PMC 20140719 pmc.key 4887326 CC BY no 0 0 10.1007/s13238-016-0264-7 4887326 27113583 264 403 6 the YfiBNR system c-di-GMP Vitamin B6 L-Trp peptidoglycan layer bioflim formation Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 416 surname:Xu;given-names:Min surname:Yang;given-names:Xuan surname:Yang;given-names:Xiu-An surname:Zhou;given-names:Lei surname:Liu;given-names:Tie-Zheng surname:Fan;given-names:Zusen surname:Jiang;given-names:Tao TITLE KEYWORDS front 7 2016 0 Structural insights into the regulatory mechanism of the Pseudomonas aeruginosa YfiBNR system species MESH: melaniev@ebi.ac.uk 2023-03-15T15:14:23Z Pseudomonas aeruginosa complex_assembly GO: melaniev@ebi.ac.uk 2023-03-15T11:33:15Z YfiBNR ABSTRACT abstract 94 YfiBNR is a recently identified bis-(3’-5’)-cyclic dimeric GMP (c-di-GMP) signaling system in opportunistic pathogens. It is a key regulator of biofilm formation, which is correlated with prolonged persistence of infection and antibiotic drug resistance. In response to cell stress, YfiB in the outer membrane can sequester the periplasmic protein YfiR, releasing its inhibition of YfiN on the inner membrane and thus provoking the diguanylate cyclase activity of YfiN to induce c-di-GMP production. However, the detailed regulatory mechanism remains elusive. Here, we report the crystal structures of YfiB alone and of an active mutant YfiBL43P complexed with YfiR with 2:2 stoichiometry. Structural analyses revealed that in contrast to the compact conformation of the dimeric YfiB alone, YfiBL43P adopts a stretched conformation allowing activated YfiB to penetrate the peptidoglycan (PG) layer and access YfiR. YfiBL43P shows a more compact PG-binding pocket and much higher PG binding affinity than wild-type YfiB, suggesting a tight correlation between PG binding and YfiB activation. In addition, our crystallographic analyses revealed that YfiR binds Vitamin B6 (VB6) or L-Trp at a YfiB-binding site and that both VB6 and L-Trp are able to reduce YfiBL43P-induced biofilm formation. Based on the structural and biochemical data, we propose an updated regulatory model of the YfiBNR system. complex_assembly GO: melaniev@ebi.ac.uk 2023-03-15T11:33:15Z YfiBNR chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T11:33:30Z bis-(3’-5’)-cyclic dimeric GMP chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T11:33:38Z c-di-GMP protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:49Z YfiB protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:00Z YfiR protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:11Z YfiN protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:11Z YfiN chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T12:55:59Z c-di-GMP evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:35:06Z crystal structures protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:49Z YfiB protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:35:14Z alone protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:35:21Z active protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:35:27Z mutant mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:35:43Z complexed with protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:00Z YfiR experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:52Z Structural analyses protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:36:03Z compact conformation oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:36:11Z dimeric protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:49Z YfiB protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:35:14Z alone mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:36:20Z stretched conformation protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:36:28Z activated protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:49Z YfiB chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T11:36:36Z peptidoglycan chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:37:01Z PG protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:00Z YfiR mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P site SO: melaniev@ebi.ac.uk 2023-03-15T11:36:51Z PG-binding pocket evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:36:57Z PG binding affinity protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:37:06Z wild-type protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:49Z YfiB protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:49Z YfiB experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T11:37:15Z crystallographic analyses protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:00Z YfiR chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T11:37:22Z Vitamin B6 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:37:05Z VB6 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:37:08Z L-Trp site SO: melaniev@ebi.ac.uk 2023-03-15T11:38:14Z YfiB-binding site chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:37:11Z VB6 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:37:14Z L-Trp mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T15:38:09Z structural and biochemical data complex_assembly GO: melaniev@ebi.ac.uk 2023-03-15T11:33:15Z YfiBNR INTRO title_1 1498 INTRODUCTION INTRO paragraph 1511 Bis-(3’-5’)-cyclic dimeric GMP (c-di-GMP) is a ubiquitous second messenger that bacteria use to facilitate behavioral adaptations to their ever-changing environment. An increase in c-di-GMP promotes biofilm formation, and a decrease results in biofilm degradation (Boehm et al.,; Duerig et al.,; Hickman et al.,; Jenal,; Romling et al.,). The c-di-GMP level is regulated by two reciprocal enzyme systems, namely, diguanylate cyclases (DGCs) that synthesize c-di-GMP and phosphodiesterases (PDEs) that hydrolyze c-di-GMP (Kulasakara et al.,; Ross et al.,; Ross et al.,). Many of these enzymes are multiple-domain proteins containing a variable N-terminal domain that commonly acts as a signal sensor or transduction module, followed by the relatively conserved GGDEF motif in DGCs or EAL/HD-GYP domains in PDEs (Hengge,; Navarro et al.,; Schirmer and Jenal,). Intriguingly, studies in diverse species have revealed that a single bacterium can have dozens of DGCs and PDEs (Hickman et al.,; Kirillina et al.,; Kulasakara et al.,; Tamayo et al.,). In Pseudomonas aeruginosa in particular, 42 genes containing putative DGCs and/or PDEs were identified (Kulasakara et al.,). The functional role of a number of downstream effectors of c-di-GMP has been characterized as affecting exopolysaccharide (EPS) production, transcription, motility, and surface attachment (Caly et al.,; Camilli and Bassler,; Ha and O’Toole,; Pesavento and Hengge,). However, due to the intricacy of c-di-GMP signaling networks and the diversity of experimental cues, the detailed mechanisms by which these signaling pathways specifically sense and integrate different inputs remain largely elusive. chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T11:33:30Z Bis-(3’-5’)-cyclic dimeric GMP chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T11:33:38Z c-di-GMP taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T12:56:28Z bacteria chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T12:55:59Z c-di-GMP chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T12:55:59Z c-di-GMP protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T12:56:36Z diguanylate cyclases protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T12:56:42Z DGCs chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T11:33:38Z c-di-GMP protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T12:56:50Z phosphodiesterases protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T12:56:57Z PDEs chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T12:55:59Z c-di-GMP structure_element SO: melaniev@ebi.ac.uk 2023-03-15T12:57:53Z N-terminal domain protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T12:57:05Z relatively conserved structure_element SO: melaniev@ebi.ac.uk 2023-03-15T12:57:11Z GGDEF motif protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T12:56:42Z DGCs structure_element SO: melaniev@ebi.ac.uk 2023-03-15T12:57:44Z EAL/HD-GYP domains protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T12:56:57Z PDEs taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T12:57:19Z bacterium protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T12:56:42Z DGCs protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T12:56:57Z PDEs species MESH: melaniev@ebi.ac.uk 2023-03-15T15:14:23Z Pseudomonas aeruginosa protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T12:56:42Z DGCs protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T12:56:57Z PDEs chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T11:33:38Z c-di-GMP chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T12:57:28Z exopolysaccharide chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T12:57:35Z EPS chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T11:33:38Z c-di-GMP INTRO paragraph 3186 Biofilm formation protects pathogenic bacteria from antibiotic treatment, and c-di-GMP-regulated biofilm formation has been extensively studied in P. aeruginosa (Evans,; Kirisits et al.,; Malone,; Reinhardt et al.,). In the lungs of cystic fibrosis (CF) patients, adherent biofilm formation and the appearance of small colony variant (SCV) morphologies of P. aeruginosa correlate with prolonged persistence of infection and poor lung function (Govan and Deretic,; Haussler et al.,; Haussler et al.,; Parsek and Singh,; Smith et al.,). Recently, Malone and coworkers identified the tripartite c-di-GMP signaling module system YfiBNR (also known as AwsXRO (Beaumont et al.,; Giddens et al.,) or Tbp (Ueda and Wood,)) by genetic screening for mutants that displayed SCV phenotypes in P. aeruginosa PAO1 (Malone et al.,; Malone et al.,). The YfiBNR system contains three protein members and modulates intracellular c-di-GMP levels in response to signals received in the periplasm (Malone et al.,). More recently, this system was also reported in other Gram-negative bacteria, such as Escherichia coli (Hufnagel et al.,; Raterman et al.,; Sanchez-Torres et al.,), Klebsiella pneumonia (Huertas et al.,) and Yersinia pestis (Ren et al.,). YfiN is an integral inner-membrane protein with two potential transmembrane helices, a periplasmic Per-Arnt-Sim (PAS) domain, and cytosolic domains containing a HAMP domain (mediate input-output signaling in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases) and a C-terminal GGDEF domain indicating a DGC’s function (Giardina et al.,; Malone et al.,). YfiN is repressed by specific interaction between its periplasmic PAS domain and the periplasmic protein YfiR (Malone et al.,). YfiB is an OmpA/Pal-like outer-membrane lipoprotein (Parsons et al.,) that can activate YfiN by sequestering YfiR (Malone et al.,) in an unknown manner. Whether YfiB directly recruits YfiR or recruits YfiR via a third partner is an open question. After the sequestration of YfiR by YfiB, the c-di-GMP produced by activated YfiN increases the biosynthesis of the Pel and Psl EPSs, resulting in the appearance of the SCV phenotype, which indicates enhanced biofilm formation (Malone et al.,). taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T12:56:28Z bacteria species MESH: melaniev@ebi.ac.uk 2023-03-15T15:14:23Z P. aeruginosa species MESH: melaniev@ebi.ac.uk 2023-03-15T15:14:23Z P. aeruginosa protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:03:59Z tripartite chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:24:01Z c-di-GMP complex_assembly GO: melaniev@ebi.ac.uk 2023-03-15T11:33:15Z YfiBNR complex_assembly GO: melaniev@ebi.ac.uk 2023-03-15T13:04:19Z AwsXRO complex_assembly GO: melaniev@ebi.ac.uk 2023-03-15T13:04:22Z Tbp experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T13:04:31Z genetic screening species MESH: melaniev@ebi.ac.uk 2023-03-15T15:14:23Z P. aeruginosa PAO1 complex_assembly GO: melaniev@ebi.ac.uk 2023-03-15T11:33:15Z YfiBNR chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T11:33:38Z c-di-GMP taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:35:04Z Gram-negative bacteria species MESH: melaniev@ebi.ac.uk 2023-03-15T15:14:23Z Escherichia coli species MESH: melaniev@ebi.ac.uk 2023-03-15T15:14:23Z Klebsiella pneumonia species MESH: melaniev@ebi.ac.uk 2023-03-15T15:14:23Z Yersinia pestis protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:11Z YfiN structure_element SO: melaniev@ebi.ac.uk 2023-03-15T13:06:10Z transmembrane helices structure_element SO: melaniev@ebi.ac.uk 2023-03-15T15:42:41Z Per-Arnt-Sim structure_element SO: melaniev@ebi.ac.uk 2023-03-15T13:06:27Z PAS structure_element SO: melaniev@ebi.ac.uk 2023-03-15T13:06:48Z HAMP domain protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T13:06:55Z histidine kinases protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T13:06:58Z adenylyl cyclases protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T13:07:00Z methyl-accepting chemotaxis proteins protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T13:07:03Z phosphatases structure_element SO: melaniev@ebi.ac.uk 2023-03-15T15:42:46Z GGDEF domain protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T13:08:50Z DGC protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:11Z YfiN protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:10:58Z repressed by structure_element SO: melaniev@ebi.ac.uk 2023-03-15T13:10:22Z PAS domain protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:00Z YfiR protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:49Z YfiB protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T13:10:50Z OmpA/Pal-like protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T13:10:56Z lipoprotein protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:11Z YfiN protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:00Z YfiR protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:49Z YfiB protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:00Z YfiR protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:00Z YfiR protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:00Z YfiR protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:49Z YfiB chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T11:33:38Z c-di-GMP protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:36:28Z activated protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:11Z YfiN chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T13:11:09Z Pel chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T13:11:16Z Psl chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T13:11:22Z EPSs INTRO paragraph 5437 It has been reported that the activation of YfiN may be induced by redox-driven misfolding of YfiR (Giardina et al.,; Malone et al.,; Malone et al.,). It is also proposed that the sequestration of YfiR by YfiB can be induced by certain YfiB-mediated cell wall stress, and mutagenesis studies revealed a number of activation residues of YfiB that were located in close proximity to the predicted first helix of the periplasmic domain (Malone et al.,). In addition, quorum sensing-related dephosphorylation of the PAS domain of YfiN may also be involved in the regulation (Ueda and Wood,; Xu et al.,). Recently, we solved the crystal structure of YfiR in both the non-oxidized and the oxidized states, revealing breakage/formation of one disulfide bond (Cys71-Cys110) and local conformational change around the other one (Cys145-Cys152), indicating that Cys145-Cys152 plays an important role in maintaining the correct folding of YfiR (Yang et al.,). protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:11Z YfiN protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:00Z YfiR protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:00Z YfiR protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:49Z YfiB protein PR: melaniev@ebi.ac.uk 2023-03-22T09:24:38Z YfiB experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T13:13:48Z mutagenesis studies structure_element SO: melaniev@ebi.ac.uk 2023-03-15T15:42:57Z activation residues protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:49Z YfiB protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:13:42Z predicted structure_element SO: melaniev@ebi.ac.uk 2023-03-15T15:43:21Z first helix structure_element SO: melaniev@ebi.ac.uk 2023-03-15T15:43:32Z periplasmic domain structure_element SO: melaniev@ebi.ac.uk 2023-03-15T15:43:41Z PAS domain protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:11Z YfiN evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T15:38:19Z crystal structure protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:00Z YfiR protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:13:56Z non-oxidized protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:11:03Z oxidized ptm MESH: melaniev@ebi.ac.uk 2023-03-15T13:14:32Z disulfide bond residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:12:24Z Cys71 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:12:31Z Cys110 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:12:12Z Cys145 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:12:18Z Cys152 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:12:12Z Cys145 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:12:18Z Cys152 protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:00Z YfiR INTRO paragraph 6386 In the present study, we solved the crystal structures of an N-terminal truncated form of YfiB (34–168) and YfiR in complex with an active mutant YfiBL43P. Most recently, Li and coworkers reported the crystal structures of YfiB (27–168) alone and YfiRC71S in complex with YfiB (59–168) (Li et al.,). Compared with the reported complex structure, YfiBL43P in our YfiB-YfiR complex structure has additional visible N-terminal residues 44–58 that are shown to play essential roles in YfiB activation and biofilm formation. Therefore, we are able to visualize the detailed allosteric arrangement of the N-terminal structure of YfiB and its important role in YfiB-YfiR interaction. In addition, we found that the YfiBL43P shows a much higher PG-binding affinity than wild-type YfiB, most likely due to its more compact PG-binding pocket. Moreover, we found that Vitamin B6 (VB6) or L-Trp can bind YfiR with an affinity in the ten millimolar range. Together with functional data, these results provide new mechanistic insights into how activated YfiB sequesters YfiR and releases the suppression of YfiN. These findings may facilitate the development and optimization of anti-biofilm drugs for the treatment of chronic infections. evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T15:38:28Z crystal structures protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:35:23Z truncated protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:49Z YfiB residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:38:46Z 34–168 protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:00Z YfiR protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:35:33Z in complex with protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:35:21Z active protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:35:27Z mutant mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T15:38:28Z crystal structures protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:49Z YfiB residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:38:52Z 27–168 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:35:14Z alone mutant MESH: melaniev@ebi.ac.uk 2023-03-15T15:39:23Z YfiRC71S protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:35:33Z in complex with protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:49Z YfiB residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:38:55Z 59–168 mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P complex_assembly GO: melaniev@ebi.ac.uk 2023-03-15T13:36:20Z YfiB-YfiR evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:37:31Z structure residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:38:57Z 44–58 protein PR: melaniev@ebi.ac.uk 2023-03-22T10:51:22Z YfiB protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:49Z YfiB complex_assembly GO: melaniev@ebi.ac.uk 2023-06-14T09:13:35Z YfiB-YfiR mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:46:46Z PG-binding affinity protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:37:06Z wild-type protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:49Z YfiB site SO: melaniev@ebi.ac.uk 2023-03-15T11:36:51Z PG-binding pocket chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T11:37:22Z Vitamin B6 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:37:20Z VB6 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:37:23Z L-Trp protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:00Z YfiR evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T15:38:39Z affinity protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:36:28Z activated protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:00Z YfiR protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:11Z YfiN RESULTS title_1 7619 RESULTS RESULTS title_2 7627 Overall structure of YfiB evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:37:28Z structure protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB RESULTS paragraph 7653 We obtained two crystal forms of YfiB (residues 34–168, lacking the signal peptide from residues 1–26 and periplasmic residues 27–33), crystal forms I and II, belonging to space groups P21 and P41, respectively. evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:38:39Z crystal forms protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:38:47Z 34–168 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:11:28Z lacking structure_element SO: melaniev@ebi.ac.uk 2023-03-15T13:39:01Z signal peptide residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:39:03Z 1–26 residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:39:06Z 27–33 13238_2016_264_Fig1_HTML.jpg Fig1 FIG fig_caption 7871 Overall structure of YfiB. (A) The overall structure of the YfiB monomer. (B) A topology diagram of the YfiB monomer. (C and D) The analytical ultracentrifugation experiment results for the wild-type YfiB and YfiBL43P evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:40:01Z structure protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:39:59Z structure protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:39:54Z monomer protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:39:54Z monomer experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T13:40:04Z analytical ultracentrifugation protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:37:06Z wild-type protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P 13238_2016_264_Fig2_HTML.jpg Fig2 FIG fig_caption 8090 Two dimeric types of YfiB dimer. (A–C) The “head to head” dimer. (D–F) The “back to back” dimer. (A) and (E) indicate the front views of the two dimers, (B) and (F) indicate the top views of the two dimers, and (C) and (D) indicate the details of the two dimeric interfaces oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:36:11Z dimeric protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:41:12Z dimer protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:41:29Z head to head oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:41:12Z dimer protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:41:35Z back to back oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:41:13Z dimer oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:41:21Z dimers oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:41:21Z dimers site SO: melaniev@ebi.ac.uk 2023-03-15T13:41:54Z dimeric interfaces RESULTS paragraph 8376 The crystal structure of YfiB monomer consists of a five-stranded β-sheet (β1-2-5-3-4) flanked by five α-helices (α1–5) on one side. In addition, there is a short helix turn connecting the β4 strand and α4 helix (Fig. 1A and 1B). Each crystal form contains three different dimeric types of YfiB, two of which are present in both, suggesting that the rest of the dimeric types may result from crystal packing. Here, we refer to the two dimeric types as “head to head” and “back to back” according to the interacting mode (Fig. 2A and 2E), with the total buried surface areas being 316.8 Å2 and 554.3 Å2, respectively. evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T15:38:19Z crystal structure protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:39:55Z monomer structure_element SO: melaniev@ebi.ac.uk 2023-03-15T13:43:04Z five-stranded β-sheet structure_element SO: melaniev@ebi.ac.uk 2023-03-15T13:43:09Z β1-2-5-3-4 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T13:43:14Z five α-helices structure_element SO: melaniev@ebi.ac.uk 2023-03-15T13:43:17Z α1–5 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T13:43:20Z helix turn structure_element SO: melaniev@ebi.ac.uk 2023-03-15T13:43:28Z β4 strand structure_element SO: melaniev@ebi.ac.uk 2023-03-15T13:43:31Z α4 helix oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:36:11Z dimeric protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:36:11Z dimeric oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:36:11Z dimeric protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:41:29Z head to head protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:41:36Z back to back RESULTS paragraph 9014 The “head to head” dimer exhibits a clamp shape. The dimerization occurs mainly via hydrophobic interactions formed by A37 and I40 on the α1 helices, L50 on the β1 strands, and W55 on the β2 strands of both molecules, making a hydrophobic interacting core (Fig. 2A–C). protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:41:29Z head to head oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:41:13Z dimer protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:11:35Z clamp shape residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:44:27Z A37 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:44:33Z I40 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T13:44:53Z α1 helices residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:44:39Z L50 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T13:44:55Z β1 strands residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:44:46Z W55 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T13:44:58Z β2 strands site SO: melaniev@ebi.ac.uk 2023-03-15T15:41:13Z hydrophobic interacting core RESULTS paragraph 9293 The “back to back” dimer presents a Y shape. The dimeric interaction is mainly hydrophilic, occurring among the main-chain and side-chain atoms of N68, L69, D70 and R71 on the α2-α3 loops and R116 and S120 on the α4 helices of both molecules, resulting in a complex hydrogen bond network (Fig. 2D–F). protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:41:36Z back to back oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:41:13Z dimer protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:46:00Z Y shape residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:46:21Z N68 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:46:27Z L69 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:46:33Z D70 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:46:38Z R71 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T13:46:42Z α2-α3 loops residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:46:47Z R116 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:46:52Z S120 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T13:46:55Z α4 helices site SO: melaniev@ebi.ac.uk 2023-06-14T09:12:22Z hydrogen bond network RESULTS title_2 9606 The YfiB-YfiR interaction complex_assembly GO: melaniev@ebi.ac.uk 2023-06-14T09:14:18Z YfiB-YfiR 13238_2016_264_Fig3_HTML.jpg Fig3 FIG fig_caption 9632 Overall structure of the YfiB-YfiR complex and the conserved surface in YfiR. (A) The overall structure