dimer of NsrR-RD is depicted in Fig 5. The dimeric interface is formed by α4-β5-α5 of RD (Fig 5A), as previously observed in other RRs. In KdpE, a network of salt bridges and other electrostatic interactions stabilize the interface within a single monomer as well as between the monomers. Majority of these interactions involve residues that are highly conserved within the OmpR/PhoB subfamily of RRs. In addition, the dimeric interface of KdpE is characterized by hydrophobic patch formed by residues Ile88 (α4), Leu91 (α4), Ala110 (α5), and Val114 (α5). Structurally, a similar set of residues is also found in NsrR: Leu94 (α4), Val110 (α5) and Ala113 (α5), respectively (depicted as spheres in Fig 5B), which are conserved to some extent on sequence level (highlighted in yellow; Fig 3). 0.99889624 oligomeric_state cleaner0 2023-07-27T13:38:00Z DUMMY: dimer 0.51494896 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR 0.630312 structure_element cleaner0 2023-07-27T13:01:58Z SO: RD 0.9989375 site cleaner0 2023-07-27T13:37:42Z SO: dimeric interface 0.9579359 structure_element cleaner0 2023-07-27T13:35:58Z SO: α4-β5-α5 0.99956626 structure_element cleaner0 2023-07-27T13:01:58Z SO: RD 0.9991442 protein_type cleaner0 2023-07-27T13:02:17Z MESH: RRs 0.95380193 protein cleaner0 2023-07-27T13:04:53Z PR: KdpE 0.9955318 bond_interaction cleaner0 2023-07-27T13:37:04Z MESH: salt bridges 0.9959799 bond_interaction cleaner0 2023-07-27T13:37:08Z MESH: electrostatic interactions 0.99902105 site cleaner0 2023-07-27T14:33:42Z SO: interface 0.99887425 oligomeric_state cleaner0 2023-07-27T13:18:43Z DUMMY: monomer 0.9986094 oligomeric_state cleaner0 2023-07-27T13:18:37Z DUMMY: monomers 0.99877894 protein_state cleaner0 2023-07-27T14:30:45Z DUMMY: highly conserved protein_type MESH: cleaner0 2023-07-27T13:03:42Z OmpR/PhoB subfamily 0.99906415 protein_type cleaner0 2023-07-27T13:02:17Z MESH: RRs 0.9990164 site cleaner0 2023-07-27T13:37:00Z SO: dimeric interface 0.55356544 protein cleaner0 2023-07-27T13:04:53Z PR: KdpE 0.9988632 site cleaner0 2023-07-27T13:36:57Z SO: hydrophobic patch 0.9995628 residue_name_number cleaner0 2023-07-27T13:36:13Z DUMMY: Ile88 0.99930334 structure_element cleaner0 2023-07-27T13:20:48Z SO: α4 0.9995664 residue_name_number cleaner0 2023-07-27T13:36:18Z DUMMY: Leu91 0.9991937 structure_element cleaner0 2023-07-27T13:20:48Z SO: α4 0.9995665 residue_name_number cleaner0 2023-07-27T13:36:23Z DUMMY: Ala110 0.9991861 structure_element cleaner0 2023-07-27T13:23:20Z SO: α5 0.99957925 residue_name_number cleaner0 2023-07-27T13:36:28Z DUMMY: Val114 0.9991819 structure_element cleaner0 2023-07-27T13:23:20Z SO: α5 0.9992605 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR 0.9995808 residue_name_number cleaner0 2023-07-27T13:36:34Z DUMMY: Leu94 0.9992924 structure_element cleaner0 2023-07-27T13:20:48Z SO: α4 0.9995852 residue_name_number cleaner0 2023-07-27T13:36:39Z DUMMY: Val110 0.99926513 structure_element cleaner0 2023-07-27T13:23:20Z SO: α5 0.9995722 residue_name_number cleaner0 2023-07-27T13:36:43Z DUMMY: Ala113 0.99918777 structure_element cleaner0 2023-07-27T13:23:20Z SO: α5 0.85403407 protein_state cleaner0 2023-07-27T13:36:48Z DUMMY: conserved pone.0149903.g005.jpg pone.0149903.g005 FIG fig_title_caption 33094 Functional dimer orientation of the RDs of NsrR. 0.99872226 oligomeric_state cleaner0 2023-07-27T13:37:59Z DUMMY: dimer 0.9926811 structure_element cleaner0 2023-07-27T13:34:52Z SO: RDs 0.99941933 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR pone.0149903.g005.jpg pone.0149903.g005 FIG fig_caption 33143 Dimeric structure of the RD of NsrR aligned to the structure of KdpE (PDB code 1ZH2, not shown). (a) The two monomers of NsrR as functional dimers are represented in a cartoon representation displayed in cyan and yellow colors. (b) Zoom-in of the dimeric interface mediated by α4-β5-α5. The monomer-monomer interactions are facilitated by hydrophobic residues (displayed as spheres), inter- and intra-domain interactions (displayed as sticks). The layout is adopted from. 0.9987577 oligomeric_state cleaner0 2023-07-27T13:37:49Z DUMMY: Dimeric 0.992486 evidence cleaner0 2023-07-27T14:55:17Z DUMMY: structure 0.9995028 structure_element cleaner0 2023-07-27T13:01:58Z SO: RD 0.9994117 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR 0.9930173 evidence cleaner0 2023-07-27T14:55:20Z DUMMY: structure 0.99721897 protein cleaner0 2023-07-27T13:04:53Z PR: KdpE 0.99883336 oligomeric_state cleaner0 2023-07-27T13:18:37Z DUMMY: monomers 0.9994098 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR 0.99882084 oligomeric_state cleaner0 2023-07-27T13:37:54Z DUMMY: dimers 0.99887955 site cleaner0 2023-07-27T13:37:41Z SO: dimeric interface structure_element SO: cleaner0 2023-07-27T13:37:36Z α4-β5-α5 0.9766981 oligomeric_state cleaner0 2023-07-27T13:18:43Z DUMMY: monomer 0.95569986 oligomeric_state cleaner0 2023-07-27T13:18:43Z DUMMY: monomer RESULTS paragraph 33626 Conserved intermolecular electrostatic interactions further stabilize the monomer-monomer interaction of KdpE and are formed between Asp97 (β5) and Arg111 (α5), Asp96 (α4–β5 loop) and Arg118 (α5), and Asp92 (α4) and Arg113 (α5). Some of these interactions can also be identified in the dimeric model of NsrR-RD. Here, Asp100 (β5) and Lys114 (α5) form an interaction within one monomer, and an intermolecular interaction can be observed between Asn95 (α4) of one monomer with Thr116 (α5) of the other monomer (Fig 3, shown in cyan). Asp99 (α4–β5 loop; Fig 3, shown in cyan) points toward the side chain of Arg121. This interaction is also observed in KdpE (Asp96 (α4–β5 loop) and Arg118 (α5)). In KdpE, Arg111 is additionally stabilized by another intra-molecular salt bridge with Glu107 (α5). Interestingly, in NsrR-RD this amino acid corresponds to Val110 (highlighted in yellow in Fig 3). As observed in this alignment, the above-mentioned arginine residue (Arg111 in KdpE) is either an arginine or a lysine residue (Lys114 in NsrR) in all RRs used in the alignment (Fig 3, shown in cyan). Interestingly, whenever an arginine is present at this position (Arg111 in KdpE), a glutamate (Glu107 in KdpE) is present as well, presumably stabilizing the arginine side chain. However, when a lysine is present at this position, the glutamate is exchanged to a hydrophobic residue contributing to the hydrophobic patch described above. Additionally, it has been shown for PhoB from E. coli and PhoP from B. subtilis that mutating the corresponding residues involved in dimerisation (residues Asp100, Val110 and Lys114 in NsrR) results in monomeric form of response regulator which has lost the ability to dimerize as well as display reduced DNA binding capabilities. 