Molecular basis for coordinating secondary metabolite production by bacterial and plant signaling molecules
2022; Elsevier BV; Volume: 298; Issue: 6 Linguagem: Inglês
10.1016/j.jbc.2022.102027
ISSN1083-351X
AutoresNannan Zhang, Jin Jei Wu, Siping Zhang, Maoran Yuan, Hang Xu, Jie Li, Ping Zhang, Mingzhu Wang, Megan L. Kempher, Xuanyu Tao, Liqun Zhang, Honghua Ge, Yong‐Xing He,
Tópico(s)Plant tissue culture and regeneration
ResumoThe production of secondary metabolites is a major mechanism used by beneficial rhizobacteria to antagonize plant pathogens. These bacteria have evolved to coordinate the production of different secondary metabolites due to the heavy metabolic burden imposed by secondary metabolism. However, for most secondary metabolites produced by bacteria, it is not known how their biosynthesis is coordinated. Here, we showed that PhlH from the rhizobacterium Pseudomonas fluorescens is a TetR-family regulator coordinating the expression of enzymes related to the biosynthesis of several secondary metabolites, including 2,4-diacetylphloroglucinol (2,4-DAPG), mupirocin, and pyoverdine. We present structures of PhlH in both its apo form and 2,4-DAPG-bound form and elucidate its ligand-recognizing and allosteric switching mechanisms. Moreover, we found that dissociation of 2,4-DAPG from the ligand-binding domain of PhlH was sufficient to allosterically trigger a pendulum-like movement of the DNA-binding domains within the PhlH dimer, leading to a closed-to-open conformational transition. Finally, molecular dynamics simulations confirmed that two distinct conformational states were stabilized by specific hydrogen bonding interactions and that disruption of these hydrogen bonds had profound effects on the conformational transition. Our findings not only reveal a well-conserved route of allosteric signal transduction in TetR-family regulators but also provide novel mechanistic insights into bacterial metabolic coregulation. The production of secondary metabolites is a major mechanism used by beneficial rhizobacteria to antagonize plant pathogens. These bacteria have evolved to coordinate the production of different secondary metabolites due to the heavy metabolic burden imposed by secondary metabolism. However, for most secondary metabolites produced by bacteria, it is not known how their biosynthesis is coordinated. Here, we showed that PhlH from the rhizobacterium Pseudomonas fluorescens is a TetR-family regulator coordinating the expression of enzymes related to the biosynthesis of several secondary metabolites, including 2,4-diacetylphloroglucinol (2,4-DAPG), mupirocin, and pyoverdine. We present structures of PhlH in both its apo form and 2,4-DAPG-bound form and elucidate its ligand-recognizing and allosteric switching mechanisms. Moreover, we found that dissociation of 2,4-DAPG from the ligand-binding domain of PhlH was sufficient to allosterically trigger a pendulum-like movement of the DNA-binding domains within the PhlH dimer, leading to a closed-to-open conformational transition. Finally, molecular dynamics simulations confirmed that two distinct conformational states were stabilized by specific hydrogen bonding interactions and that disruption of these hydrogen bonds had profound effects on the conformational transition. Our findings not only reveal a well-conserved route of allosteric signal transduction in TetR-family regulators but also provide novel mechanistic insights into bacterial metabolic coregulation. With the increasing demand for crop production, contamination of soil and water by chemical fertilizers and pesticides has become a challenging global environmental problem (1Tengerdy R.P. Szakács G. Perspectives in agrobiotechnology.J. Biotechnol. 1998; 66: 91-99Crossref PubMed Scopus (29) Google Scholar). The use of plant growth–promoting rhizobacteria (PGPR), which are biocontrol agents that colonize plant roots and suppress the growth of phytopathogens, has been proven to be an environment-friendly and sustainable way of increasing crop yields (2Haas D. Défago G. Biological control of soil-borne pathogens by fluorescent pseudomonads.Nat. Rev. Microbiol. 