of the YfiB-YfiR complex. The YfiBL43P molecules are shown in cyan and yellow. The YfiR molecules are shown in green and magenta. Two interacting regions are highlighted by red rectangles. (B) Structural superposition of apo YfiB and YfiR-bound YfiBL43P. To illustrate the differences between apo YfiB and YfiR-bound YfiBL43P, the apo YfiB is shown in pink, except residues 34–70 are shown in red, whereas the YfiR-bound YfiBL43P is shown in cyan, except residues 44–70 are shown in blue. (C) Close-up view of the differences between apo YfiB and YfiR-bound YfiBL43P. The residues proposed to contribute to YfiB activation are illustrated in sticks. The key residues in apo YfiB are shown in red and those in YfiBL43P are shown in blue. (D) Close-up views showing interactions in regions I and II. YfiBL43P and YfiR are shown in cyan and green, respectively. (E and F) The conserved surface in YfiR contributes to the interaction with YfiB. (G) The residues of YfiR responsible for interacting with YfiB are shown in green sticks, and the proposed YfiN-interacting residues are shown in yellow sticks. The red sticks, which represent the YfiB-interacting residues, are also responsible for the proposed interactions with YfiN evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T15:38:44Z structure complex_assembly GO: melaniev@ebi.ac.uk 2023-03-15T13:36:21Z YfiB-YfiR site SO: melaniev@ebi.ac.uk 2023-03-15T14:48:07Z conserved surface protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:01Z YfiR evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T15:38:47Z structure complex_assembly GO: melaniev@ebi.ac.uk 2023-03-15T13:36:21Z YfiB-YfiR mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:01Z YfiR experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T14:30:57Z Structural superposition protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:51:30Z apo protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:51:38Z YfiR-bound mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:51:30Z apo protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:51:38Z YfiR-bound mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:51:30Z apo protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:22:46Z 34–70 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:51:38Z YfiR-bound mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:22:50Z 44–70 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:51:30Z apo protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:51:38Z YfiR-bound mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P protein PR: melaniev@ebi.ac.uk 2023-03-22T09:25:27Z YfiB protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:51:30Z apo protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:25:17Z regions I and II mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:01Z YfiR site SO: melaniev@ebi.ac.uk 2023-03-15T14:48:07Z conserved surface protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:01Z YfiR protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:25:22Z residues protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:01Z YfiR protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB site SO: melaniev@ebi.ac.uk 2023-03-15T15:41:34Z YfiN-interacting residues site SO: melaniev@ebi.ac.uk 2023-03-15T15:41:39Z YfiB-interacting residues protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:12Z YfiN RESULTS paragraph 10966 To gain structural insights into the YfiB-YfiR interaction, we co-expressed YfiB (residues 34–168) and YfiR (residues 35–190, lacking the signal peptide), but failed to obtain the complex, in accordance with a previous report in which no stable complex of YfiB-YfiR was observed (Malone et al.,). It has been reported that single mutants of Q39, L43, F48 and W55 contribute to YfiB activation leading to the induction of the SCV phenotype in P. aeruginosa PAO1 (Malone et al.,). It is likely that these residues may be involved in the conformational changes of YfiB that are related to YfiR sequestration (Fig. 3C). Therefore, we constructed two such single mutants of YfiB (YfiBL43P and YfiBF48S). As expected, both mutants form a stable complex with YfiR. Finally, we crystalized YfiR in complex with the YfiBL43P mutant and solved the structure at 1.78 Å resolution by molecular replacement using YfiR and YfiB as models. complex_assembly GO: melaniev@ebi.ac.uk 2023-06-14T09:15:02Z YfiB-YfiR experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T15:39:41Z co-expressed protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:38:47Z 34–168 protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:01Z YfiR residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:22:55Z 35–190 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:11:42Z lacking structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:25:25Z signal peptide protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:11:57Z no stable complex_assembly GO: melaniev@ebi.ac.uk 2023-03-15T13:36:21Z YfiB-YfiR experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T15:39:45Z single mutants of residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:23:15Z Q39 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:27:28Z L43 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:20:18Z F48 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:44:46Z W55 protein PR: melaniev@ebi.ac.uk 2023-03-22T09:25:43Z YfiB species MESH: melaniev@ebi.ac.uk 2023-03-15T15:14:23Z P. aeruginosa PAO1 protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB protein PR: melaniev@ebi.ac.uk 2023-03-22T09:25:55Z YfiR experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T14:13:09Z constructed two such single mutants protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P mutant MESH: melaniev@ebi.ac.uk 2023-03-15T15:39:30Z YfiBF48S protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:12:40Z stable protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:12:45Z complex with protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:01Z YfiR experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T14:13:16Z crystalized protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:01Z YfiR protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:35:33Z in complex with mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:35:27Z mutant evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T15:38:51Z structure experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T14:13:06Z molecular replacement protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:01Z YfiR protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB RESULTS paragraph 11897 The YfiB-YfiR complex is a 2:2 heterotetramer (Fig. 3A) in which the YfiR dimer is clamped by two separated YfiBL43P molecules with a total buried surface area of 3161.2 Å2. The YfiR dimer in the complex is identical to the non-oxidized YfiR dimer alone (Yang et al.,), with only Cys145-Cys152 of the two disulfide bonds well formed, suggesting Cys71-Cys110 disulfide bond formation is not essential for forming YfiB-YfiR complex. The N-terminal structural conformation of YfiBL43P, from the foremost N-terminus to residue D70, is significantly altered compared with that of the apo YfiB. The majority of the α1 helix (residues 34–43) is invisible on the electron density map, and the α2 helix and β1 and β2 strands are rearranged to form a long loop containing two short α-helix turns (Fig. 3B and 3C), thus embracing the YfiR dimer. The observed changes in conformation of YfiB and the results of mutagenesis suggest a mechanism by which YfiB sequesters YfiR. complex_assembly GO: melaniev@ebi.ac.uk 2023-03-15T13:36:21Z YfiB-YfiR oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T15:41:01Z heterotetramer protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:01Z YfiR oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:41:13Z dimer mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:01Z YfiR oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:41:13Z dimer protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:13:56Z non-oxidized protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:01Z YfiR oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:41:13Z dimer protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:35:14Z alone residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:12:12Z Cys145 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:12:18Z Cys152 ptm MESH: melaniev@ebi.ac.uk 2023-03-15T14:12:38Z disulfide bonds residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:12:24Z Cys71 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:12:31Z Cys110 ptm MESH: melaniev@ebi.ac.uk 2023-03-15T13:14:32Z disulfide bond complex_assembly GO: melaniev@ebi.ac.uk 2023-03-15T13:36:21Z YfiB-YfiR mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:46:33Z D70 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:51:30Z apo protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB structure_element SO: melaniev@ebi.ac.uk 2023-03-15T14:14:03Z α1 helix residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:14:23Z 34–43 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:12:58Z electron density map structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:25:30Z α2 helix structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:25:33Z β1 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:25:36Z β2 strands structure_element SO: melaniev@ebi.ac.uk 2023-03-15T14:19:31Z loop structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:25:41Z α-helix turns protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:01Z YfiR oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:41:13Z dimer protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T15:39:51Z mutagenesis protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:01Z YfiR RESULTS paragraph 12877 The YfiB-YfiR interface can be divided into two regions (Fig. 3A and 3D). Region I is formed by numerous main-chain and side-chain hydrophilic interactions between residues E45, G47 and E53 from the N-terminal extended loop of YfiB and residues S57, R60, A89 and H177 from YfiR (Fig. 3D-I(i)). Additionally, three hydrophobic anchoring sites exist in region I. The residues F48 and W55 of YfiB are inserted into the hydrophobic cores mainly formed by the main chain and side chain carbon atoms of residues S57/Q88/A89/N90 and R60/R175/H177 of YfiR, respectively; and F57 of YfiB is inserted into the hydrophobic pocket formed by L166/I169/V176/P178/L181 of YfiR (Fig. 3D-I(ii)). In region II, the side chains of R96, E98 and E157 from YfiB interact with the side chains of E163, S146 and R171 from YfiR, respectively. Additionally, the main chains of I163 and V165 from YfiB form hydrogen bonds with the main chains of L166 and A164 from YfiR, respectively, and the main chain of P166 from YfiB interacts with the side chain of R185 from YfiR (Fig. 3D-II). These two regions contribute a robust hydrogen-bonding network to the YfiB-YfiR interface, resulting in a tightly bound complex. site SO: melaniev@ebi.ac.uk 2023-03-15T14:23:18Z YfiB-YfiR interface structure_element SO: melaniev@ebi.ac.uk 2023-03-15T14:20:25Z Region I residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:19:07Z E45 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:19:15Z G47 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:19:22Z E53 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T14:19:31Z loop protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:19:52Z S57 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:19:58Z R60 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:20:04Z A89 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:20:10Z H177 protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:01Z YfiR site SO: melaniev@ebi.ac.uk 2023-03-15T15:41:44Z hydrophobic anchoring sites structure_element SO: melaniev@ebi.ac.uk 2023-03-15T14:20:25Z region I residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:20:18Z F48 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:44:46Z W55 protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB site SO: melaniev@ebi.ac.uk 2023-03-15T15:41:49Z hydrophobic cores residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:19:52Z S57 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:20:43Z Q88 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:20:04Z A89 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:20:53Z N90 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:19:58Z R60 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:21:05Z R175 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:20:10Z H177 protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:01Z YfiR residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:21:14Z F57 protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB site SO: melaniev@ebi.ac.uk 2023-03-15T14:23:36Z hydrophobic pocket residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:21:23Z L166 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:21:29Z I169 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:21:36Z V176 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:21:43Z P178 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:21:49Z L181 protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:01Z YfiR structure_element SO: melaniev@ebi.ac.uk 2023-03-15T14:21:57Z region II residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:22:08Z R96 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:22:15Z E98 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:22:23Z E157 protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:22:33Z E163 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:22:40Z S146 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:22:48Z R171 protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:01Z YfiR residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:22:55Z I163 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:23:02Z