0.99631524 bond_interaction cleaner0 2023-07-27T13:37:09Z MESH: electrostatic interactions 0.9952254 oligomeric_state cleaner0 2023-07-27T13:18:43Z DUMMY: monomer 0.98631454 oligomeric_state cleaner0 2023-07-27T13:18:43Z DUMMY: monomer 0.99910814 protein cleaner0 2023-07-27T13:04:53Z PR: KdpE 0.99952424 residue_name_number cleaner0 2023-07-27T13:38:33Z DUMMY: Asp97 0.9993235 structure_element cleaner0 2023-07-27T13:31:29Z SO: β5 0.99950457 residue_name_number cleaner0 2023-07-27T13:38:39Z DUMMY: Arg111 0.99937016 structure_element cleaner0 2023-07-27T13:23:20Z SO: α5 0.9995074 residue_name_number cleaner0 2023-07-27T13:38:43Z DUMMY: Asp96 0.9988633 structure_element cleaner0 2023-07-27T13:38:27Z SO: α4–β5 loop 0.9994844 residue_name_number cleaner0 2023-07-27T13:38:49Z DUMMY: Arg118 0.9993494 structure_element cleaner0 2023-07-27T13:23:20Z SO: α5 0.9995092 residue_name_number cleaner0 2023-07-27T13:38:54Z DUMMY: Asp92 0.99934167 structure_element cleaner0 2023-07-27T13:20:48Z SO: α4 0.9995054 residue_name_number cleaner0 2023-07-27T13:38:59Z DUMMY: Arg113 0.9993774 structure_element cleaner0 2023-07-27T13:23:20Z SO: α5 0.99893016 oligomeric_state cleaner0 2023-07-27T13:37:50Z DUMMY: dimeric protein PR: cleaner0 2023-07-27T12:47:45Z NsrR structure_element SO: cleaner0 2023-07-27T13:01:58Z RD 0.99952006 residue_name_number cleaner0 2023-07-27T13:39:13Z DUMMY: Asp100 0.9993284 structure_element cleaner0 2023-07-27T13:31:29Z SO: β5 0.9995098 residue_name_number cleaner0 2023-07-27T13:39:18Z DUMMY: Lys114 0.99942315 structure_element cleaner0 2023-07-27T13:23:20Z SO: α5 0.9988727 oligomeric_state cleaner0 2023-07-27T13:18:43Z DUMMY: monomer 0.9995421 residue_name_number cleaner0 2023-07-27T13:39:23Z DUMMY: Asn95 0.9994081 structure_element cleaner0 2023-07-27T13:20:48Z SO: α4 0.9987913 oligomeric_state cleaner0 2023-07-27T13:18:43Z DUMMY: monomer 0.9995415 residue_name_number cleaner0 2023-07-27T13:39:29Z DUMMY: Thr116 0.9993672 structure_element cleaner0 2023-07-27T13:23:20Z SO: α5 0.99874914 oligomeric_state cleaner0 2023-07-27T13:18:43Z DUMMY: monomer 0.9995491 residue_name_number cleaner0 2023-07-27T13:39:34Z DUMMY: Asp99 0.9990548 structure_element cleaner0 2023-07-27T13:38:28Z SO: α4–β5 loop 0.9995134 residue_name_number cleaner0 2023-07-27T13:19:10Z DUMMY: Arg121 0.9983334 protein cleaner0 2023-07-27T13:04:53Z PR: KdpE 0.99952364 residue_name_number cleaner0 2023-07-27T13:38:44Z DUMMY: Asp96 0.998955 structure_element cleaner0 2023-07-27T13:38:28Z SO: α4–β5 loop 0.99951935 residue_name_number cleaner0 2023-07-27T13:38:49Z DUMMY: Arg118 0.9993787 structure_element cleaner0 2023-07-27T13:23:20Z SO: α5 0.9982463 protein cleaner0 2023-07-27T13:04:53Z PR: KdpE 0.9995041 residue_name_number cleaner0 2023-07-27T13:38:39Z DUMMY: Arg111 0.9941443 bond_interaction cleaner0 2023-07-27T13:40:59Z MESH: salt bridge 0.9995147 residue_name_number cleaner0 2023-07-27T13:40:07Z DUMMY: Glu107 0.9993493 structure_element cleaner0 2023-07-27T13:23:20Z SO: α5 protein PR: cleaner0 2023-07-27T12:47:45Z NsrR structure_element SO: cleaner0 2023-07-27T13:01:58Z RD 0.9995579 residue_name_number cleaner0 2023-07-27T13:36:40Z DUMMY: Val110 0.9959351 experimental_method cleaner0 2023-07-27T14:41:12Z MESH: alignment 0.99778134 residue_name cleaner0 2023-07-27T13:40:32Z SO: arginine 0.999509 residue_name_number cleaner0 2023-07-27T13:38:39Z DUMMY: Arg111 0.99797183 protein cleaner0 2023-07-27T13:04:53Z PR: KdpE 0.9978527 residue_name cleaner0 2023-07-27T13:40:32Z SO: arginine 0.99764544 residue_name cleaner0 2023-07-27T13:27:21Z SO: lysine 0.99949944 residue_name_number cleaner0 2023-07-27T13:39:18Z DUMMY: Lys114 0.99928856 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR 0.9987174 protein_type cleaner0 2023-07-27T13:02:17Z MESH: RRs 0.95484114 experimental_method cleaner0 2023-07-27T14:41:18Z MESH: alignment 0.9974968 residue_name cleaner0 2023-07-27T13:40:32Z SO: arginine 0.99949026 residue_name_number cleaner0 2023-07-27T13:38:39Z DUMMY: Arg111 0.9955577 protein cleaner0 2023-07-27T13:04:53Z PR: KdpE 0.9974166 residue_name cleaner0 2023-07-27T13:40:38Z SO: glutamate 0.9995074 residue_name_number cleaner0 2023-07-27T13:40:08Z DUMMY: Glu107 0.99625236 protein cleaner0 2023-07-27T13:04:53Z PR: KdpE 0.997759 residue_name cleaner0 2023-07-27T13:40:33Z SO: arginine 0.99775827 residue_name cleaner0 2023-07-27T13:27:21Z SO: lysine 0.99813676 residue_name cleaner0 2023-07-27T13:40:39Z SO: glutamate 0.99848545 site cleaner0 2023-07-27T14:33:53Z SO: hydrophobic patch 0.9991761 protein cleaner0 2023-07-27T13:28:32Z PR: PhoB 0.99839574 species cleaner0 2023-07-27T13:21:53Z MESH: E. coli 0.999171 protein cleaner0 2023-07-27T13:04:37Z PR: PhoP 0.99854344 species cleaner0 2023-07-27T14:27:14Z MESH: B. subtilis 0.99686015 experimental_method cleaner0 2023-07-27T14:41:21Z MESH: mutating 0.99954164 residue_name_number cleaner0 2023-07-27T13:39:13Z DUMMY: Asp100 0.9995635 residue_name_number cleaner0 2023-07-27T13:36:40Z DUMMY: Val110 0.99952424 residue_name_number cleaner0 2023-07-27T13:39:18Z DUMMY: Lys114 0.99932265 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR 0.99883074 oligomeric_state cleaner0 2023-07-27T14:32:29Z DUMMY: monomeric 0.99803215 protein_type cleaner0 2023-07-27T12:47:38Z MESH: response regulator protein_state DUMMY: cleaner0 2023-07-27T14:42:08Z lost the ability to dimerize chemical CHEBI: cleaner0 2023-07-27T12:49:58Z DNA RESULTS title_2 35450 Overall Structure of C-terminal DNA-binding effector domain of NsrR 0.9876565 evidence cleaner0 2023-07-27T14:55:28Z DUMMY: Structure 0.9935832 structure_element cleaner0 2023-07-27T12:49:02Z SO: DNA-binding effector domain 0.99934393 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR RESULTS paragraph 35518 The structure of NsrR-ED from S. agalactiae was determined using experimental phases from a single-wavelength anomalous dispersion dataset from the rectangular plate-shaped crystal derivatized with platinum at a resolution of 1.6 Å in space group P21212. The Rwork and Rfree values after refinement were 0.18 and 0.22, respectively. Ramachandran validation was done using MolProbity. Almost all residues (99.48%, 193 amino acids) were in the preferred or allowed regions, while 0.52% (1 amino acid) were localized in the disallowed region. The latter is Glu128 (last residue of the linker region) of chain B that is involved in crystal contacts and, therefore, likely adopts an unfavorable conformation. The structure contained a few ethylene glycol molecules introduced by the cryo-protecting procedure. The data collection and refinement statistics are listed in Table 1. 