2005; 3: 307-319Crossref PubMed Scopus (1773) Google Scholar, 3Siddiqui Z.A. PGPR: Biocontrol and Biofertilization. Springer, Dordrecht2006Crossref Scopus (42) Google Scholar). The production of secondary metabolites, such as antibiotics and siderophores, is a major mechanism used by PGPR to antagonize plant pathogens (4Haas D. Keel C. Regulation of antibiotic production in root-colonizing Peudomonas spp. And relevance for biological control of plant disease.Annu. Rev. Phytopathol. 2003; 41: 117-153Crossref PubMed Scopus (601) Google Scholar, 5Lucke M. Correa M.G. Levy A. The role of secretion systems, effectors, and secondary metabolites of beneficial rhizobacteria in interactions with plants and microbes.Front. Plant Sci. 2020; 11: 589416Crossref PubMed Scopus (37) Google Scholar). Typically, a PGPR strain has the potential to produce multiple secondary metabolites via distinct metabolic pathways involving different gene clusters. As secondary metabolism is a very energy-consuming process and exerts a fitness cost to the producers, spontaneous mutants that do not secrete the secondary metabolites can invade the population by out-competing the WT strains and eventually lead to the population collapse (6Diggle S.P. Griffin A.S. Campbell G.S. West S.A. Cooperation and conflict in quorum-sensing bacterial populations.Nature. 2007; 450: 411-414Crossref PubMed Scopus (592) Google Scholar). To circumvent this problem, bacteria usually coordinate the biosynthetic processes of different secondary metabolites in response to their physiological status and various environmental cues, i.e., promote the coproduction of certain secondary metabolites while inhibiting the production of another (7Tsunematsu Y. Ishikawa N. Wakana D. Goda Y. Noguchi H. Moriya H. et al.Distinct mechanisms for spiro-carbon formation reveal biosynthetic pathway crosstalk.Nat. Chem. Biol. 2013; 9: 818-825Crossref PubMed Scopus (101) Google Scholar). In this way, bacteria can save its energy by only producing the secondary metabolites that are useful at a specific time, thereby preventing the invasion of the secondary metabolite nonproducing mutants and maintaining the stability of the whole bacterial population. However, in most cases, it is not very clear how PGPR coordinates the production of different secondary metabolites, and a better understanding of this process will aid in designing and developing more effective and potent biocontrol agents in agricultural practice. Pseudomonas fluorescens is a well-known PGPR that antagonizes invading phytopathogens by producing a wide array of secondary metabolites such as 2,4-diacetylphloroglucinol (2,4-DAPG), mupirocin, pyoluteorin, pyrrolnitrin, rhizoxin analogs, orfamide A, hydrogen cyanide, and pyoverdine (4Haas D. Keel C. Regulation of antibiotic production in root-colonizing Peudomonas spp. And relevance for biological control of plant disease.Annu. Rev. Phytopathol. 2003; 41: 117-153Crossref PubMed Scopus (601) Google Scholar, 8El-Sayed A.K. Hothersall J. Cooper S.M. Stephens E. Simpson T.J. Thomas C.M. Characterization of the mupirocin biosynthesis gene cluster from Pseudomonas fluorescens NCIMB 10586.Chem. 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Thomashow L.S. Farrand S.K. Activation of the phz operon of Pseudomonas fluorescens 2-79 requires the LuxR homolog PhzR, N-(3-OH-Hexanoyl)-L-homoserine lactone produced by the LuxI homolog PhzI, and a cis-acting phz box.J. Bacteriol. 2005; 187: 6517-6527Crossref PubMed Scopus (76) Google Scholar). More importantly, the GacA/GacS two-component system plays a major role in posttranscriptionally regulating the production of secondary metabolites of P. fluorescens, although the inducing signal of GacA/GacS is still unknown (11Latour X. The evanescent GacS signal.Microorganisms. 2020; https://doi.org/10.3390/microorganisms8111746Crossref Scopus (18) Google Scholar, 12Laville J. Voisard C. Keel C. Maurhofer M. Défago G. Haas D. Global control in Pseudomonas fluorescens mediating antibiotic synthesis and suppression of black root rot of tobacco.Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1562-1566Crossref PubMed Scopus (304) Google Scholar). Metabolic coregulation has been reported to operate between the 2,4-DAPG and pyoluteorin biosynthesis, but the coordination mechanism for the production of other secondary metabolites is still not well understood. Among the diverse secondary metabolites produced by P. fluorescens, 2,4-DAPG is a major contributor to the suppression of soil-borne pathogens including Fusarium oxysporum, Septoria tritici, Thielaviopsis basicola, and Rhizoctonia solani (13Raaijmakers J.M. Weller D.M. Exploiting genotypic diversity of 2,4-diacetylphloroglucinol-producing Pseudomonas spp.: characterization of superior root-colonizing P. Fluorescens strain Q8r1-96.Appl. Environ. Microbiol. 2001; 67: 2545-2554Crossref PubMed Scopus (197) Google Scholar, 14Raaijmakers J.M. Weller D.M. Natural plant protection by 2,4-diacetylphloroglucinol-producing Pseudomonas spp. In take-all decline soils.Mol. Plant Microbe Interact. 1998; 11: 144-152Crossref Scopus (386) Google Scholar). Two pairs of oppositely transcribed operons, phlF-phlACBDE and phlG-phlH, are involved in 2,4-DAPG biosynthesis (15Bangera M.G. Thomashow L.S. Identification and characterization of a gene cluster for synthesis of the polyketide antibiotic 2, 4-diacetylphloroglucinol from Pseudomonas fluorescens Q2-87.J. Bacteriol. 1999; 181: 3155-3163Crossref PubMed Google Scholar). The phlABCD gene cluster encodes catalytic enzymes that convert malonyl-CoA into 2,4-DAPG through multiple steps (16Achkar J. Xian M. Zhao H. Frost J.W. Biosynthesis of phloroglucinol.J. Am. Chem. Soc. 2005; 127: 5332-5333Crossref PubMed Scopus (139) Google Scholar, 17Pavkov-Keller T. Schmidt N.G. Żądło-Dobrowolska A. Kroutil W. Gruber K. Structure and catalytic mechanism of a bacterial friedel-crafts acylase.Chembiochem. 2019; 20: 88-95Crossref PubMed Scopus (21) Google Scholar), while phlE encodes a transmembrane permease implicated in 2,4-DAPG resistance (18Abbas A. McGuire J.E. Crowley D. Baysse C. Dow M. O'Gara F. The putative permease PhlE of Pseudomonas fluorescens F113 has a role in 2,4-diacetylphloroglucinol resistance and in general stress tolerance.Microbiology. 2004; 150: 2443-2450Crossref PubMed Scopus (46) Google Scholar). The TetR-family regulator PhlF transcriptionally represses the expression of the 2,4-DAPG biosynthetic operon phlACBDE, and this repression is released by 2,4-DAPG, resulting in the positive-feedback regulation of 2,4-DAPG biosynthesis (19Schnider-Keel U. Seematter A. Maurhofer M. Blumer C. Duffy B. Gigot-Bonnefoy C. et al.Autoinduction of 2,4-diacetylphloroglucinol biosynthesis in the biocontrol agent Pseudomonas fluorescens CHA0 and repression by the bacterial metabolites salicylate and pyoluteorin.J. Bacteriol. 2000; 182: 1215-1225Crossref PubMed Scopus (269) Google Scholar). The PhlH regulator, which also belongs to the TetR family, regulates the expression of the 2,4-DAPG hydrolase phlG in response to 2,4-DAPG, thus providing a negative-feedback regulation of 2,4-DAPG biosynthesis (20Yan X. Yang R. Zhao R.-X. Han J.-T. Jia W.-J. Li D.-Y. et al.Transcriptional regulator PhlH modulates 2,4-diacetylphloroglucinol biosynthesis in response to the biosynthetic intermediate and end product.Appl. Environ. Microbiol. 2017; 83e01419-17Crossref Scopus (24) Google Scholar). This regulatory mechanism is likely to be crucial for the competitive root colonization of P. fluorescens, since triggering 2,4-DAPG degradation in response to its intracellular concentration could relieve the metabolic burden caused by 2,4-DAPG biosynthesis (21Yu X.-Q. Yan X. Zhang M.-Y. Zhang L.-Q. He Y.-X. Flavonoids repress the production of antifungal 2,4-DAPG but potentially facilitate root colonization of the rhizobacterium Pseudomonas fluorescens.Environ. Microbiol. 2020; 22: 5073-5089Crossref PubMed Scopus (12) Google Scholar). Like other TetR-family regulators, PhlH contains an N-terminal DNA-binding domain (DBD) and a larger C-terminal ligand-binding domain (LBD). In contrast to the conserved DBD, the LBD of PhlH exhibits little sequence identity with other well-characterized TetR-family regulators, consistent with the notion that the ligands sensed by TetR-family regulators show considerable diversity. More recently, PhlH was also found to recognize several plant-derived flavonoids including phloretin, which reduced the production of 2,4-DAPG in P. fluorescens (21Yu X.