V165 protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:21:23Z L166 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:23:22Z A164 protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:01Z YfiR residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:23:26Z P166 protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:23:09Z R185 protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:01Z YfiR site SO: melaniev@ebi.ac.uk 2023-03-15T14:24:13Z hydrogen-bonding network site SO: melaniev@ebi.ac.uk 2023-03-15T14:23:18Z YfiB-YfiR interface RESULTS paragraph 14067 Based on the observations that two separated YfiBL43P molecules form a 2:2 complex structure with YfiR dimer, we performed an analytical ultracentrifugation experiment to check the oligomeric states of wild-type YfiB and YfiBL43P. The results showed that wild-type YfiB exists in both monomeric and dimeric states in solution, while YfiBL43P primarily adopts the monomer state in solution (Fig. 1C–D). This suggests that the N-terminus of YfiB plays an important role in forming the dimeric YfiB in solution and that the conformational change of residue L43 is associated with the stretch of the N-terminus and opening of the dimer. Therefore, it is possible that both dimeric types might exist in solution. For simplicity, we only discuss the “head to head” dimer in the following text. mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:27:48Z structure protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:01Z YfiR oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:41:13Z dimer experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T14:27:12Z analytical ultracentrifugation protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:37:06Z wild-type protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:37:06Z wild-type protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T15:41:06Z monomeric oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:36:11Z dimeric mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:39:55Z monomer protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:36:11Z dimeric protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:27:28Z L43 oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:41:13Z dimer oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:36:11Z dimeric protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:41:29Z head to head oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:41:13Z dimer RESULTS title_2 14862 The PG-binding site of YfiB site SO: melaniev@ebi.ac.uk 2023-03-15T14:28:20Z PG-binding site protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB 13238_2016_264_Fig4_HTML.jpg Fig4 FIG fig_caption 14890 The PG-binding site in YfiB. (A) Structural superposition of the PG-binding sites of the H. influenzae Pal/PG-P complex and YfiR-bound YfiBL43P complexed with sulfate ions. (B) Close-up view showing the key residues of Pal interacting with the m-Dap5 ε-carboxylate group of PG-P. Pal is shown in wheat and PG-P is in magenta. (C) Close-up view showing the key residues of YfiR-bound YfiBL43P interacting with a sulfate ion. YfiR-bound YfiBL43P is shown in cyan; the sulfate ion, in green; and the water molecule, in yellow. (D) Structural superposition of the PG-binding sites of apo YfiB and YfiR-bound YfiBL43P, the key residues are shown in stick. Apo YfiB is shown in yellow and YfiR-bound YfiBL43P in cyan. (E and F) MST data and analysis for binding affinities of (E) YfiB wild-type and (F) YfiBL43P with PG. (G) The sequence alignment of P. aeruginosa and E. coli sources of YfiB, Pal and the periplasmic domain of OmpA site SO: melaniev@ebi.ac.uk 2023-03-15T14:28:20Z PG-binding site protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T14:30:57Z Structural superposition site SO: melaniev@ebi.ac.uk 2023-03-15T14:31:43Z PG-binding sites species MESH: melaniev@ebi.ac.uk 2023-03-15T15:14:23Z H. influenzae complex_assembly GO: melaniev@ebi.ac.uk 2023-03-15T14:31:27Z Pal/PG-P protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:51:38Z YfiR-bound mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:35:43Z complexed with chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T14:32:02Z sulfate protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T14:31:16Z Pal chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:37:28Z m-Dap5 ε-carboxylate chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T14:32:13Z PG-P protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T14:31:16Z Pal chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T14:32:13Z PG-P protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:51:38Z YfiR-bound mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T14:32:02Z sulfate protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:51:38Z YfiR-bound mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T14:32:02Z sulfate chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T14:31:56Z water experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T14:30:57Z Structural superposition site SO: melaniev@ebi.ac.uk 2023-03-15T14:31:43Z PG-binding sites protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:51:30Z apo protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:51:38Z YfiR-bound mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:51:30Z Apo protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:51:39Z YfiR-bound mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T14:46:26Z MST evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T15:38:57Z binding affinities protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:37:06Z wild-type mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T14:32:25Z PG experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T15:39:57Z sequence alignment species MESH: melaniev@ebi.ac.uk 2023-03-15T15:14:23Z P. aeruginosa species MESH: melaniev@ebi.ac.uk 2023-03-15T15:14:23Z E. coli protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T14:31:16Z Pal structure_element SO: melaniev@ebi.ac.uk 2023-03-15T15:43:32Z periplasmic domain protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T14:32:45Z OmpA RESULTS paragraph 15823 PG-associated lipoprotein (Pal) is highly conserved in Gram-negative bacteria and anchors to the outer membrane through an N-terminal lipid attachment and to PG layer through its periplasmic domain, which is implicated in maintaining outer membrane integrity. Previous homology modeling studies suggested that YfiB contains a Pal-like PG-binding site (Parsons et al.,), and the mutation of two residues at this site, D102 and G105, reduces the ability for biofilm formation and surface attachment (Malone et al.,). In the YfiB-YfiR complex, one sulfate ion is found at the bottom of each YfiBL43P molecule (Fig. 3A) and forms a strong hydrogen bond with D102 of YfiBL43P (Fig. 4A and 4C). Structural superposition between YfiBL43P and Haemophilus influenzae Pal complexed with biosynthetic peptidoglycan precursor (PG-P), UDP-N-acetylmuramyl-L-Ala-α-D-Glu-m-Dap-D-Ala-D-Ala (m-Dap is meso-diaminopimelate) (PDB code: 2aiz) (Parsons et al.,), revealed that the sulfate ion is located at the position of the m-Dap5 ϵ-carboxylate group in the Pal/PG-P complex (Fig. 4A). In the Pal/PG-P complex structure, the m-Dap5 ϵ-carboxylate group interacts with the side-chain atoms of D71 and the main-chain amide of D37 (Fig. 4B). Similarly, in the YfiR-bound YfiBL43P structure, the sulfate ion interacts with the side-chain atoms of D102 (corresponding to D71 in Pal) and R117 (corresponding to R86 in Pal) and the main-chain amide of N68 (corresponding to D37 in Pal). Moreover, a water molecule was found to bridge the sulfate ion and the side chains of N67 and D102, strengthening the hydrogen bond network (Fig. 4C). In addition, sequence alignment of YfiB with Pal and the periplasmic domain of OmpA (proteins containing PG-binding site) showed that N68 and D102 are highly conserved (Fig. 4G, blue stars), suggesting that these residues contribute to the PG-binding ability of YfiB. protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T14:40:05Z PG-associated lipoprotein protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T14:31:16Z Pal protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:40:31Z highly conserved taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:35:11Z Gram-negative bacteria structure_element SO: melaniev@ebi.ac.uk 2023-03-15T15:43:32Z periplasmic domain experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T14:40:51Z homology modeling protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:50Z YfiB site SO: melaniev@ebi.ac.uk 2023-03-15T14:40:00Z Pal-like PG-binding site experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T14:40:40Z mutation of two residues residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:38:29Z D102 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:38:35Z G105 complex_assembly GO: melaniev@ebi.ac.uk 2023-03-15T13:36:21Z YfiB-YfiR chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T14:32:02Z sulfate mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:38:29Z D102 mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T14:30:57Z Structural superposition mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P species MESH: melaniev@ebi.ac.uk 2023-03-15T15:14:23Z Haemophilus influenzae protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T14:31:16Z Pal protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:35:43Z complexed with chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:37:36Z peptidoglycan precursor chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T14:32:13Z PG-P chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:37:39Z UDP-N-acetylmuramyl-L-Ala-α-D-Glu-m-Dap-D-Ala-D-Ala chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:37:42Z m-Dap chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:37:46Z meso-diaminopimelate chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T14:32:02Z sulfate chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:37:50Z m-Dap5 ϵ-carboxylate complex_assembly GO: melaniev@ebi.ac.uk 2023-03-15T15:36:46Z Pal/PG-P complex_assembly GO: melaniev@ebi.ac.uk 2023-03-15T15:36:50Z Pal/PG-P evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:39:46Z structure chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:37:53Z m-Dap5 ϵ-carboxylate residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:38:43Z D71 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:38:50Z D37 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:51:39Z YfiR-bound mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:39:49Z structure chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T14:32:02Z sulfate residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:38:29Z D102 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:38:43Z D71 protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T14:31:16Z Pal residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:38:59Z R117 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:39:05Z R86 protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T14:31:16Z Pal residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:46:21Z N68 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:38:50Z D37 protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T14:31:16Z Pal chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T14:31:56Z water chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T14:32:02Z sulfate residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:39:26Z N67 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:38:29Z D102 site SO: melaniev@ebi.ac.uk 2023-06-14T09:16:41Z hydrogen bond network experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T14:40:44Z sequence alignment protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:51Z YfiB protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T14:31:16Z Pal structure_element SO: melaniev@ebi.ac.uk 2023-03-15T15:43:32Z periplasmic domain protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T14:39:31Z OmpA site SO: melaniev@ebi.ac.uk 2023-03-15T14:28:20Z PG-binding site residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:46:21Z N68 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:38:29Z D102 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:40:32Z highly conserved protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:51Z YfiB RESULTS paragraph 17719 Interestingly, superposition of apo YfiB with YfiR-bound YfiBL43P revealed that the PG-binding region is largely altered mainly due to different conformation of the N68 containing loop. Compared to YfiBL43P, the N68-containing loop of the apo YfiB flips away about 7 Å, and D102 and R117 swing slightly outward; thus, the PG-binding pocket is enlarged with no sulfate ion or water bound (Fig. 4D). Therefore, we proposed that the PG-binding ability of inactive YfiB might be weaker than that of active YfiB. To validate this, we performed a microscale thermophoresis (MST) assay to measure the binding affinities of PG to wild-type YfiB and YfiBL43P, respectively. The results indicated that the PG-binding affinity of YfiBL43P is 65.5 μmol/L, which is about 16-fold stronger than that of wild-type YfiB (Kd = 1.1 mmol/L) (Fig. 4E–F). As the experiment is performed in the absence of YfiR, it suggests that an increase in the PG-binding affinity of YfiB is not a result of YfiB-YfiR interaction and is highly coupled to the activation of YfiB characterized by a stretched N-terminal conformation. experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T14:47:10Z superposition protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:51:30Z apo protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:51Z YfiB protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:51:39Z YfiR-bound mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P site SO: melaniev@ebi.ac.uk 2023-03-15T15:41:54Z PG-binding region protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:47:00Z different conformation residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:46:21Z N68 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T14:19:31Z loop mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:46:21Z N68 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T14:19:31Z loop protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:51:30Z apo protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:51Z YfiB residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:38:29Z D102 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:38:59Z R117 site SO: melaniev@ebi.ac.uk 2023-03-15T11:36:51Z PG-binding pocket chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T14:32:02Z sulfate chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T14:31:56Z water chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:27:19Z PG protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:46:54Z inactive protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:51Z YfiB protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:35:21Z active protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:51Z YfiB experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T14:46:20Z microscale thermophoresis experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T14:46:26Z MST evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:46:39Z binding affinities chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:38:00Z PG protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:37:06Z wild-type protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:51Z YfiB mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:46:46Z PG-binding affinity mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:37:06Z wild-type protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:51Z YfiB protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:17:35Z in the absence of protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:02Z YfiR evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:46:46Z PG-binding affinity protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:51Z YfiB complex_assembly GO: melaniev@ebi.ac.uk 2023-06-14T09:17:01Z YfiB-YfiR protein PR: melaniev@ebi.ac.uk 2023-03-15T16:17:54Z YfiB protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:18:26Z stretched N-terminal conformation RESULTS title_2 18822 The conserved surface in YfiR is functional for binding YfiB and YfiN site SO: melaniev@ebi.ac.uk 2023-03-15T14:48:07Z conserved surface protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:02Z YfiR protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:51Z YfiB protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:12Z YfiN RESULTS paragraph 18892 Calculation using the ConSurf Server (http://consurf.tau.ac.il/), which estimates the evolutionary conservation of amino acid positions and visualizes information on the structure surface, revealed a conserved surface on YfiR that contributes to the interaction with YfiB (Fig. 3E and 3F). Interestingly, the majority of this conserved surface contributes to the interaction with YfiB (Fig. 3E and 3F). Malone JG et al. have reported that F151, E163, I169 and Q187, located near the C-terminus of YfiR, comprise a putative YfiN binding site (Malone et al.,). Interestingly, these residues are part of the conserved surface of YfiR (Fig. 3G). F151, E163 and I169 form a hydrophobic core while, Q187 is located at the end of the α6 helix. E163 and I169 are YfiB-interacting residues of YfiR, in which E163 forms a hydrogen bond with R96 of YfiB (Fig. 3D-II) and I169 is involved in forming the L166/I169/V176/P178/L181 hydrophobic core for anchoring F57 of YfiB (Fig. 3D-I(ii)). Collectively, a part of the YfiB-YfiR interface overlaps with the proposed YfiR-YfiN interface, suggesting alteration in the association-disassociation equilibrium of YfiR-YfiN and hence the ability of YfiB to sequester YfiR. experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T15:40:03Z ConSurf Server evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T15:39:06Z evolutionary conservation site SO: melaniev@ebi.ac.uk 2023-03-15T15:42:00Z structure surface site SO: melaniev@ebi.ac.uk 2023-03-15T14:48:07Z conserved surface protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:02Z YfiR protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:51Z YfiB site SO: melaniev@ebi.ac.uk 2023-03-15T14:48:07Z conserved surface protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:51Z YfiB residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:23:32Z F151 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:22:33Z E163 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:21:29Z I169 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:23:36Z Q187 protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:02Z YfiR site SO: melaniev@ebi.ac.uk 2023-03-15T15:42:08Z YfiN binding site site SO: melaniev@ebi.ac.uk 2023-03-15T14:48:07Z conserved surface protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:02Z YfiR residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:23:45Z F151 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:22:33Z E163 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:21:29Z I169 site SO: melaniev@ebi.ac.uk 2023-03-15T14:51:31Z hydrophobic core residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:23:49Z Q187 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:25:52Z α6 helix residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:22:33Z E163 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:21:29Z I169 site SO: melaniev@ebi.ac.uk 2023-03-15T15:42:11Z YfiB-interacting residues protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:02Z YfiR residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:22:33Z E163 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:22:08Z R96 protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:51Z YfiB residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:21:29Z I169 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:21:23Z L166 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:21:29Z I169 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:21:36Z V176 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:21:43Z P178 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:21:49Z L181 site SO: melaniev@ebi.ac.uk 2023-03-15T14:51:31Z hydrophobic core residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:21:14Z F57 protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:51Z YfiB site SO: melaniev@ebi.ac.uk 2023-03-15T14:23:18Z YfiB-YfiR interface site SO: melaniev@ebi.ac.uk 2023-03-15T15:42:18Z YfiR-YfiN interface protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:02Z YfiR protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:12Z YfiN protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:51Z YfiB protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:02Z YfiR RESULTS title_2 20101 YfiR binds small molecules protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:02Z YfiR RESULTS paragraph 20128 Previous studies indicated that YfiR constitutes a YfiB-independent sensing device that can activate YfiN in response to the redox status of the periplasm, and we have reported YfiR structures in both the non-oxidized and the oxidized states earlier, revealing that the Cys145-Cys152 disulfide bond plays an essential role in maintaining the correct folding of YfiR (Yang et al.,). However, whether YfiR is involved in other regulatory mechanisms is still an open question. protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:02Z YfiR protein PR: melaniev@ebi.ac.uk 2023-03-22T09:27:42Z YfiB protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:12Z YfiN protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:02Z YfiR evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:53:31Z structures protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:13:56Z non-oxidized protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:53:28Z oxidized residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:12:12Z Cys145 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:12:18Z Cys152 ptm MESH: melaniev@ebi.ac.uk 2023-03-15T13:14:32Z disulfide bond protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:02Z YfiR protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:02Z YfiR 13238_2016_264_Fig5_HTML.jpg Fig5 FIG fig_caption 20602 Overall Structures of VB6-bound and Trp-bound YfiR. (A) Superposition of the overall structures of VB6-bound and Trp-bound YfiR. (B) Close-up views showing the key residues of YfiR that bind VB6 and L-Trp. The electron densities of VB6 and Trp are countered at 3.0σ and 2.3σ, respectively, in |Fo|-|Fc| maps. (C) Superposition of the hydrophobic pocket of YfiR with VB6, L-Trp and F57 of YfiB evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:55:21Z Structures protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:58:52Z VB6-bound protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:59:00Z Trp-bound protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:02Z YfiR experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T14:55:52Z Superposition evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:55:16Z structures protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:58:52Z VB6-bound protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:59:00Z Trp-bound protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:02Z YfiR protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:02Z YfiR chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T14:55:49Z VB6 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T14:55:46Z L-Trp evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:55:13Z electron densities chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T14:55:38Z VB6 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T14:55:35Z Trp evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:55:19Z |Fo|-|Fc| maps experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T14:55:24Z Superposition site SO: melaniev@ebi.ac.uk 2023-03-15T14:23:36Z hydrophobic pocket protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:02Z YfiR chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T14:55:44Z VB6 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T14:55:41Z L-Trp residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:21:15Z F57 protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:51Z YfiB RESULTS paragraph 21003 Intriguingly, a Dali search (Holm and Rosenstrom,) indicated that the closest homologs of YfiR shared the characteristic of being able to bind several structurally similar small molecules, such as L-Trp, L-Phe, B-group vitamins and their analogs, encouraging us to test whether YfiR can recognize these molecules. For this purpose, we co-crystallized YfiR or soaked YfiR crystals with different small molecules, including L-Trp and B-group vitamins. Fortunately, we found obvious small-molecule density in the VB6-bound and Trp-bound YfiR crystal structures (Fig. 5A and 5B), and in both structures, the YfiR dimers resemble the oxidized YfiR structure in which both two disulfide bonds are well formed (Yang et al.,). experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T14:59:05Z Dali search protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:02Z YfiR chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T14:59:16Z L-Trp chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T14:59:19Z L-Phe protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:02Z YfiR experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T14:59:10Z co-crystallized protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:02Z YfiR experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T14:59:13Z soaked protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:02Z YfiR evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:59:33Z crystals chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T14:59:22Z L-Trp evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:59:31Z small-molecule density protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:58:52Z VB6-bound protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:59:00Z Trp-bound protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:02Z YfiR evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T15:38:28Z crystal structures evidence DUMMY: melaniev@ebi.ac.uk 2023-06-14T09:23:30Z structures protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:03Z YfiR oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:41:21Z dimers protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:59:45Z oxidized protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:03Z YfiR evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:59:29Z structure ptm MESH: melaniev@ebi.ac.uk 2023-03-15T14:12:38Z disulfide bonds 13238_2016_264_Fig6_HTML.jpg Fig6 FIG fig_caption 21723 Functional analysis of VB6 and L-Trp. (A and B) The effect of increasing concentrations of VB6 or L-Trp on YfiBL43P-induced attachment (bars). The relative optical density is represented as curves. Wild-type YfiB is used as negative control. (C and D) BIAcore data and analysis for binding affinities of (C) VB6 and (D) L-Trp with YfiR. (E–G) ITC data and analysis for titration of (E) YfiB wild-type, (F) YfiBL43P, and (G) YfiBL43P/F57A into YfiR chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:01:47Z VB6 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:01:50Z L-Trp experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T15:01:56Z effect of increasing concentrations chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:01:59Z VB6 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:02:02Z L-Trp mutant MESH: melaniev@ebi.ac.uk 2023-03-22T09:28:17Z YfiBL43P evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T15:02:05Z relative optical density protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:37:06Z Wild-type protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:51Z YfiB experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T15:02:11Z BIAcore evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T15:02:15Z binding affinities chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:02:18Z VB6 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:02:21Z L-Trp protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:03Z YfiR experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T15:02:26Z ITC experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T15:02:30Z titration protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:51Z YfiB protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:37:06Z wild-type mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P mutant MESH: melaniev@ebi.ac.uk 2023-03-15T15:06:07Z F57A protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:03Z YfiR RESULTS paragraph 22173 Structural analyses revealed that the VB6 and L-Trp molecules are bound at the periphery of the YfiR dimer, but not at the dimer interface. Interestingly, VB6 and L-Trp were found to occupy the same hydrophobic pocket, formed by L166/I169/V176/P178/L181 of YfiR, which is also a binding pocket for F57 of YfiB, as observed in the YfiB-YfiR complex (Fig. 5C). To evaluate the importance of F57 in YfiBL43P-YfiR interaction, the binding affinities of YfiBL43P and YfiBL43P/F57A for YfiR were measured by isothermal titration calorimetry (ITC). The results showed Kd values of 1.4 × 10−7 mol/L and 5.3 × 10−7 mol/L for YfiBL43P and YfiBL43P/F57A, respectively, revealing that the YfiBL43P/F57A mutant caused a 3.8-fold reduction in the binding affinity compared with the YfiBL43P mutant (Fig. 6F and 6G). experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:52Z Structural analyses chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:06:21Z VB6 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:06:23Z L-Trp protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T15:05:24Z bound at protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:03Z YfiR oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:41:13Z dimer site SO: melaniev@ebi.ac.uk 2023-03-15T15:05:55Z dimer interface chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:06:26Z VB6 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:06:28Z L-Trp site SO: melaniev@ebi.ac.uk 2023-03-15T14:23:36Z hydrophobic pocket residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:21:23Z L166 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:21:29Z I169 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:21:36Z V176 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:21:43Z P178 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:21:49Z L181 protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:03Z YfiR site SO: melaniev@ebi.ac.uk 2023-03-15T15:05:48Z binding pocket residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:21:15Z F57 protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:51Z YfiB complex_assembly GO: melaniev@ebi.ac.uk 2023-03-15T13:36:21Z YfiB-YfiR residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:21:15Z F57 complex_assembly GO: melaniev@ebi.ac.uk 2023-06-14T09:18:10Z YfiBL43P-YfiR evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T15:06:11Z binding affinities mutant MESH: melaniev@ebi.ac.uk 2023-03-22T09:28:32Z YfiBL43P mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P mutant MESH: melaniev@ebi.ac.uk 2023-03-15T15:06:07Z F57A protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:03Z YfiR experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T15:05:08Z isothermal titration calorimetry experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T15:02:26Z ITC evidence DUMMY: melaniev@ebi.ac.uk 2023-03-22T09:29:03Z Kd mutant MESH: melaniev@ebi.ac.uk 2023-03-22T09:28:47Z YfiBL43P mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P mutant MESH: melaniev@ebi.ac.uk 2023-03-15T15:06:07Z F57A mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P mutant MESH: melaniev@ebi.ac.uk 2023-03-15T15:06:07Z F57A protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:35:27Z mutant evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T15:06:13Z binding affinity mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:35Z YfiBL43P protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:35:27Z mutant RESULTS paragraph 22983 In parallel, to better understand the putative functional role of VB6 and L-Trp, yfiB was deleted in a PAO1 wild-type strain, and a construct expressing the YfiBL43P mutant was transformed into the PAO1 ΔyfiB strain to trigger YfiBL43P-induced biofilm formation. Growth and surface attachment assays were carried out for the yfiB-L43P strain in the presence of increasing concentrations of VB6 or L-Trp. As shown in Fig. 6A and 6B, the over-expression of YfiBL43P induced strong surface attachment and much slower growth of the yfiB-L43P strain, and as expected, a certain amount of VB6 or L-Trp (4–6 mmol/L for VB6 and 6–10 mmol/L for L-Trp) could reduce the surface attachment. Interestingly, at a concentration higher than 8 mmol/L, VB6 lost its ability to inhibit biofilm formation, implying that the VB6-involving regulatory mechanism is highly complicated and remains to be further investigated. chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:11:44Z VB6 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:11:48Z L-Trp gene GENE: melaniev@ebi.ac.uk 2023-03-15T15:11:04Z yfiB experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T15:12:47Z deleted species MESH: melaniev@ebi.ac.uk 2023-03-15T15:14:23Z PAO1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:37:06Z wild-type experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T15:12:38Z construct expressing mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:36Z YfiBL43P protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:35:27Z mutant experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T15:12:44Z transformed into species MESH: melaniev@ebi.ac.uk 2023-03-15T15:14:23Z PAO1 mutant MESH: melaniev@ebi.ac.uk 2023-03-15T15:12:11Z ΔyfiB mutant MESH: melaniev@ebi.ac.uk 2023-03-22T09:29:21Z YfiBL43P experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T15:12:35Z Growth and surface attachment assays mutant MESH: melaniev@ebi.ac.uk 2023-03-15T15:11:30Z yfiB-L43P experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T15:12:41Z increasing concentrations chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:11:57Z VB6 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:12:00Z L-Trp experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T15:12:33Z over-expression mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:36Z YfiBL43P mutant MESH: melaniev@ebi.ac.uk 2023-03-15T15:11:40Z yfiB-L43P chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:12:15Z VB6 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:12:18Z L-Trp chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:12:20Z VB6 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:12:23Z L-Trp chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:12:26Z VB6 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:29:34Z VB6 RESULTS paragraph 23891 Of note, both VB6 and L-Trp have been reported to correlate with biofilm formation in certain Gram-negative bacteria (Grubman et al.,; Shimazaki et al.,). In Helicobacter pylori in particular, VB6 biosynthetic enzymes act as novel virulence factors, and VB6 is required for full motility and virulence (Grubman et al.,). In E. coli, mutants with decreased tryptophan synthesis show greater biofilm formation, and matured biofilm is degraded by L-tryptophan addition (Shimazaki et al.,). However, the detailed mechanism remains elusive. chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:14:14Z VB6 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:14:12Z L-Trp taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:35:11Z Gram-negative bacteria species MESH: melaniev@ebi.ac.uk 2023-03-15T15:14:23Z Helicobacter pylori chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:14:07Z VB6 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:14:04Z VB6 species MESH: melaniev@ebi.ac.uk 2023-03-15T15:14:23Z E. coli chemical CHEBI: melaniev@ebi.ac.uk 2023-06-15T15:01:48Z tryptophan chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:14:01Z L-tryptophan RESULTS paragraph 24430 To answer the question whether competition of VB6 or L-Trp for the YfiB F57-binding pocket of YfiR plays an essential role in inhibiting biofilm formation, we measured the binding affinities of VB6 and L-Trp for YfiR via BIAcore experiments. The results showed relatively weak Kd values of 35.2 mmol/L and 76.9 mmol/L for VB6 and L-Trp, respectively (Fig. 6C and 6D). Based on our results, we concluded that VB6 or L-Trp can bind to YfiR, however, VB6 or L-Trp alone may have little effects in interrupting the YfiB-YfiR interaction, the mechanism by which VB6 or L-Trp inhibits biofilm formation remains unclear and requires further investigation. chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:17:51Z VB6 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:17:54Z L-Trp protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:51Z YfiB site SO: melaniev@ebi.ac.uk 2023-03-15T15:17:58Z F57-binding pocket protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:03Z YfiR evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T15:17:40Z binding affinities chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:17:43Z VB6 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:17:46Z L-Trp protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:03Z YfiR experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T15:17:30Z BIAcore evidence DUMMY: melaniev@ebi.ac.uk 2023-03-22T09:30:32Z Kd chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:17:37Z VB6 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:17:34Z L-Trp chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:17:22Z VB6 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:17:19Z L-Trp protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:03Z YfiR chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:17:16Z VB6 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:17:14Z L-Trp protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:35:14Z alone complex_assembly GO: melaniev@ebi.ac.uk 2023-06-14T09:18:52Z YfiB-YfiR chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:16:51Z VB6 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T15:17:03Z L-Trp DISCUSS title_1 25080 DISCUSSION DISCUSS paragraph 25091 Previous studies suggested that in response to cell stress, YfiB in the outer membrane sequesters the periplasmic protein YfiR, releasing its inhibition of YfiN on the inner membrane and thus inducing the diguanylate cyclase activity of YfiN to allow c-di-GMP production (Giardina et al.,; Malone et al.,; Malone et al.,). However, the pattern of interaction between these proteins and the detailed regulatory mechanism remain unknown due to a lack of structural information. protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:51Z YfiB protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:03Z YfiR protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:12Z YfiN protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:12Z YfiN chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:30:47Z c-di-GMP DISCUSS paragraph 25567 Here, we report the crystal structures of YfiB alone and an active mutant YfiBL43P in complex with YfiR, indicating that YfiR forms a 2:2 complex with YfiB via a region composed of conserved residues. Our structural data analysis shows that the activated YfiB has an N-terminal portion that is largely altered, adopting a stretched conformation compared with the compact conformation of the apo YfiB. The apo YfiB structure constructed beginning at residue 34 has a compact conformation of approximately 45 Å in length. In addition to the preceding 8 aa loop (from the lipid acceptor Cys26 to Gly34), the full length of the periplasmic portion of apo YfiB can reach approximately 60 Å. It was reported that the distance between the outer membrane and the cell wall is approximately 50 Å and that the thickness of the PG layer is approximately 70 Å (Matias et al.,). Thus, YfiB alone represents an inactive form that may only partially insert into the PG matrix. By contrast, YfiR-bound YfiBL43P (residues 44–168) has a stretched conformation of approximately 55 Å in length. In addition to the 17 preceding intracellular residues (from the lipid acceptor Cys26 to Leu43), the length of the intracellular portion of active YfiB may extend over 100 Å, assuming a fully stretched conformation. Provided that the diameter of the widest part of the YfiB dimer is approximately 64 Å, which is slightly smaller than the smallest diameter of the PG pore (70 Å) (Meroueh et al.,), the YfiB dimer should be able to penetrate the PG layer. evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T15:38:28Z crystal structures protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:51Z YfiB protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:35:14Z alone protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:35:21Z active protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:35:27Z mutant mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:36Z YfiBL43P protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:35:34Z in complex with protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:03Z YfiR protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:03Z YfiR protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T15:24:18Z complex with protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:51Z YfiB experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T15:24:00Z structural data analysis protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:36:28Z