0.99821424 evidence cleaner0 2023-07-27T14:55:31Z DUMMY: structure protein PR: cleaner0 2023-07-27T12:47:45Z NsrR structure_element SO: cleaner0 2023-07-27T13:02:09Z ED 0.9984174 species cleaner0 2023-07-27T12:56:39Z MESH: S. agalactiae 0.9618526 experimental_method cleaner0 2023-07-27T14:42:34Z MESH: single-wavelength anomalous dispersion dataset 0.7794421 chemical cleaner0 2023-07-27T13:42:13Z CHEBI: platinum 0.9981863 evidence cleaner0 2023-07-27T14:55:34Z DUMMY: Rwork 0.9977349 evidence cleaner0 2023-07-27T14:55:37Z DUMMY: Rfree evidence DUMMY: cleaner0 2023-07-27T13:41:43Z Ramachandran validation 0.9995865 residue_name_number cleaner0 2023-07-27T13:42:01Z DUMMY: Glu128 0.99935395 structure_element cleaner0 2023-07-27T13:52:42Z SO: linker region 0.9993657 structure_element cleaner0 2023-07-27T13:43:05Z SO: chain B 0.9984049 evidence cleaner0 2023-07-27T14:55:41Z DUMMY: structure 0.9827248 chemical cleaner0 2023-07-27T13:42:16Z CHEBI: ethylene glycol RESULTS paragraph 36393 The C-terminal effector DNA-binding domain of NsrR is about 13 kDa in size and consists of residues 129–243 (including 21 amino acid residues of the expression tag). Monomer A contains residue 129–224 and monomer B contain residues 128–225. For Asp147 of chain A and Glu174 of chain B, poor electron density was observed for the side chains and, thus, these side chains were removed during refinement. The asymmetric unit contains two copies of NsrR-ED related by two-fold rotational symmetry. An overlay revealed that both monomers display high similarity in their overall structure with an rmsd of 0.5 Å over 95 Cα atoms. We therefore describe for the overall structure only monomer A. 0.9993509 structure_element cleaner0 2023-07-27T13:42:27Z SO: effector DNA-binding domain 0.99940264 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR 0.9978315 residue_range cleaner0 2023-07-27T13:42:37Z DUMMY: 129–243 0.99845517 oligomeric_state cleaner0 2023-07-27T13:18:43Z DUMMY: Monomer 0.99803346 structure_element cleaner0 2023-07-27T13:42:49Z SO: A 0.99786425 residue_range cleaner0 2023-07-27T13:42:44Z DUMMY: 129–224 0.9984761 oligomeric_state cleaner0 2023-07-27T13:18:43Z DUMMY: monomer 0.9977214 structure_element cleaner0 2023-07-27T13:42:53Z SO: B 0.9979051 residue_range cleaner0 2023-07-27T13:42:47Z DUMMY: 128–225 0.99954945 residue_name_number cleaner0 2023-07-27T14:07:39Z DUMMY: Asp147 0.99925065 structure_element cleaner0 2023-07-27T13:42:59Z SO: chain A 0.99954873 residue_name_number cleaner0 2023-07-27T14:08:04Z DUMMY: Glu174 0.99925137 structure_element cleaner0 2023-07-27T13:43:04Z SO: chain B 0.9981958 evidence cleaner0 2023-07-27T14:55:44Z DUMMY: electron density protein PR: cleaner0 2023-07-27T12:47:45Z NsrR structure_element SO: cleaner0 2023-07-27T13:02:09Z ED 0.99870884 experimental_method cleaner0 2023-07-27T14:42:54Z MESH: overlay 0.9979359 oligomeric_state cleaner0 2023-07-27T13:18:37Z DUMMY: monomers 0.9912305 evidence cleaner0 2023-07-27T14:55:49Z DUMMY: structure 0.9986547 evidence cleaner0 2023-07-27T13:18:49Z DUMMY: rmsd 0.9728925 evidence cleaner0 2023-07-27T14:55:52Z DUMMY: structure 0.9981357 oligomeric_state cleaner0 2023-07-27T13:18:43Z DUMMY: monomer 0.9901218 structure_element cleaner0 2023-07-27T13:43:14Z SO: A RESULTS paragraph 37089 The ED domain of NsrR consists of six β-strands and three α-helices in a β6-β7-β8-β9-α6-α7-α8-β10-β11 topology (the secondary structure elements are counted in continuation of those of the RD). The effector domain starts with a 4-stranded antiparallel β-sheet, followed by three α-helices and eventually ends in a C-terminal β-hairpin (Fig 6). The two β-sheets sandwich the three α-helices. 0.99951637 structure_element cleaner0 2023-07-27T13:02:09Z SO: ED 0.9992681 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR 0.9784997 structure_element cleaner0 2023-07-27T13:44:02Z SO: β-strands 0.99849993 structure_element cleaner0 2023-07-27T13:43:54Z SO: α-helices structure_element SO: cleaner0 2023-07-27T13:43:36Z β6-β7-β8-β9-α6-α7-α8-β10-β11 0.9995215 structure_element cleaner0 2023-07-27T13:01:58Z SO: RD 0.9992093 structure_element cleaner0 2023-07-27T13:02:03Z SO: effector domain 0.99837077 structure_element cleaner0 2023-07-27T13:44:07Z SO: 4-stranded antiparallel β-sheet 0.9986458 structure_element cleaner0 2023-07-27T13:43:54Z SO: α-helices 0.99908113 structure_element cleaner0 2023-07-27T13:43:43Z SO: β-hairpin 0.998061 structure_element cleaner0 2023-07-27T13:43:47Z SO: β-sheets 0.99907786 structure_element cleaner0 2023-07-27T13:43:53Z SO: α-helices pone.0149903.g006.jpg pone.0149903.g006 FIG fig_title_caption 37534 Structure of the C-terminal effector domain of NsrR. 0.99674857 evidence cleaner0 2023-07-27T14:55:57Z DUMMY: Structure 0.9992944 structure_element cleaner0 2023-07-27T13:02:03Z SO: effector domain 0.99928445 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR pone.0149903.g006.jpg pone.0149903.g006 FIG fig_caption 37587 Cartoon representation of the C-terminal effector domain of NsrR (green; recognition helix in cyan). The structural areas with the highest variations compared to the effector domains of DrrB (pink, 1P2F), MtrA (grey, 2GWR), and PhoB (blue, 1GXQ) are marked. The transactivation loop of MtrA is missing in the structure, therefore, the two termini are connected by a dashed line. 0.99799705 structure_element cleaner0 2023-07-27T13:02:03Z SO: effector domain 0.99933964 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR 0.999251 structure_element cleaner0 2023-07-27T13:06:32Z SO: recognition helix 0.9877807 structure_element cleaner0 2023-07-27T14:49:18Z SO: effector domains 0.9993525 protein cleaner0 2023-07-27T13:04:43Z PR: DrrB 0.99932384 protein cleaner0 2023-07-27T13:04:26Z PR: MtrA 0.9993337 protein cleaner0 2023-07-27T13:28:32Z PR: PhoB 0.99938977 structure_element cleaner0 2023-07-27T13:46:56Z SO: transactivation loop 0.99929297 protein cleaner0 2023-07-27T13:04:26Z PR: MtrA protein_state DUMMY: cleaner0 2023-07-27T14:32:10Z missing 0.99806935 evidence cleaner0 2023-07-27T14:56:08Z DUMMY: structure RESULTS paragraph 37966 The characteristic feature of the OmpR/PhoB subfamily of RRs is a winged helix-turn-helix (wHTH) fold that is adopted by the α7-loop-α8 segment in full-length and single effector domain structures of RRs of this subfamily. The structure of NsrR-ED also contains such a wHTH motif built up by helices α7 and α8 (Fig 6). The second helix of the wHTH motif is important for DNA-binding and, therefore, is termed “recognition helix” (shown in cyan in Fig 6). Furthermore, a helix within the HTH motif, named “positioning helix”, is important for proper orientation and positioning of the loop between these two helices and is referred to as “transactivation loop” (also called α-loop; Fig 6). In the structure of NsrR-ED, helix α8 is identified as the recognition helix, α7 as the positioning helix, and the loop region between helices α7-α8 as transactivation loop as observed in other RRs (Fig 6). The 16-residue long, solvent-exposed recognition helix α8 of NsrR-ED contains four positively charged residues that can potentially interact with DNA. These are Arg198, Arg200, Lys201, and Lys202. When comparing the sequence of NsrR with PhoB, KdpE, and MtrA, the alignment (Fig 3, colored in blue) emphasizes the variations at these positions, except for Arg200, which is conserved throughout the lantibiotic resistance RRs. Additionally, Lys202 is also highly conserved throughout the family of RRs except PhoB, clearly reflecting differences in the