-Q. Yan X. Zhang M.-Y. Zhang L.-Q. He Y.-X. Flavonoids repress the production of antifungal 2,4-DAPG but potentially facilitate root colonization of the rhizobacterium Pseudomonas fluorescens.Environ. Microbiol. 2020; 22: 5073-5089Crossref PubMed Scopus (12) Google Scholar). Therefore, it appears that PhlH can sense molecular signals from both bacteria and plants and precisely balance the costs and benefits of 2,4-DAPG production in complex and dynamic niches. However, little is known regarding the mechanism on how PhlH recognizes its cognate molecular signals and what the physiological consequences of these signals are. Here using quantitative proteomics, we found the TetR-family regulator PhlH from P. fluorescens was involved in coordinating the expression of proteins involved in the biosynthesis of 2,4-DAPG, mupirocin, and pyoverdine. More importantly, we presented the crystal structures of P. fluorescens PhlH in both its apo form and 2,4-DAPG–bound form, illustrating its ligand-recognizing and allosteric switching mechanisms. Our work not only reveals a novel role of PhlH in coordinating different secondary metabolic pathways but provides important mechanistic insights into the ligand-triggered conformational switching of TetR-family regulators. To explore the physiological role of PhlH, we performed quantitative protein expression profiling of the WT and phlH in-frame deletion mutant (ΔphlH) strains. Differentially expressed proteins were screened based on the criteria of fold change greater than 1.5 and p-value less than 0.05, leading to the identification of 29 upregulated and 45 downregulated proteins in the ΔphlH strain (Fig. 1A and Table S1). Based on Gene Ontology enrichment analysis, differentially expressed proteins involved in antibiotic biosynthesis and porin activity were significantly enriched (p < 0.010; Fisher's exact test) in ΔphlH (Fig. 1B). The expression of the DAPG hydrolase PhlG increased over 32-fold in the ΔphlH strain compared to the WT strain, consistent with our previous finding that PhlH acts as a repressor of the phlG gene (20Yan X. Yang R. Zhao R.-X. Han J.-T. Jia W.-J. Li D.-Y. et al.Transcriptional regulator PhlH modulates 2,4-diacetylphloroglucinol biosynthesis in response to the biosynthetic intermediate and end product.Appl. Environ. Microbiol. 2017; 83e01419-17Crossref Scopus (24) Google Scholar). Interestingly, proteins involved in pyoverdine biosynthesis including PvdA, PvdD, PvdE, PvdH, PvdL, and MbtH (22Ochsner U.A. Wilderman P.J. Vasil A.I. Vasil M.L. GeneChip expression analysis of the iron starvation response in Pseudomonas aeruginosa: identification of novel pyoverdine biosynthesis genes.Mol. Microbiol. 2002; 45: 1277-1287Crossref PubMed Scopus (331) Google Scholar, 23Drake E.J. Cao J. Qu J. Shah M.B. Straubinger R.M. Gulick A.M. The 1.8 A crystal structure of PA2412, an MbtH-like protein from the pyoverdine cluster of Pseudomonas aeruginosa.J. Biol. Chem. 2007; 282: 20425-20434Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) were upregulated in the ΔphlH strain, though to a lesser extent. In addition, the ΔphlH strain showed decreased expression of an acyl-CoA dehydrogenase implicated in catalyzing a step of fatty acid β-oxidation (24Wells G. Palethorpe S. Pesci E.C. PsrA controls the synthesis of the Pseudomonas aeruginosa quinolone signal via repression of the FadE homolog, PA0506.PLoS One. 2017; 12e0189331Crossref Scopus (19) Google Scholar) and 12 proteins involved in mupirocin biosynthesis, including MmpA, MmpC, MmpD, MupC, MupE, MupJ, MupK, MupP, MupQ, MupV, MupW, and MupZ(8); however, these proteins only had fold changes around 1.5, suggesting that PhlH indirectly influence fatty acid β-oxidation and production of pyoverdine and mupirocin. To investigate how PhlH recognizes its cognate ligands, we solved the crystal structure of PhlH in a complex with 2,4-DAPG at 2.1 Å resolution. Similar to typical TetR-family regulators, PhlH forms a homodimer with each subunit consisting of a DBD (helices α1-α3) and an LBD (helices α4-α9). The DBD of PhlH contains a canonical DNA-binding helix-turn-helix motif (helices