activated protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:51Z YfiB structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:26:07Z N-terminal portion protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:36:21Z stretched conformation protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:36:03Z compact conformation protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:51:30Z apo protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:51Z YfiB protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:51:30Z apo protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:51Z YfiB evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T15:24:06Z structure residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:22:41Z 34 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:36:03Z compact conformation residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:23:03Z preceding 8 aa structure_element SO: melaniev@ebi.ac.uk 2023-03-15T14:19:31Z loop residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-15T15:24:34Z Cys26 to Gly34 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:18:54Z full length protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:51:30Z apo protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:51Z YfiB protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:51Z YfiB protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:35:14Z alone protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:46:54Z inactive protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:51:39Z YfiR-bound mutant MESH: melaniev@ebi.ac.uk 2023-03-15T11:35:36Z YfiBL43P residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-15T15:24:40Z 44–168 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:36:21Z stretched conformation residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:23:07Z 17 preceding intracellular residues residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-15T15:24:37Z Cys26 to Leu43 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:35:21Z active protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:51Z YfiB protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:19:01Z fully stretched conformation protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:51Z YfiB oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:41:13Z dimer protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:51Z YfiB oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:41:13Z dimer 13238_2016_264_Fig7_HTML.jpg Fig7 FIG fig_caption 27106 Regulatory model of the YfiBNR tripartite system. The periplasmic domain of YfiB and the YfiB-YfiR complex are depicted according to the crystal structures. The lipid acceptor Cys26 is indicated as blue ball. The loop connecting Cys26 and Gly34 of YfiB is modeled. The PAS domain of YfiN is shown as pink oval. Once activated by certain cell stress, the dimeric YfiB transforms from a compact conformation to a stretched conformation, allowing the periplasmic domain of the membrane-anchored YfiB to penetrate the cell wall and sequester the YfiR dimer, thus relieving the repression of YfiN complex_assembly GO: melaniev@ebi.ac.uk 2023-03-15T11:33:15Z YfiBNR protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:03:59Z tripartite structure_element SO: melaniev@ebi.ac.uk 2023-03-15T15:43:32Z periplasmic domain protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:51Z YfiB complex_assembly GO: melaniev@ebi.ac.uk 2023-03-15T13:36:21Z YfiB-YfiR evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T15:38:28Z crystal structures residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T15:27:57Z Cys26 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T14:19:31Z loop residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T15:27:49Z Cys26 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T15:27:52Z Gly34 protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:51Z YfiB structure_element SO: melaniev@ebi.ac.uk 2023-03-15T15:43:41Z PAS domain protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:12Z YfiN protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:36:28Z activated oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:36:11Z dimeric protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:51Z YfiB protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:36:03Z compact conformation protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:36:21Z stretched conformation structure_element SO: melaniev@ebi.ac.uk 2023-03-15T15:43:32Z periplasmic domain protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:19:09Z membrane-anchored protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:51Z YfiB protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:03Z YfiR oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:41:13Z dimer protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:12Z YfiN DISCUSS paragraph 27698 These results, together with our observation that activated YfiB has a much higher cell wall binding affinity, and previous mutagenesis data showing that (1) both PG binding and membrane anchoring are required for YfiB activity and (2) activating mutations possessing an altered N-terminal loop length are dominant over the loss of PG binding (Malone et al.,), suggest an updated regulatory model of the YfiBNR system (Fig. 7). In this model, in response to a particular cell stress that is yet to be identified, the dimeric YfiB is activated from a compact, inactive conformation to a stretched conformation, which possesses increased PG binding affinity. This allows the C-terminal portion of the membrane-anchored YfiB to reach, bind and penetrate the cell wall and sequester the YfiR dimer. The YfiBNR system provides a good example of a delicate homeostatic system that integrates multiple signals to regulate the c-di-GMP level. Homologs of the YfiBNR system are functionally conserved in P. aeruginosa (Malone et al.,; Malone et al.,), E. coli (Hufnagel et al.,; Raterman et al.,; Sanchez-Torres et al.,), K. pneumonia (Huertas et al.,) and Y. pestis (Ren et al.,), where they affect c-di-GMP production and biofilm formation. The mechanism by which activated YfiB relieves the repression of YfiN may be applicable to the YfiBNR system in other bacteria and to analogous outside-in signaling for c-di-GMP production, which in turn may be relevant to the development of drugs that can circumvent complicated antibiotic resistance. protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:36:28Z activated protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:51Z YfiB evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T15:39:17Z cell wall binding affinity chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:31:40Z PG protein PR: melaniev@ebi.ac.uk 2023-03-22T09:32:09Z YfiB structure_element SO: melaniev@ebi.ac.uk 2023-03-15T14:19:31Z loop chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:31:59Z PG complex_assembly GO: melaniev@ebi.ac.uk 2023-03-15T11:33:15Z YfiBNR oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:36:11Z dimeric protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:52Z YfiB protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:36:28Z activated protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:20:26Z compact protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T14:46:54Z inactive protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:20:32Z conformation protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:36:21Z stretched conformation chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:31:50Z PG structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:26:12Z C-terminal portion protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:20:37Z membrane-anchored protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:52Z YfiB protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:03Z YfiR oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T13:41:13Z dimer complex_assembly GO: melaniev@ebi.ac.uk 2023-03-15T11:33:15Z YfiBNR chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T11:33:38Z c-di-GMP complex_assembly GO: melaniev@ebi.ac.uk 2023-03-15T11:33:15Z YfiBNR protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:20:48Z functionally conserved species MESH: melaniev@ebi.ac.uk 2023-03-15T15:14:23Z P. aeruginosa species MESH: melaniev@ebi.ac.uk 2023-03-15T15:14:23Z E. coli species MESH: melaniev@ebi.ac.uk 2023-03-15T15:14:23Z K. pneumonia species MESH: melaniev@ebi.ac.uk 2023-03-15T15:14:23Z Y. pestis chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:32:20Z c-di-GMP protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T11:36:28Z activated protein PR: melaniev@ebi.ac.uk 2023-03-15T11:33:52Z YfiB protein PR: melaniev@ebi.ac.uk 2023-03-15T11:34:12Z YfiN complex_assembly GO: melaniev@ebi.ac.uk 2023-03-15T11:33:15Z YfiBNR taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T12:56:28Z bacteria chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:32:34Z c-di-GMP METHODS title_1 29236 MATERIALS AND METHODS METHODS title_2 29258 Protein expression and purification METHODS paragraph 29294 P. aeruginosa YfiR (residues 35–190, lacking the predicted N-terminal periplasmic localization signaling peptide) and YfiB (residues 34–168, lacking the signal peptide from residues 1–26 and periplasmic residues 27–33) were cloned into ORF1 of the pETDuet-1 (Merck Millipore, Darmstadt, Germany) vector via the BamHI and HindIII restriction sites, with a constructed N-terminal His6 and a TEV cleavage site, respectively. In addition, YfiB (residues 34–168) was ligated into the NdeI and XhoI restriction sites of ORF2 in the previously constructed YfiR expression vector. Site-directed mutagenesis was carried out using a QuikChange kit (Agilent Technologies, Santa Clara, CA), following the manufacturer’s instructions. METHODS paragraph 30028 The proteins were over-expressed in the E. coli BL21-CodonPlus(DE3)-RIPL strain. Protein expression was induced by adding 0.5–1 mmol/L IPTG at an OD600 of approximately 0.8. The cell cultures were then incubated for an additional 4.5 h at 37°C. The cells were subsequently harvested by centrifugation and stored at −80°C. METHODS paragraph 30356 Cell suspensions were thawed and homogenized using a high-pressure homogenizer (JNBIO, Beijing, China). YfiR was first purified by Ni affinity chromatography and then incubated with His6-tagged TEV protease overnight. The His6-TEV cleavage site was subsequently removed by incubation with Ni-NTA resin. Finally, YfiR was purified with a HiTrap STM column (GE Healthcare), followed by a Superdex 200 (GE Healthcare) column. YfiB was purified with Ni affinity chromatography, followed by a Superdex 200 (GE Healthcare) column. The YfiB-YfiR complex was first purified by Ni affinity chromatography, then by a Superdex 200 (GE Healthcare) column, and finally by a HiTrap STM column (GE Healthcare). All of the purified fractions were collected and concentrated to ~40 mg/mL in 20 mmol/L Tris-HCl (pH 8.0) and 200 mmol/L NaCl, frozen in liquid nitrogen and stored at −80°C. METHODS title_2 31229 Crystallization and data collection METHODS paragraph 31265 Crystal screening was performed with commercial screening kits (Hampton Research, CA, USA) using the sitting-drop vapor diffusion method, and positive hits were optimized using the hanging-drop vapor diffusion method at 293 K. Crystals of the YfiB protein were obtained and optimized in buffer containing 0.2 mol/L lithium sulfate monohydrate, 0.1 mol/L Tris-HCl (pH 8.0) and 30% w/v polyethylene glycol 4000. After being soaked for a few seconds in cryoprotection solution (well solution complemented with 25% xylitol), the crystals were cooled by plunging them into liquid nitrogen. Diffraction-quality crystals of the YfiB-YfiR complex were grown in buffer containing 0.2 mol/L ammonium sulfate, 0.1 mol/L Tris-HCl (pH 8.0) and 12% w/v polyethylene glycol 8000. The crystals were cryoprotected with 8% (w/v) polyethylene glycol 8000 and 0.1 mol/L Tris-HCl (pH 7.5) supplemented with saturated sucrose prior to being flash frozen. Crystals of the native YfiR were obtained and optimized in 0.1 mol/L HEPES (pH 7.5) and 1.8 mol/L ammonium sulfate. VB6-bound YfiR crystals were obtained by soaking the native YfiR crystals in 2 mmol/L VB6 molecules. Trp-bound YfiR crystals were obtained by co-crystalizing the YfiR protein and 4 mmol/L L-Trp molecules in 0.2 mol/L NaCl, 0.1 mol/L BIS-TRIS (pH 5.5), and 25% w/v polyethylene glycol 3350. For cryoprotection, both the VB6-bound and the L-Trp-bound YfiR crystals were soaked in 2.5 mol/L lithium sulfate monohydrate for a few seconds before data collection. Diffraction data for the YfiB crystal belonging to space group P21 was collected in house, the data for the YfiB crystal belonging to space group P41 and for the Trp-bound YfiR crystal were collected on beamline BL17U at the Shanghai Synchrotron Radiation Facility (SSRF), and the data for the VB6-bound YfiR crystal were collected on beamline BL18U at SSRF. Finally, the data for the YfiB-YfiR complex crystal were collected on beamline BL-1A at the Photon Factory in Japan. The diffraction data were processed with the HKL2000 software program (Otwinowski and Minor,). METHODS title_2 33343 Structure determination and refinement Tab1.xml Tab1 TABLE table_caption 33382 Data collection, phasing and refinement statistics Tab1.xml Tab1 TABLE table <?xml version="1.0" encoding="UTF-8"?