sequences of DNA to be bound. protein_type MESH: cleaner0 2023-07-27T13:03:42Z OmpR/PhoB subfamily 0.99827945 protein_type cleaner0 2023-07-27T13:02:17Z MESH: RRs 0.9990485 structure_element cleaner0 2023-07-27T13:04:09Z SO: winged helix-turn-helix 0.99674976 structure_element cleaner0 2023-07-27T13:04:14Z SO: wHTH 0.9984329 structure_element cleaner0 2023-07-27T13:46:34Z SO: α7-loop-α8 segment 0.999053 protein_state cleaner0 2023-07-27T12:50:19Z DUMMY: full-length structure_element SO: cleaner0 2023-07-27T13:02:03Z effector domain 0.99596786 evidence cleaner0 2023-07-27T14:56:16Z DUMMY: structures 0.99909353 protein_type cleaner0 2023-07-27T13:02:17Z MESH: RRs 0.9975446 evidence cleaner0 2023-07-27T14:56:12Z DUMMY: structure protein PR: cleaner0 2023-07-27T12:47:45Z NsrR structure_element SO: cleaner0 2023-07-27T13:02:09Z ED structure_element SO: cleaner0 2023-07-27T13:04:14Z wHTH 0.98942417 structure_element cleaner0 2023-07-27T14:49:22Z SO: helices 0.9994035 structure_element cleaner0 2023-07-27T13:45:32Z SO: α7 0.99931467 structure_element cleaner0 2023-07-27T13:47:07Z SO: α8 structure_element SO: cleaner0 2023-07-27T13:46:02Z helix structure_element SO: cleaner0 2023-07-27T13:04:14Z wHTH 0.8894096 chemical cleaner0 2023-07-27T12:49:58Z CHEBI: DNA 0.9991862 structure_element cleaner0 2023-07-27T13:06:32Z SO: recognition helix 0.92855954 structure_element cleaner0 2023-07-27T13:24:14Z SO: helix structure_element SO: cleaner0 2023-07-27T13:46:19Z HTH 0.9990045 structure_element cleaner0 2023-07-27T13:46:49Z SO: positioning helix 0.99816555 structure_element cleaner0 2023-07-27T14:49:26Z SO: loop 0.9992594 structure_element cleaner0 2023-07-27T13:46:55Z SO: transactivation loop 0.9993288 structure_element cleaner0 2023-07-27T14:49:30Z SO: α-loop 0.99809307 evidence cleaner0 2023-07-27T14:56:19Z DUMMY: structure protein PR: cleaner0 2023-07-27T12:47:45Z NsrR structure_element SO: cleaner0 2023-07-27T13:02:09Z ED 0.99365133 structure_element cleaner0 2023-07-27T13:24:14Z SO: helix 0.99941874 structure_element cleaner0 2023-07-27T13:47:06Z SO: α8 0.99918497 structure_element cleaner0 2023-07-27T13:06:32Z SO: recognition helix 0.9993969 structure_element cleaner0 2023-07-27T13:45:33Z SO: α7 0.99906194 structure_element cleaner0 2023-07-27T13:46:50Z SO: positioning helix 0.84965134 structure_element cleaner0 2023-07-27T14:49:36Z SO: loop region 0.9968534 structure_element cleaner0 2023-07-27T13:47:42Z SO: α7-α8 0.9992547 structure_element cleaner0 2023-07-27T13:46:56Z SO: transactivation loop 0.9991375 protein_type cleaner0 2023-07-27T13:02:17Z MESH: RRs 0.95358604 protein_state cleaner0 2023-07-27T14:31:06Z DUMMY: solvent-exposed 0.99926454 structure_element cleaner0 2023-07-27T13:06:32Z SO: recognition helix 0.9992513 structure_element cleaner0 2023-07-27T13:47:07Z SO: α8 0.58672327 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR structure_element SO: cleaner0 2023-07-27T13:02:09Z ED 0.97925687 chemical cleaner0 2023-07-27T12:49:58Z CHEBI: DNA 0.99947995 residue_name_number cleaner0 2023-07-27T13:48:03Z DUMMY: Arg198 0.999479 residue_name_number cleaner0 2023-07-27T13:48:08Z DUMMY: Arg200 0.99947435 residue_name_number cleaner0 2023-07-27T13:48:12Z DUMMY: Lys201 0.9994831 residue_name_number cleaner0 2023-07-27T13:48:17Z DUMMY: Lys202 0.99930596 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR 0.9993229 protein cleaner0 2023-07-27T13:28:32Z PR: PhoB 0.99927527 protein cleaner0 2023-07-27T13:04:53Z PR: KdpE 0.9993191 protein cleaner0 2023-07-27T13:04:26Z PR: MtrA 0.9937802 experimental_method cleaner0 2023-07-27T14:42:59Z MESH: alignment 0.9994955 residue_name_number cleaner0 2023-07-27T13:48:09Z DUMMY: Arg200 0.9980679 protein_state cleaner0 2023-07-27T13:48:38Z DUMMY: conserved 0.99790066 protein_type cleaner0 2023-07-27T13:48:33Z MESH: lantibiotic resistance RRs 0.9994874 residue_name_number cleaner0 2023-07-27T13:48:18Z DUMMY: Lys202 0.99876195 protein_state cleaner0 2023-07-27T13:48:41Z DUMMY: highly conserved 0.99916935 protein_type cleaner0 2023-07-27T13:02:17Z MESH: RRs 0.9992446 protein cleaner0 2023-07-27T13:28:32Z PR: PhoB 0.96336824 chemical cleaner0 2023-07-27T12:49:58Z CHEBI: DNA RESULTS title_2 39484 Comparison with structures of other effector domains 0.9963588 evidence cleaner0 2023-07-27T14:56:24Z DUMMY: structures 0.7678622 structure_element cleaner0 2023-07-27T14:49:40Z SO: effector domains RESULTS paragraph 39537 We performed a DALI search to identify structurally related proteins to NsrR-ED. Here the structure of the effector domain of PhoB from E. coli (PDB code: 1GXQ) (Z-score of 13.7) is structurally the most closely related. Similar to the PhoB effector domain, a 9-residues long loop (amino acid 182–189) is also present in the structure of NsrR-ED that connects helices α7 and α8. The rmsd between the three helices of the effector domain (including the two helices forming the wHTH motif) of PhoB and NsrR-ED is 1.6 Å over 47 Cα atoms, clearly indicating that NsrR belongs to the OmpR/PhoB family of RRs. 0.9987474 experimental_method cleaner0 2023-07-27T14:43:29Z MESH: DALI search protein PR: cleaner0 2023-07-27T12:47:45Z NsrR structure_element SO: cleaner0 2023-07-27T13:02:09Z ED 0.99845695 evidence cleaner0 2023-07-27T14:56:27Z DUMMY: structure 0.9990599 structure_element cleaner0 2023-07-27T13:02:03Z SO: effector domain 0.99923027 protein cleaner0 2023-07-27T13:28:32Z PR: PhoB 0.99851304 species cleaner0 2023-07-27T13:21:53Z MESH: E. coli 0.99834114 evidence cleaner0 2023-07-27T13:34:58Z DUMMY: Z-score 0.9986601 protein cleaner0 2023-07-27T13:28:32Z PR: PhoB 0.9989355 structure_element cleaner0 2023-07-27T13:02:03Z SO: effector domain 0.96873546 structure_element cleaner0 2023-07-27T14:49:49Z SO: loop 0.9973235 residue_range cleaner0 2023-07-27T13:49:51Z DUMMY: 182–189 0.99845123 evidence cleaner0 2023-07-27T14:56:30Z DUMMY: structure protein PR: cleaner0 2023-07-27T12:47:45Z NsrR structure_element SO: cleaner0 2023-07-27T13:02:09Z ED 0.9939365 structure_element cleaner0 2023-07-27T14:49:58Z SO: helices 0.99922574 structure_element cleaner0 2023-07-27T13:45:33Z SO: α7 0.9988863 structure_element cleaner0 2023-07-27T13:47:07Z SO: α8 0.99877197 evidence cleaner0 2023-07-27T13:18:49Z DUMMY: rmsd 0.99845666 structure_element cleaner0 2023-07-27T13:02:03Z SO: effector domain 0.6872202 structure_element cleaner0 2023-07-27T14:49:54Z SO: helices structure_element SO: cleaner0 2023-07-27T13:04:14Z wHTH 0.99910176 protein cleaner0 2023-07-27T13:28:32Z PR: PhoB protein PR: cleaner0 2023-07-27T12:47:45Z NsrR structure_element SO: cleaner0 2023-07-27T13:02:09Z ED 0.99917275 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR protein_type MESH: cleaner0 2023-07-27T13:49:46Z OmpR/PhoB family of RRs RESULTS paragraph 40153 Therefore, we superimposed the Cα traces of the effector domain of NsrR (NsrR-ED) with other previously determined effector domains from the OmpR/PhoB family such as DrrB, MtrA and of only the effector domain structure of PhoB from E. coli. Overall, the structures are very similar with rmsd’s ranging from 1.7 to 2.6 Å (Table 2). The highest variations (Fig 6) are visible in in the loop regions α7-α8, which corresponds to the transactivation loop. Interestingly, this region also shows low sequence conservation (Fig 3). In many RRs this transactivation loop along with the recognition helix α8, form inter-domain contacts in the inactive state and are only exposed upon activation of the RRs via a conformational change where the N- and C-terminal domains move away from each other. 