α2 and α3) with the α3 helix recognizing DNA. The two LBDs mediate dimerization through the α6 helix and α8-α9 helices, which form a 4-helix bundle in the dimer. A DALI search revealed that PhlH has the highest structural similarity with phloretin-bound TtgR from Pseudomonas putida (PDB code: 2UXI), with a Z-score of 5.8 and root mean square deviation (RMSD) of 4.44 Å for the 268 equivalent Cα positions, despite the fact that PhlH shares little sequence identity (∼18%) with TtgR. Notably, the distance between the DNA recognition helices (α3, residues 54–57) is ∼46 Å, which is significantly larger than the 34 Å major groove distance of B-DNA, confirming that 2,4-DAPG is an allosteric ligand precluding PhlH from binding DNA. A prominent feature of PhlH is a long interior tunnel-like cavity, surrounded by helices α4, α5, α6, α7, and α8, in a vertical orientation with respect to the axis of the dimer (Fig. 2A). The tunnel is ∼30 Å long and fully buried, with mainly hydrophobic residues lining the tunnel and pointing their side chains toward the lumen. The ligands 2,4-DAPG are well-defined and located at both ends of the tunnel. Consistent with the hydrophobic nature of 2,4-DAPG, all residues involved in 2,4-DAPG binding are hydrophobic, including V80, L85, A145, L148, A169, and F173 in the α4, α7, and α8 helices from one protomer and F178′, L186′, V190′ in the α8 helix in the other protomer (Fig. 2B). Of note, the residue F173 interacts with 2,4-DAPG via π-π stacking, which could be essential for the binding affinity between PhlH and 2,4-DAPG. The fact that the interior tunnel of PhlH is not fully occupied by 2,4-DAPG suggests that PhlH should have the potential to accommodate other hydrophobic ligands of larger sizes. Indeed, PhlH was recently reported to recognize several plant-derived flavonoids including phloretin (21Yu X.-Q. Yan X. Zhang M.-Y. Zhang L.-Q. He Y.-X. Flavonoids repress the production of antifungal 2,4-DAPG but potentially facilitate root colonization of the rhizobacterium Pseudomonas fluorescens.Environ. Microbiol. 2020; 22: 5073-5089Crossref PubMed Scopus (12) Google Scholar). Despite multiple attempts, no cocrystals of PhlH and flavonoids were obtained. Instead, we used the molecular docking method to interrogate the interaction mode of PhlH and phloretin (Fig. 2C). It was revealed that the phloroglucinol moiety of phloretin almost overlapped with the 2,4-DAPG molecule, suggesting a very similar binding mode shared between phloretin and 2,4-DAPG. However, as the phloroglucinol moiety lacks two acetyl groups compared to 2,4-DAPG, it makes fewer contacts with the hydrophobic residues involved in 2,4-DAPG binding, including F173, L186, and V190. This may explain the weaker binding affinity of phloretin with PhlH compared to 2,4-DAPG (21Yu X.-Q. Yan X. Zhang M.-Y. Zhang L.-Q. He Y.-X. Flavonoids repress the production of antifungal 2,4-DAPG but potentially facilitate root colonization of the rhizobacterium Pseudomonas fluorescens.Environ. Microbiol. 2020; 22: 5073-5089Crossref PubMed Scopus (12) Google Scholar). In addition, the phenol moiety of phloretin points to the interior of the PhlH ligand-binding tunnel, making contact with L119, L122, L177, and I181, due to the long, hydrophobic nature of the PhlH interior tunnel. To evaluate the role of the hydrophobic ligand-binding tunnel in PhlH–DNA association, single-site mutants of the ligand-binding residues (V80A, L85A, L148A, F173A, F178A, L186A, and V190A) were constructed, and electrophoretic mobility shift assays (EMSAs) were performed to monitor the ligand-induced protein–DNA dissociation. Mutated residues involved in ligand binding can decrease the affinity of PhlH and a ligand, thus leading to the increased strength of PhlH–DNA interactions in the presence of ligands. As shown in Figure 3, the L85A, L186A, F173A, and V190A mutants displayed increased binding affinities to the phlG operator sequence in the presence of 2,4-DAPG, indicating that these four residues are important for binding 2,4-DAPG. Using isothermal titration calorimetry (ITC) assay, it was further confirmed that these mutants had significantly reduced binding affinities toward 2,4-DAPG compared to the