> <table frame="hsides" rules="groups"><thead><tr><th align="left"> <bold>Data collection</bold> </th><th align="left">YfiB (crystal form I)</th><th align="left">YfiB (crystal form II)</th><th align="left">VB6-bound YfiR</th><th align="left">Trp-bound YfiR</th><th align="left">YfiBL43P-YfiR</th></tr></thead><tbody><tr><td align="left">Space group</td><td align="left"> <italic>P</italic>21</td><td align="left"> <italic>P</italic>41</td><td align="left"> <italic>P</italic>43212</td><td align="left"> <italic>P</italic>43212</td><td align="left"> <italic>P</italic>1</td></tr><tr><td align="left">Wavelength (Å)</td><td align="left">1.54187</td><td align="left">0.9791</td><td align="left">0.97861</td><td align="left">0.9791</td><td align="left">1.10000</td></tr><tr><td align="left">Resolution (Å)^a </td><td align="left">50.0–2.15 (2.19–2.15)</td><td align="left">50.0–2.80 (2.85–2.8)</td><td align="left">50.0–2.4 (2.44–2.4)</td><td align="left">50.0–2.5 (2.54–2.5)</td><td align="left">50–1.78 (1.86–1.78)</td></tr><tr><td align="left" colspan="6">Cell dimensions</td></tr><tr><td align="left"> a, b, c (Å)</td><td align="left">65.85, 90.45, 66.30</td><td align="left">46.95, 46.95, 154.24</td><td align="left">120.24, 120.24, 84.99</td><td align="left">120.88, 120.88, 88.46</td><td align="left">49.50, 58.57, 69.86</td></tr><tr><td align="left"> α, β, γ (°)</td><td align="left">90, 113.87, 90</td><td align="left">90, 90, 90</td><td align="left">90, 90, 90</td><td align="left">90, 90, 90</td><td align="left">72.93, 96.98, 90.19</td></tr><tr><td align="left"> Unique reflections</td><td align="left">37,625 (1866)</td><td align="left">8,105 (412)</td><td align="left">24,776 (1202)</td><td align="left">23170 (1132)</td><td align="left">67,774 (6615)</td></tr><tr><td align="left"> <italic> I/</italic>σ<italic>I</italic> </td><td align="left">19.59 (2.62)</td><td align="left">12.36 (4.15)</td><td align="left">20.17 (2.4)</td><td align="left">39.5 (4.68)</td><td align="left">17.75 (1.89)</td></tr><tr><td align="left"> Completeness (%)</td><td align="left">97.1 (95.4)</td><td align="left">97.8 (100)</td><td align="left">99.1 (98.8)</td><td align="left">99.9 (100)</td><td align="left">96.5 (94.6)</td></tr><tr><td align="left"> <italic>R</italic> _merge (%)</td><td align="left">6.5 (44.5)</td><td align="left">14.6 (49.7)</td><td align="left">8.9 (56.8)</td><td align="left">9.4 (89.2)</td><td align="left">5.6 (46.3)</td></tr><tr><td align="left"> <italic> R</italic> _meas (%)</td><td align="left">7.4 (51.6)</td><td align="left">15.4 (52.0)</td><td align="left">9.6 (61.7)</td><td align="left">9.6 (90.8)</td><td align="left">6.6 (55.1)</td></tr><tr><td align="left"> CC1/2^b </td><td align="left">0.747</td><td align="left">0.952</td><td align="left">0.899</td><td align="left">0.974</td><td align="left">0.849</td></tr><tr><td align="left" colspan="6"> <bold>Refinement</bold> </td></tr><tr><td align="left"> <italic> R</italic> _work (%)</td><td align="left">20.14</td><td align="left">19.17</td><td align="left">17.82</td><td align="left">18.66</td><td align="left">17.90</td></tr><tr><td align="left"> <italic> R</italic> _free(%)</td><td align="left">26.29</td><td align="left">26.49</td><td align="left">19.81</td><td align="left">23.05</td><td align="left">20.61</td></tr><tr><td align="left" colspan="6">Average B factors (Ų)</td></tr><tr><td align="left"> Protein</td><td align="left">25.54</td><td align="left">42.70</td><td align="left">38.68</td><td align="left">35.03</td><td align="left">32.54</td></tr><tr><td align="left"> VB6</td><td align="left">-</td><td align="left">-</td><td align="left">44.08</td><td align="left">-</td><td align="left">-</td></tr><tr><td align="left"> Trp</td><td align="left">-</td><td align="left">-</td><td align="left">-</td><td align="left">87.51</td><td align="left">-</td></tr><tr><td align="left"> SO₄ ²⁻ </td><td align="left">37.16</td><td align="left">66.52</td><td align="left">51.55</td><td align="left">41.93</td><td align="left">45.51</td></tr><tr><td align="left"> H₂O</td><td align="left">32.91</td><td align="left">36.09</td><td align="left">40.58</td><td align="left">34.75</td><td align="left">43.52</td></tr><tr><td align="left" colspan="6">Root mean square deviations</td></tr><tr><td align="left"> Bond lengths (Å)</td><td align="left">0.009</td><td align="left">0.009</td><td align="left">0.007</td><td align="left">0.007</td><td align="left">0.007</td></tr><tr><td align="left"> Bond angles (°)</td><td align="left">1.085</td><td align="left">1.132</td><td align="left">1.021</td><td align="left">0.977</td><td align="left">1.110</td></tr><tr><td align="left" colspan="6">Ramachandran plot</td></tr><tr><td align="left"> Most favored (%)</td><td align="left">92.6</td><td align="left">87.7</td><td align="left">96.5</td><td align="left">98.1</td><td align="left">94.2</td></tr><tr><td align="left"> Additionally allowed (%)</td><td align="left">7.4</td><td align="left">12.3</td><td align="left">3.5</td><td align="left">1.9</td><td align="left">5.8</td></tr><tr><td align="left"> Generously allowed (%)</td><td align="left">0</td><td align="left">0</td><td align="left">0</td><td align="left">0</td><td align="left">0</td></tr><tr><td align="left"> Disallowed</td><td align="left">0</td><td align="left">0</td><td align="left">0</td><td align="left">0</td><td align="left">0</td></tr></tbody></table> 33433 Data collection YfiB (crystal form I) YfiB (crystal form II) VB6-bound YfiR Trp-bound YfiR YfiBL43P-YfiR Space group P21 P41 P43212 P43212 P1 Wavelength (Å) 1.54187 0.9791 0.97861 0.9791 1.10000 Resolution (Å)a 50.0–2.15 (2.19–2.15) 50.0–2.80 (2.85–2.8) 50.0–2.4 (2.44–2.4) 50.0–2.5 (2.54–2.5) 50–1.78 (1.86–1.78) Cell dimensions  a, b, c (Å) 65.85, 90.45, 66.30 46.95, 46.95, 154.24 120.24, 120.24, 84.99 120.88, 120.88, 88.46 49.50, 58.57, 69.86  α, β, γ (°) 90, 113.87, 90 90, 90, 90 90, 90, 90 90, 90, 90 72.93, 96.98, 90.19  Unique reflections 37,625 (1866) 8,105 (412) 24,776 (1202) 23170 (1132) 67,774 (6615)  I/σI 19.59 (2.62) 12.36 (4.15) 20.17 (2.4) 39.5 (4.68) 17.75 (1.89)  Completeness (%) 97.1 (95.4) 97.8 (100) 99.1 (98.8) 99.9 (100) 96.5 (94.6)  Rmerge (%) 6.5 (44.5) 14.6 (49.7) 8.9 (56.8) 9.4 (89.2) 5.6 (46.3)  Rmeas (%) 7.4 (51.6) 15.4 (52.0) 9.6 (61.7) 9.6 (90.8) 6.6 (55.1)  CC1/2b 0.747 0.952 0.899 0.974 0.849 Refinement  Rwork (%) 20.14 19.17 17.82 18.66 17.90  Rfree(%) 26.29 26.49 19.81 23.05 20.61 Average B factors (Å2)  Protein 25.54 42.70 38.68 35.03 32.54  VB6 - - 44.08 - -  Trp - - - 87.51 -  SO42− 37.16 66.52 51.55 41.93 45.51  H2O 32.91 36.09 40.58 34.75 43.52 Root mean square deviations  Bond lengths (Å) 0.009 0.009 0.007 0.007 0.007  Bond angles (°) 1.085 1.132 1.021 0.977 1.110 Ramachandran plot  Most favored (%) 92.6 87.7 96.5 98.1 94.2  Additionally allowed (%) 7.4 12.3 3.5 1.9 5.8  Generously allowed (%) 0 0 0 0 0  Disallowed 0 0 0 0 0 Tab1.xml Tab1 TABLE table_foot 35033 a Numbers in parentheses are for the highest resolution shell Tab1.xml Tab1 TABLE table_foot 35096 b The values of CC1/2 are for the highest resolution shell METHODS paragraph 35156 The two YfiB crystal structures respectively belonging to space groups P21 and P41 were both solved by molecular replacement (Lebedev et al.,) using the putative MotB-like protein DVU_2228 from D. vulgaris as a model (PDB code: 3khn) at 2.15 Å and 2.8 Å resolution, respectively. Both the VB6-bound and the Trp-bound YfiR crystals belonging to space group P43212, with a dimer in the asymmetric unit, were solved by molecular replacement (Lebedev et al.,) using native YfiR as a model (PDB code: 4YN7) at 2.4 Å and 2.5 Å resolution, respectively. The YfiB-YfiR crystal belonging to space group P1, with a 2:2 heterotetramer in the asymmetric unit, was solved by molecular replacement using YfiR and YfiB as models. Electron density maps were calculated using PHENIX (Adams et al.,). Model building was performed using COOT (Emsley et al.,) and refined with PHENIX (Adams et al.,; Afonine et al.,). The final structures were analyzed with PROCHECK (Laskowski et al.,). Data collection and refinement statistics are presented in Table 1. The figures depicting structures were prepared using PyMOL (http://www.pymol.org). Atomic coordinates and structure factors have been deposited in the RCSB Protein Data Bank (http://www.pdb.org) under accession codes 5EAZ, 5EB0, 5EB1, 5EB2 and 5EB3. METHODS title_2 36447 Analytical ultracentrifugation METHODS paragraph 36478 Sedimentation velocity measurements were performed on a Beckman ProteomeLab XL-I at 25°C. All protein samples were diluted to an OD280 of 0.7 in 20 mmol/L Tris (pH 8.0) and 200 mmol/L NaCl. Data were collected at 60,000 rpm. (262,000 ×g) every 3 min at a wavelength of 280 nm. Interference sedimentation coefficient distributions, or c(M), were calculated from the sedimentation velocity data using SEDFIT (Schuck,). METHODS title_2 36897 PG preparation METHODS paragraph 36912 PG was extracted from the E. coli DH5α strain by following a method described previously (Desmarais et al.,). Briefly, cells were cultured until they reached an OD600 of 0.7–0.8 and then collected at 5,000 ×g, 4°C. The collected bacteria were dripped into the boiling 6% (w/v) SDS and stirred at 500 rpm in a boiling water bath for 3 h before incubating overnight at room temperature. The large PG polymers were collected by ultracentrifugation at 130,000 ×g for 1 h at room temperature and washed repeatedly to remove SDS. The pellet was treated with Pronase E (200 μg/mL final concentration) for 3 h at 60°C followed by SDS to remove contaminating proteins and washed three times to remove the SDS by ultracentrifugation. Next, the samples were treated with lysozyme (200 μg/mL final concentration) for 16 h at 37°C. Finally, the purified PG is obtained by treating the samples in a boiling water bath for 10 min and centrifuging it at 13,000 ×g to remove the contaminating lysozyme. METHODS title_2 37909 Microscale thermophoresis (MST) METHODS paragraph 37941 Purified YfiB wild-type and it mutant YfiBL43P were fluorescently labeled using the NanoTemper blue protein-labeling kit according to the manufacturer’s protocol. This resulted in coupling of the fluorescent dye NT-495. PG was titrated in 1:1 dilutions starting at 1 mmol/L. To determine of the Kd values, 10 μL labeled protein was mixed with 10 μL PG at various concentrations in Hepes buffer (20 mmol/L Hepes, 200 mmol/L NaCl, 0.005% Tween-20, pH 7.5). After 10 min of incubation, all binding reaction mixtures were loaded into the MST-grade glass capillaries (NanoTemper Technologies), and thermophoresis was measured with a NanoTemper Monolith-NT115 system (20% light-emitting diode, 20% IR laser power). METHODS title_2 38654 Deletion of the yfiB genes METHODS paragraph 38681 The yfiB deletion construct was produced by SOE-PCR (Hmelo et al.,) and contained homologous flanking regions to the target gene. This construct was ligated into the pEX18Gm vector between the HindIII and the KpnI sites. The resulting vector was then used to delete yfiB by two-step allelic exchange (Hmelo et al.,). After being introduced into PAO1 via biparental mating with E. coli SM10 (λpir), single crossovers were selected on Vogel-Bonner Minimal Medium (VBMM), which was used for counter-selection against E. coli (P. aeruginosa can utilize citrate as a sole carbon source and energy source, whereas E. coli cannot), containing 50 μg/mL gentamycin. Restreaking was then performed on no-salt Luria-Bertani (NSLB) agar that contained 15% sucrose to force the resolution of double crossovers. Deletion of yfiB in the strains was confirmed by colony PCR. METHODS paragraph 39546 For complementation experiments, yfiB wild-type and L43P mutant genes were cloned into the pJN105 vector via the EcoRI and XbaI restriction sites, respectively. The plasmids were then individually transformed into the PAO1 ΔyfiB strain using the rapid electroporation method described in Choi KH et al. (Choi et al.,). Transformants were selected on LB plates containing 50 μg/mL gentamycin. For induction, arabinose was added to a final concentration of 0.2%. METHODS title_2 40012 Attachment assays METHODS paragraph 40030 The attachment assays were carried out using the MBECTM (Minimum Biofilm Eradication Concentration, Innovotech, Inc.) biofilm inoculator, which consists of a plastic lid with 96 pegs and 96 individual wells. The MBEC plates containing 150 μL LB medium/well were inoculated with 1% overnight cultures of the yfiB-L43P strain and incubated overnight at 37°C without shaking. VB6, L-Trp and arabinose were added as appropriate. The peg lids were washed with distilled water, and the attached cell material was then stained with 0.1% crystal violet solution (5% methanol, 5% isopropanol) before further washing to remove excess dye. The crystal violet was re-dissolved in 20% acetic acid solution, and the absorbance was measured at 600 nm. Assays were performed with 12 wells/strain and repeated independently for each experiment. METHODS title_2 40860 BIAcore analysis METHODS paragraph 40877 The interaction kinetics of YfiR with VB6 and L-Trp were examined on a SPR machine Biacore 3000 (GE Healthcare) at 25°C. The running buffer (20 mmol/L HEPES, pH 7.5, 150 mmol/L NaCl, 0.005% (v/v) Tween-20) was vacuum filtered, and degassed immediately prior to use. YfiR at 10 μg/mL in 10 mmol/L sodium acetate (pH 5.5) was immobilized to 3000 response units on the carboxymethylated dextran surface-modified chip (CM5 chip). The binding affinities were evaluated over a range of 2.5–40 mmol/L concentrations. Meanwhile, for both binding assays, the concentration of 10 mmol/L was repeated as an internal control. All of the data collected were analyzed using BIAevaluation software version 4.1. METHODS title_2 41577 ITC assays METHODS paragraph 41588 ITC experiments were performed in a buffer composed of 20 mmol/L Tris (pH 8.0) and 150 mmol/L NaCl at 25°C using an iTC200 calorimeter (GE Healthcare). YfiB wild-type or its mutants (YfiBL43P, YfiBL43P/F57A) (0.4 mmol/L, in the syringe) was titrated into YfiR (0.04 mmol/L, in the cell), respectively. The titration sequence included a single 0.5 µL injection, followed by 19 injections of 2 µL each, with a 2-min interval between injections and a stirring rate of 1000 rpm. The calorimetric data were then analyzed with OriginLab software (GE Healthcare). METHODS footnote 42153 Min Xu, Xuan Yang and Xiu-An Yang have contributed equally to this work. 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