0.9987494 experimental_method cleaner0 2023-07-27T14:43:43Z MESH: superimposed 0.9971052 structure_element cleaner0 2023-07-27T13:02:03Z SO: effector domain 0.99924135 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR protein PR: cleaner0 2023-07-27T12:47:45Z NsrR structure_element SO: cleaner0 2023-07-27T13:02:09Z ED 0.9866476 structure_element cleaner0 2023-07-27T14:50:01Z SO: effector domains protein_type MESH: cleaner0 2023-07-27T13:50:19Z OmpR/PhoB family 0.99935406 protein cleaner0 2023-07-27T13:04:43Z PR: DrrB 0.9993337 protein cleaner0 2023-07-27T13:04:26Z PR: MtrA 0.9747043 structure_element cleaner0 2023-07-27T13:02:03Z SO: effector domain 0.99701023 evidence cleaner0 2023-07-27T14:56:48Z DUMMY: structure 0.9992217 protein cleaner0 2023-07-27T13:28:32Z PR: PhoB 0.9985159 species cleaner0 2023-07-27T13:21:53Z MESH: E. coli 0.9981047 evidence cleaner0 2023-07-27T14:56:50Z DUMMY: structures 0.9984321 evidence cleaner0 2023-07-27T13:18:49Z DUMMY: rmsd 0.9979315 structure_element cleaner0 2023-07-27T14:50:08Z SO: loop 0.9983066 structure_element cleaner0 2023-07-27T13:50:38Z SO: α7-α8 0.99934065 structure_element cleaner0 2023-07-27T13:46:56Z SO: transactivation loop 0.9992601 protein_type cleaner0 2023-07-27T13:02:17Z MESH: RRs 0.9994088 structure_element cleaner0 2023-07-27T13:46:56Z SO: transactivation loop 0.9993139 structure_element cleaner0 2023-07-27T13:06:32Z SO: recognition helix 0.99946314 structure_element cleaner0 2023-07-27T13:47:07Z SO: α8 0.9992349 protein_state cleaner0 2023-07-27T12:50:31Z DUMMY: inactive 0.9992236 protein_type cleaner0 2023-07-27T13:02:17Z MESH: RRs RESULTS title_2 40956 Linker region 0.99827135 structure_element cleaner0 2023-07-27T13:52:42Z SO: Linker region RESULTS paragraph 40970 The linkers that connect the RDs and EDs in response regulators are highly variable with respect to both length and sequence. The exact boundaries of these linkers are difficult to predict from sequence alignments in the absence of structural information of the distinct RR. Linker lengths in OmpR/PhoB proteins of unknown structure have been estimated by comparing the number of residues between conserved landmark residues in the regulatory and effector domains to those from structurally characterized family members. Such analysis has indicated that linker lengths vary from 5 to 20 residues. Similar to the OmpR/PhoB family, the lantibiotic resistance-associated family of response regulators also displays diverse linker regions, which are recognized in sequence alignments by the introduction of gaps (Fig 3). Interestingly, two arginine residues (Arg120 and Arg121 in NsrR; Fig 3, shown in purple) at the end of the RDs seem to be strictly conserved throughout the family of response regulators in both the OmpR/PhoB and lantibiotic resistance-associated RRs, indicating a conserved similarity. As seen in the structures of MtrA and KdpE, this arginine residue residing at the end of α5 participates in the active state dimer interface of the RD through a salt bridge interaction with an aspartate residue. This aspartate residue is identified in NsrR as Asp99 (see above). Arginine 121 of NsrR points towards this Asp99 residue however, the distance for a salt bridge interaction is too large. 0.99914396 structure_element cleaner0 2023-07-27T14:50:18Z SO: linkers 0.9995012 structure_element cleaner0 2023-07-27T13:34:52Z SO: RDs 0.99951756 structure_element cleaner0 2023-07-27T14:50:23Z SO: EDs 0.998411 protein_type cleaner0 2023-07-27T13:03:37Z MESH: response regulators 0.99304634 protein_state cleaner0 2023-07-27T14:31:40Z DUMMY: highly variable 0.9991146 structure_element cleaner0 2023-07-27T14:50:26Z SO: linkers 0.99842864 experimental_method cleaner0 2023-07-27T14:43:57Z MESH: sequence alignments protein_state DUMMY: cleaner0 2023-07-27T12:59:04Z absence of 0.9994678 protein_type cleaner0 2023-07-27T12:58:46Z MESH: RR 0.9903804 structure_element cleaner0 2023-07-27T14:50:30Z SO: Linker 0.9952068 protein_type cleaner0 2023-07-27T13:50:54Z MESH: OmpR/PhoB proteins 0.99726564 structure_element cleaner0 2023-07-27T14:50:37Z SO: regulatory and effector domains 0.9916016 protein_type cleaner0 2023-07-27T13:51:15Z MESH: OmpR/PhoB family 0.9810604 protein_type cleaner0 2023-07-27T13:51:07Z MESH: lantibiotic resistance-associated family of response regulators 0.9988881 structure_element cleaner0 2023-07-27T14:50:45Z SO: linker regions 0.9986211 experimental_method cleaner0 2023-07-27T14:43:59Z MESH: sequence alignments 0.99692875 residue_name cleaner0 2023-07-27T13:40:33Z SO: arginine 0.9995346 residue_name_number cleaner0 2023-07-27T13:19:05Z DUMMY: Arg120 0.99953735 residue_name_number cleaner0 2023-07-27T13:19:10Z DUMMY: Arg121 0.99940956 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR 0.99954176 structure_element cleaner0 2023-07-27T13:34:52Z SO: RDs 0.9988153 protein_state cleaner0 2023-07-27T14:31:45Z DUMMY: strictly conserved 0.9983387 protein_type cleaner0 2023-07-27T13:03:37Z MESH: response regulators protein_type MESH: cleaner0 2023-07-27T13:51:41Z OmpR/PhoB and lantibiotic resistance-associated RRs 0.98540246 protein_state cleaner0 2023-07-27T14:31:49Z DUMMY: conserved 0.99819034 evidence cleaner0 2023-07-27T14:56:55Z DUMMY: structures 0.99942976 protein cleaner0 2023-07-27T13:04:26Z PR: MtrA 0.99939 protein cleaner0 2023-07-27T13:04:53Z PR: KdpE 0.9974917 residue_name cleaner0 2023-07-27T13:40:33Z SO: arginine 0.9993438 structure_element cleaner0 2023-07-27T13:23:20Z SO: α5 0.9989869 protein_state cleaner0 2023-07-27T12:50:25Z DUMMY: active 0.9989382 site cleaner0 2023-07-27T13:52:09Z SO: dimer interface 0.9995241 structure_element cleaner0 2023-07-27T13:01:58Z SO: RD 0.99504364 bond_interaction cleaner0 2023-07-27T13:41:00Z MESH: salt bridge 0.99703443 residue_name cleaner0 2023-07-27T13:26:45Z SO: aspartate 0.9972184 residue_name cleaner0 2023-07-27T13:26:45Z SO: aspartate 0.99941874 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR 0.999471 residue_name_number cleaner0 2023-07-27T13:39:35Z DUMMY: Asp99 0.9929842 residue_name_number cleaner0 2023-07-27T13:51:51Z DUMMY: Arginine 121 0.9994128 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR 0.99952173 residue_name_number cleaner0 2023-07-27T13:39:35Z DUMMY: Asp99 0.99290204 bond_interaction cleaner0 2023-07-27T13:41:00Z MESH: salt bridge RESULTS paragraph 42477 Although we aimed at crystallizing full-length NsrR, this endeavor failed due to proteolytic cleavage within the linker region during the time period of crystallization. Nonetheless, the structures of NsrR-RD and NsrR-ED together provide the required structural knowledge to predict the linker region that joins the receiver and effector domains. The linker region of NsrR consists of approximately nine residues (Fig 3), comprising 120RRSQQFIQQ128 (underlined residues are neither present in the structure of RD nor in ED of NsrR) and contains two positively charged amino acids. 