WT PhlH (Table 1 and Fig. S1). For the V80A, L148A, and F178A mutants, 2,4-DAPG could still trigger protein–DNA unbinding, but to a lesser extent, compared with the case of the WT PhlH, suggesting these three residues played a less prominent role in binding 2,4-DAPG. On the other hand, phloretin showed little induction of DNA dissociation for the L85A and L186A mutants and slightly compromised induction effects for the V80A and L148A mutants (Fig. 3), supporting our docking result that phloretin and 2,4-DAPG occupy the same hydrophobic tunnel of PhlH. However, the F173A and V190A mutants still showed phloretin-induced DNA dissociation, consistent with the docking result that these two residues had weaker interactions with phloretin than with 2,4-DAPG. Interestingly, phloretin barely induced DNA dissociation from the F178 mutant, indicating that the F178 residue plays a more significant role in binding phloretin than 2,4-DAPG. Collectively, these results confirmed that the ligand-binding tunnel is essential for ligand-induced DNA dissociation from PhlH but also suggested slightly different binding modes for different ligands such as 2,4-DAPG and phloretin.Table 1ITC analysis of the interactions between PhlH mutant proteins and 2,4-DAPGProteinKa (M−1)Kd (μΜ)ΔG (kcal mol−1)ΔH (kcal mol−1)TΔS (kcal mol−1)PhlH1.17 × 1058.5−6.8−12.2−5.4F173A4.94 × 10420.2−6.5−44.0−37.5L85A2.56 × 10439−6.0−5.30.7L186A6.50 × 10415.4−6.5−2.14.4V190A4.12 × 10424.3−6.3−4.81.5H76A9.14 × 10410.9−6.7−4.12.6R124A7.54 × 10413.2−7.0−34.5−27.5 Open table in a new tab To unravel the ligand-induced allosteric switching mechanism of PhlH, we solved the crystal structure of PhlH in the apo form at 2.4 Å resolution. Superposition of the apo-PhlH and 2,4-DAPG-bound PhlH dimers resulted in an RMSD of 3.0 Å for 306 Cα atoms, indicating a large conformational change between these two structures (Fig. 4A). A further inspection revealed that the LBD showed a much larger conformational change than the DBD (RMSD of 0.7 Å for 97 Cα atoms versus 0.28 Å for 42 Cα atoms) upon 2,4-DAPG binding (Fig. 4B). In the absence of 2,4-DAPG, the α7 helix and α8 helix which participate in 2,4-DAPG binding were partially unfolded in the N terminus and C terminus, respectively, whereas upon 2,4-DAPG binding, these two helices became intact, well-folded long α helices, this was most likely due to their hydrophobic interaction with 2,4-DAPG via residues F173, L186, L191, and V190. It seemed that 2,4-DAPG acted as a hydrophobic core that facilitated proper folding of the α7 and α8 helices, and these helices were essential for 2,4-DAPG binding. Notably, the long α4 helix comprising residues (K64-R91) in the apo-PhlH structure kinked and was drawn closer to 2,4-DAPG in the 2,4-DAPG-bound structure, with residue Val80 forming a hydrophobic interaction with 2,4-DAPG. This led to the displacement of the α4 helix, which acted like a pendulum that forced the DNA recognition helix α3 to move in the same direction. Further analysis of the PhlH dimeric assembly revealed that 2,4-DAPG binding resulted in a larger dimeric interface of ∼2250 Å2, compared with the buried interface of ∼1850 Å2 in the apo structure, indicating 2,4-DAPG binding not only influenced the conformation of the PhlH protomer but had a substantial impact on the mode of dimeric assembly as well. The increased dimeric interface was mostly attributed to tighter α6-α6 packing (Fig. 4A), suggesting that the intersubunit coupling was strengthened upon 2,4-DAPG binding. As a consequence, the 2,4-DAPG-bound PhlH dimer displayed a DBD-open conformation, with 46 Å distance between the two DNA recognition helices α3, whereas the apo-PhlH dimer showed a DBD-closed conformation, with the separation distance of the α3 helices perfectly matching the 34 Å separation between the major grooves of an ideal B-DNA (Fig. 4A). The above structural comparison suggests that 2,4-DAPG mechanically triggers a closed to open conformational transition of PhlH. It is intriguing that the ligand-free form of PhlH exhibits a closed, DNA-binding competent configuration, which is in sharp contrast with the previous notion that the vast majority of available TetR-family crystal