0.99736965 experimental_method cleaner0 2023-07-27T14:44:04Z MESH: crystallizing 0.99897003 protein_state cleaner0 2023-07-27T12:50:19Z DUMMY: full-length 0.99939334 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR 0.9992441 structure_element cleaner0 2023-07-27T13:52:42Z SO: linker region 0.9977715 experimental_method cleaner0 2023-07-27T14:44:12Z MESH: crystallization 0.9983114 evidence cleaner0 2023-07-27T14:57:00Z DUMMY: structures protein PR: cleaner0 2023-07-27T12:47:45Z NsrR 0.50491655 structure_element cleaner0 2023-07-27T13:01:58Z SO: RD 0.49194387 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR 0.5739222 structure_element cleaner0 2023-07-27T13:02:09Z SO: ED 0.9992206 structure_element cleaner0 2023-07-27T13:52:41Z SO: linker region 0.9984927 structure_element cleaner0 2023-07-27T14:50:52Z SO: receiver and effector domains 0.9992281 structure_element cleaner0 2023-07-27T13:52:42Z SO: linker region 0.9993925 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR 0.91002107 structure_element cleaner0 2023-07-27T13:52:45Z SO: 120RRSQQFIQQ128 0.9981325 evidence cleaner0 2023-07-27T14:57:02Z DUMMY: structure 0.9994654 structure_element cleaner0 2023-07-27T13:01:58Z SO: RD 0.9994773 structure_element cleaner0 2023-07-27T13:02:09Z SO: ED 0.9993216 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR RESULTS title_2 43058 DNA-binding mode of NsrR using a full-length model 0.97402394 chemical cleaner0 2023-07-27T12:49:58Z CHEBI: DNA 0.9993411 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR 0.99909955 protein_state cleaner0 2023-07-27T12:50:19Z DUMMY: full-length RESULTS paragraph 43109 Since the structures of both domains of NsrR were determined, we used this structural information together with the available crystal structures of related proteins to create a model of the full-length NsrR in its active and inactive state. 0.9957717 evidence cleaner0 2023-07-27T14:57:08Z DUMMY: structures 0.9993992 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR 0.92059565 evidence cleaner0 2023-07-27T14:57:11Z DUMMY: structural information 0.9983585 evidence cleaner0 2023-07-27T14:57:14Z DUMMY: crystal structures 0.99907106 protein_state cleaner0 2023-07-27T12:50:19Z DUMMY: full-length 0.99939525 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR 0.99906856 protein_state cleaner0 2023-07-27T12:50:25Z DUMMY: active 0.99910116 protein_state cleaner0 2023-07-27T12:50:31Z DUMMY: inactive RESULTS paragraph 43350 To achieve this, we first carefully analyzed the outcome of the Dali search for each domain and identified structurally highly similar proteins (based on Z-scores and rmsd values) and choose the full-length structures previously reported. This resulted in a list of possible templates for modeling the full-length structure of NsrR (Table 2). In solution, RRs exist in equilibrium between the active and inactive state, which is shifted towards the active state upon phosphorylation of the ED. This results in oligomerization of the RR and a higher affinity towards DNA. 0.9985951 experimental_method cleaner0 2023-07-27T14:44:17Z MESH: Dali search 0.99855995 evidence cleaner0 2023-07-27T14:57:27Z DUMMY: Z-scores 0.9982128 evidence cleaner0 2023-07-27T13:18:49Z DUMMY: rmsd 0.9991496 protein_state cleaner0 2023-07-27T12:50:19Z DUMMY: full-length 0.99840325 evidence cleaner0 2023-07-27T14:57:17Z DUMMY: structures 0.9990954 protein_state cleaner0 2023-07-27T12:50:19Z DUMMY: full-length 0.99808913 evidence cleaner0 2023-07-27T14:57:32Z DUMMY: structure 0.9993893 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR 0.99833775 protein_type cleaner0 2023-07-27T13:02:17Z MESH: RRs 0.9991147 protein_state cleaner0 2023-07-27T12:50:25Z DUMMY: active 0.99910897 protein_state cleaner0 2023-07-27T12:50:31Z DUMMY: inactive 0.99906796 protein_state cleaner0 2023-07-27T12:50:25Z DUMMY: active 0.99414575 ptm cleaner0 2023-07-27T12:50:12Z MESH: phosphorylation 0.999181 structure_element cleaner0 2023-07-27T13:02:09Z SO: ED 0.9882895 protein_type cleaner0 2023-07-27T12:58:46Z MESH: RR 0.9954269 chemical cleaner0 2023-07-27T12:49:58Z CHEBI: DNA RESULTS paragraph 43921 Based on the above-mentioned criteria, the structure of MtrA from M. tuberculosis, crystallized in an inactive and non-phosphorylated state, seemed best suited for modeling purposes. Furthermore, the linker between the two domains of MtrA contains nine amino acids and is of similar length as the linker of NsrR. We aligned the NsrR-RD and -ED to the corresponding MtrA domains and evaluated the structure. This mimics the closed inactive conformation of NsrR (Fig 7A; the missing linker is represented as dotted line). 0.9976841 evidence cleaner0 2023-07-27T14:57:20Z DUMMY: structure 0.99903095 protein cleaner0 2023-07-27T13:04:26Z PR: MtrA 0.9985315 species cleaner0 2023-07-27T13:28:25Z MESH: M. tuberculosis 0.99837327 experimental_method cleaner0 2023-07-27T14:44:21Z MESH: crystallized 0.9992687 protein_state cleaner0 2023-07-27T12:50:31Z DUMMY: inactive 0.99901205 protein_state cleaner0 2023-07-27T14:31:56Z DUMMY: non-phosphorylated 0.9976125 structure_element cleaner0 2023-07-27T13:53:09Z SO: linker 0.9992268 protein cleaner0 2023-07-27T13:04:26Z PR: MtrA 0.9992353 structure_element cleaner0 2023-07-27T13:53:04Z SO: linker 0.9994473 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR 0.99869484 experimental_method cleaner0 2023-07-27T14:44:24Z MESH: aligned 0.99834764 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR 0.99888676 structure_element cleaner0 2023-07-27T13:01:58Z SO: RD 0.99809986 structure_element cleaner0 2023-07-27T13:02:09Z SO: ED 0.96828985 protein cleaner0 2023-07-27T13:04:26Z PR: MtrA 0.99627304 evidence cleaner0 2023-07-27T14:57:22Z DUMMY: structure 0.99923456 protein_state cleaner0 2023-07-27T13:05:25Z DUMMY: closed 0.9991221 protein_state cleaner0 2023-07-27T12:50:31Z DUMMY: inactive 0.9994073 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR 0.9783392 protein_state cleaner0 2023-07-27T14:32:09Z DUMMY: missing 0.9979387 structure_element cleaner0 2023-07-27T13:53:06Z SO: linker pone.0149903.g007.jpg pone.0149903.g007 FIG fig_title_caption 44441 Model of full-length NsrR in its inactive state and active state. 0.9991148 protein_state cleaner0 2023-07-27T12:50:19Z DUMMY: full-length 0.99936193 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR 0.9992078 protein_state cleaner0 2023-07-27T12:50:31Z DUMMY: inactive 0.9992238 protein_state cleaner0 2023-07-27T12:50:25Z DUMMY: active pone.0149903.g007.jpg pone.0149903.g007 FIG fig_caption 44507 The RD domain of NsrR is highlighted in yellow and the ED domain in green with the “recognition helix” colored in cyan. (a) Inactive state conformation: Both domains of NsrR were aligned to the structure of MtrA (not shown), which adopts a closed inactive conformation, to obtain a model of full-length NsrR. Phe101 and Asp187 stabilize this closed conformation. The missing linker is represented by a dotted line. (b) Active state conformation: A model of full-length NsrR in active conformation based on the alignment of both the domains of NsrR to the structure of DNA bound structure of KdpE (PDB code: 4KNY), adopting an active open conformation, where the other molecule of NsrR is shown in shades of blue with the recognition helix colored in green. 0.99949074 structure_element cleaner0 2023-07-27T13:01:58Z SO: RD 0.99939835 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR 0.99950325 structure_element cleaner0 2023-07-27T13:02:09Z SO: ED 0.9961858 structure_element cleaner0 2023-07-27T13:06:32Z SO: recognition helix 0.99931276 protein_state cleaner0 2023-07-27T12:50:31Z DUMMY: Inactive 0.9993789 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR 0.9916055 experimental_method cleaner0 2023-07-27T14:44:36Z MESH: aligned 0.9903324 evidence cleaner0 2023-07-27T14:57:37Z DUMMY: structure 0.99943084 protein cleaner0 2023-07-27T13:04:26Z PR: MtrA 0.99928015 protein_state cleaner0 2023-07-27T13:05:25Z DUMMY: closed 0.99893826 protein_state cleaner0 2023-07-27T12:50:31Z DUMMY: inactive 0.9991382 protein_state cleaner0 2023-07-27T12:50:19Z DUMMY: full-length 0.9993907 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR 0.999585 residue_name_number cleaner0 2023-07-27T13:29:21Z DUMMY: Phe101 0.99958366 residue_name_number cleaner0 2023-07-27T14:08:18Z DUMMY: Asp187 0.9992489 protein_state cleaner0 2023-07-27T13:05:25Z DUMMY: closed protein_state DUMMY: cleaner0 2023-07-27T14:32:10Z missing 0.9982326 structure_element cleaner0 2023-07-27T14:51:00Z SO: linker 0.99928844 protein_state cleaner0 2023-07-27T12:50:25Z DUMMY: Active 0.9992003 protein_state cleaner0 2023-07-27T12:50:19Z DUMMY: full-length 0.9993948 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR 0.99929166 protein_state cleaner0 2023-07-27T12:50:25Z DUMMY: active 0.99759716 experimental_method cleaner0 2023-07-27T14:44:41Z MESH: alignment 0.9993851 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR 0.99301624 evidence cleaner0 2023-07-27T14:57:39Z DUMMY: structure 0.9988744 protein_state cleaner0 2023-07-27T13:53:38Z DUMMY: DNA bound 0.9658328 evidence cleaner0 2023-07-27T14:57:42Z DUMMY: structure 0.8901941 protein cleaner0 2023-07-27T13:04:53Z PR: KdpE protein_state DUMMY: cleaner0 2023-07-27T12:50:25Z active protein_state DUMMY: cleaner0 2023-07-27T13:05:19Z open 0.9993364 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR 0.9990963 structure_element cleaner0 2023-07-27T13:06:32Z SO: recognition helix RESULTS paragraph 45268 In MtrA, the two domains interact via the α4-β5-α5 interface of the receiver domain and the end of α7, α7-α8 loop and α8 of the effector domain. Both interfaces have been shown to form functionally important contact areas in the active state within members of the OmpR/PhoB subfamily. In our model of full-length NsrR, a similar orientation between the domains is observed, contributing to the inter-domain interactions. The inactive conformation of MtrA is supported by the orientation of the side chain of Tyr102, which points away from the active Asp56 residue, while the side chain of Tyr102 interacts with Asp190 of the RD of MtrA, thereby stabilizing its closed conformation. In the model of NsrR, similar amino acids are present, Phe101 (switch residue) and Asp188 (Fig 3, represented by orange boxes) forming a likewise similar network of interaction. 0.9994369 protein cleaner0 2023-07-27T13:04:26Z PR: MtrA 0.9988864 site cleaner0 2023-07-27T13:34:36Z SO: α4-β5-α5 interface 0.99934936 structure_element cleaner0 2023-07-27T12:48:56Z SO: receiver domain 0.9994467 structure_element cleaner0 2023-07-27T13:45:33Z SO: α7 0.99927455 structure_element cleaner0 2023-07-27T13:54:04Z SO: α7-α8 loop 0.9993687 structure_element cleaner0 2023-07-27T13:47:07Z SO: α8 0.9989896 structure_element cleaner0 2023-07-27T13:02:03Z SO: effector domain 0.99842715 site cleaner0 2023-07-27T14:34:06Z SO: interfaces 0.9991906 protein_state cleaner0 2023-07-27T12:50:25Z DUMMY: active protein_type MESH: cleaner0 2023-07-27T13:03:42Z OmpR/PhoB subfamily 0.9991438 protein_state cleaner0 2023-07-27T12:50:19Z DUMMY: full-length 0.99945253 protein cleaner0 2023-07-27T12:47:45Z PR: NsrR 0.99926037 protein_state cleaner0 2023-07-27T12:50:32Z DUMMY: inactive 0.9994436 protein cleaner0 2023-07-27T13:04:26Z PR: MtrA 0.9995383 residue_name_number cleaner0 2023-07-27T13:54:29Z DUMMY: Tyr102 0.99907994 protein_state cleaner0 2023-07-27T12:50:25Z DUMMY: active 0.9995233 residue_name_number cleaner0 2023-07-27T13:54:43Z DUMMY: Asp56 0.9995345 residue_name_number cleaner0 2023-07-27T13:54:30Z DUMMY: Tyr102 0.99953544 residue_name_number cleaner0 2023-07-27T13:54:37Z DUMMY: Asp190 0.9995652 structure_element cleaner0 2023-07-27T13:01:58Z SO: RD 0.999433 protein cleaner0 2023-07-27T13:04:26Z PR: MtrA 0.9992292 protein_state cleaner0 2023-07-27T13:05:25Z DUMMY: closed 0.9994388 protein cleaner0 2023-07-27T12:47:46Z PR: NsrR 0.99956363 residue_name_number cleaner0 2023-07-27T13:29:21Z DUMMY: Phe101 0.9895373 site cleaner0 2023-07-27T14:34:51Z SO: switch residue 0.9995402 residue_name_number cleaner0 2023-07-27T13:54:50Z DUMMY: Asp188 RESULTS paragraph 46155 Next, we were interested in the active conformation of the NsrR protein adopting an active “open” conformation in the dimeric state. We compared and aligned the NsrR-RD and ED on the dimeric structure of KdpE that was solved in the DNA-bound state (Fig 7B). 0.9992353 protein_state cleaner0 2023-07-27T12:50:25Z DUMMY: active 0.99932516 protein cleaner0 2023-07-27T12:47:46Z PR: NsrR 0.9992791 protein_state cleaner0 2023-07-27T12:50:25Z DUMMY: active 0.99926335 protein_state cleaner0 2023-07-27T13:05:19Z DUMMY: open 0.998841 oligomeric_state cleaner0 2023-07-27T13:37:50Z DUMMY: dimeric 0.9955654 experimental_method cleaner0 2023-07-27T14:44:48Z MESH: compared and aligned 0.998359 protein cleaner0 2023-07-27T12:47:46Z PR: NsrR 0.9993024 structure_element cleaner0 2023-07-27T13:01:58Z SO: RD 0.99938285 structure_element cleaner0 2023-07-27T13:02:09Z SO: ED 0.99880266 oligomeric_state cleaner0 2023-07-27T13:37:50Z DUMMY: dimeric 0.9975605 evidence cleaner0 2023-07-27T14:57:45Z DUMMY: structure 0.998285 protein cleaner0 2023-07-27T13:04:53Z PR: KdpE 0.9948185 experimental_method cleaner0 2023-07-27T14:44:52Z MESH: solved 0.9988813 protein_state cleaner0 2023-07-27T13:06:03Z DUMMY: DNA-bound RESULTS paragraph 46417 Also the linker region of KdpE is of similar length as of NsrR, which suggests that the distance in the DNA-bound state between the RD and ED of NsrR will be similar to that in the KdpE active dimer. We superimposed the ED of NsrR with the DNA-binding domain of KdpE resulting in a reasonably well-aligned structure (rmsd of 2.6Å over 86 Cα atoms; Table 2). As a result, a highly positive groove is created by the two ED domains of NsrR which likely represents the DNA binding site as observed in KdpE. A prediction of the putative promoter sequence that NsrR binds via the BPROM online server was performed (S3 Fig). A promoter region was identified upstream of the nsr operon. However, the regulation of the predicted promoter and the DNA binding by NsrR has to be confirmed. 