structures including TetR, with no effector bound, correspond to the open conformation (25Yu Z. Reichheld S.E. Savchenko A. Parkinson J. Davidson A.R. A comprehensive analysis of structural and sequence conservation in the TetR family transcriptional regulators.J. Mol. Biol. 2010; 400: 847-864Crossref PubMed Scopus (104) Google Scholar, 26Cuthbertson L. Nodwell J.R. The TetR family of regulators.Microbiol. Mol. Biol. Rev. 2013; 77: 440-475Crossref PubMed Scopus (329) Google Scholar, 27Lara J. Diacovich L. Trajtenberg F. Larrieux N. Malchiodi E.L. Fernández M.M. et al.Mycobacterium tuberculosis FasR senses long fatty acyl-CoA through a tunnel and a hydrophobic transmission spine.Nat. Commun. 2020; 11: 3703Crossref PubMed Scopus (9) Google Scholar). For those TetR-family regulators, the presence of cognate operator DNA rather than ligand dissociation induces an open to closed conformational switching. Therefore, PhlH is considerably different from these TetR-family regulators in the aspect that dissociation of 2,4-DAPG from the LBD was sufficient to allosterically trigger a pendulum-like movement of the DBD within the PhlH dimer, leading to a conformational transition from closed to open. Since PhlH adopts two distinct conformations, we speculated that specific hydrogen-bonding interactions may be involved in stabilizing these two separate conformations and may play an important role in the allosteric signal transduction. Consistently, in the 2,4-DAPG bound, DBD-open conformation, the H76 residue in the α4 helix forms a hydrogen bond with K144 in the α7 helix, whereas in the ligand-free, DBD-closed conformation, this interaction was completely disrupted. Additionally, the side-chain of the R124 residue from the α6 helix seems to be involved in maintaining the closed conformation by forming hydrogen bonds with the main-chain carboxyls of A34 from the α1 helix, I181 from the α8 helix, and L127 from the α6′ helix, yet it is dislocated and only forms hydrogen bonds with the main-chain carboxyls of A34 and E35 in the 2,4-DAPG bound, open conformation (Fig. 4C). Multiple sequence alignment further indicates the H76, K144, and R124 residues are well-conserved in the PhlH proteins from different Pseudomonas species (Fig. S2), suggesting that the abovementioned hydrogen bonding interactions are likely to take an important part in maintaining the ligand-binding and the DNA-binding conformations of PhlH. To investigate the conformational dynamics of PhlH, all atom molecular dynamics (MD) were performed on the structure of 2,4-DAPG-bound PhlH for 1000 ns. To characterize the low-energy conformational states of the simulation system, we computed the potential of mean force (PMF) free energy profiles (Fig. 5). The separation distance between the two DNA recognition helices α3 (dH3), H76:ND1-K144:NZ distance (dHK) were selected as reaction coordinates. The 2D PMF profile revealed a single low-energy state (dH3 = ∼47 Å, dHK= ∼2.8 Å). This conformational state corresponds to the open, DNA binding-incompetent conformation, as indicated by the 47 Å distance between the α3 helices (Fig. 5A). In this low-energy state, the hydrogen binding between H76 and K144 is preserved, corroborating our hypothesis that these two residues are essential for locking PhlH in the open conformation. To further explore the dynamics of the conformational transition, we removed the 2,4-DAPG molecule from the structure and performed a 1000 ns MD simulation. Interestingly, the 2D PMF profile revealed an additional low energy state (dH3 = ∼47 Å, dHK= ∼5.0 Å), which corresponds to an intermediate, DNA binding-incompetent state, with the H76-K144 hydrogen-bond interrupted (Fig. 5B). This suggests that dissociation of DAPG first results in disruption of the H76-K144 interaction and may subsequently trigger a pendulum-like movement of the DBDs. To sample a larger conformational space of PhlH with 2,4-DAPG removed, we performed a 1000 ns gaussian accelerated molecular dynamics (GaMD), an enhanced sampling computational technique that can accelerate simulations by orders of magnitude (28Miao Y. Feher V.A. McCammon J.A. Gaussian acceler
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