0.99928784 structure_element cleaner0 2023-07-27T13:52:42Z SO: linker region 0.9978363 protein cleaner0 2023-07-27T13:04:53Z PR: KdpE 0.99921405 protein cleaner0 2023-07-27T12:47:46Z PR: NsrR 0.9989531 protein_state cleaner0 2023-07-27T13:06:03Z DUMMY: DNA-bound 0.99952734 structure_element cleaner0 2023-07-27T13:01:58Z SO: RD 0.9995184 structure_element cleaner0 2023-07-27T13:02:09Z SO: ED 0.9993623 protein cleaner0 2023-07-27T12:47:46Z PR: NsrR 0.9984419 protein cleaner0 2023-07-27T13:04:53Z PR: KdpE 0.9992557 protein_state cleaner0 2023-07-27T12:50:25Z DUMMY: active 0.9987226 oligomeric_state cleaner0 2023-07-27T13:38:00Z DUMMY: dimer 0.9987375 experimental_method cleaner0 2023-07-27T14:44:57Z MESH: superimposed 0.9994961 structure_element cleaner0 2023-07-27T13:02:09Z SO: ED 0.9992569 protein cleaner0 2023-07-27T12:47:46Z PR: NsrR 0.9989898 structure_element cleaner0 2023-07-27T13:55:17Z SO: DNA-binding domain 0.9977089 protein cleaner0 2023-07-27T13:04:53Z PR: KdpE 0.99800843 evidence cleaner0 2023-07-27T14:57:51Z DUMMY: structure 0.998494 evidence cleaner0 2023-07-27T13:18:49Z DUMMY: rmsd 0.9923591 site cleaner0 2023-07-27T14:34:55Z SO: highly positive groove 0.99947745 structure_element cleaner0 2023-07-27T13:02:09Z SO: ED 0.9993467 protein cleaner0 2023-07-27T12:47:46Z PR: NsrR 0.9987176 site cleaner0 2023-07-27T13:55:37Z SO: DNA binding site 0.9975803 protein cleaner0 2023-07-27T13:04:53Z PR: KdpE 0.99932516 protein cleaner0 2023-07-27T12:47:46Z PR: NsrR gene GENE: cleaner0 2023-07-27T13:02:41Z nsr chemical CHEBI: cleaner0 2023-07-27T12:49:59Z DNA 0.99938023 protein cleaner0 2023-07-27T12:47:46Z PR: NsrR RESULTS title_2 47199 Conclusion RESULTS paragraph 47210 In numerous pathogenic bacteria such as S. agalactiae, S. aureus, and C. difficile that apparently do not produce a lantibiotic, a gene cluster is present to provide resistance against lantibiotics such as nisin, nukacin ISK-1, lacticin 481 gallidermin, actagardine, or mersacidin. The regulation of the expression of these genes is mediated by two-component systems. The structure of the response regulator NsrR from S. agalactiae presented in this study is the first structural information available for the subgroup of lantibiotic resistance-associated RRs. 0.9978606 taxonomy_domain cleaner0 2023-07-27T12:51:05Z DUMMY: bacteria 0.9981645 species cleaner0 2023-07-27T12:56:39Z MESH: S. agalactiae 0.9976611 species cleaner0 2023-07-27T12:55:51Z MESH: S. aureus 0.9971164 species cleaner0 2023-07-27T13:24:38Z MESH: C. difficile 0.9989114 chemical cleaner0 2023-07-27T12:52:05Z CHEBI: lantibiotic 0.99796283 chemical cleaner0 2023-07-27T12:48:05Z CHEBI: lantibiotics 0.9993581 chemical cleaner0 2023-07-27T12:52:00Z CHEBI: nisin 0.9985107 chemical cleaner0 2023-07-27T14:28:19Z CHEBI: nukacin ISK-1 0.99897635 chemical cleaner0 2023-07-27T13:01:07Z CHEBI: lacticin 481 0.9987948 chemical cleaner0 2023-07-27T12:53:41Z CHEBI: gallidermin 0.9993537 chemical cleaner0 2023-07-27T14:28:23Z CHEBI: actagardine 0.9993405 chemical cleaner0 2023-07-27T14:28:26Z CHEBI: mersacidin 0.99779874 evidence cleaner0 2023-07-27T14:57:54Z DUMMY: structure 0.9980495 protein_type cleaner0 2023-07-27T12:47:38Z MESH: response regulator 0.99939275 protein cleaner0 2023-07-27T12:47:46Z PR: NsrR 0.9983414 species cleaner0 2023-07-27T12:56:39Z MESH: S. agalactiae protein_type MESH: cleaner0 2023-07-27T13:32:11Z lantibiotic resistance-associated RRs SUPPL title_1 47771 Supporting Information REF title 47794 References 95 105 surname:Cotter;given-names:PD surname:Ross;given-names:RP surname:Hill;given-names:C 10.1038/nrmicro2937 23268227 REF Nature Reviews Microbiology ref 11 2012 47805 Bacteriocins—a viable alternative to antibiotics? 385 392 surname:Bierbaum;given-names:G surname:Szekat;given-names:C surname:Josten;given-names:M surname:Heidrich;given-names:C surname:Kempter;given-names:C surname:Jung;given-names:G 8593044 REF Applied and environmental microbiology ref 62 1996 47857 Engineering of a novel thioether bridge and role of modified residues in the 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surname:Zaschke;given-names:J surname:Wagner;given-names:M surname:Abts;given-names:A surname:Fey;given-names:I 10.1002/mbo3.205 25176038 REF MicrobiologyOpen ref 3 2014 48246 The C‐terminus of nisin is important for the ABC transporter NisFEG to confer immunity in Lactococcus lactis 1772 1779 surname:Wiedemann;given-names:I surname:Breukink;given-names:E surname:Van Kraaij;given-names:C surname:Kuipers;given-names:OP surname:Bierbaum;given-names:G surname:De Kruijff;given-names:B 11038353 REF Journal of Biological Chemistry ref 276 2001 48357 Specific Binding of Nisin to the Peptidoglycan Precursor Lipid II Combines Pore Formation and Inhibition of Cell Wall Biosynthesis for Potent Antibiotic Activity 814 825 surname:Engelke;given-names:G surname:Gutowski-Eckel;given-names:Z surname:Kiesau;given-names:P surname:Siegers;given-names:K surname:Hammelmann;given-names:M surname:Entian;given-names:K 8161176 REF Applied and environmental microbiology ref 60 1994 48519 Regulation of nisin biosynthesis and immunity in Lactococcus lactis 6F3 281 291 surname:Kuipers;given-names:OP surname:Beerthuyzen;given-names:MM surname:Siezen;given-names:RJ surname:VOS;given-names:WM 7689965 REF European Journal of Biochemistry ref 216 1993 48591 Characterization of the nisin gene cluster nisABTCIPR of Lactococcus lactis 106 113 surname:Guder;given-names:A surname:Schmitter;given-names:T surname:Wiedemann;given-names:I surname:Sahl H-;given-names:G surname:Bierbaum;given-names:G 11772616 REF Applied and environmental microbiology ref 68 2002 48667 Role of the single regulator MrsR1 and the two-component system MrsR2/K2 in the regulation of mersacidin production and immunity 145 154 surname:Alkhatib;given-names:Z surname:Abts;given-names:A surname:Mavaro;given-names:A surname:Schmitt;given-names:L surname:Smits;given-names:SH 10.1016/j.jbiotec.2012.01.032 22329892 REF Journal of biotechnology ref 159 2012 48796 Lantibiotics: how do producers become self-protected? 633 684 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