(C) PLOS One
This story was originally published by PLOS One and is unaltered.
. . . . . . . . . .



Structural and functional characterization of AfsR, an SARP family transcriptional activator of antibiotic biosynthesis in Streptomyces [1]

['Yiqun Wang', 'State Key Laboratory Of Microbial Metabolism', 'School Of Life Sciences', 'Biotechnology', 'Shanghai Jiao Tong University', 'Shanghai', 'Xu Yang', 'Feng Yu', 'Zixin Deng', 'Shuangjun Lin']

Date: 2024-03

Streptomyces antibiotic regulatory proteins (SARPs) are widely distributed activators of antibiotic biosynthesis. Streptomyces coelicolor AfsR is an SARP regulator with an additional nucleotide-binding oligomerization domain (NOD) and a tetratricopeptide repeat (TPR) domain. Here, we present cryo-electron microscopy (cryo-EM) structures and in vitro assays to demonstrate how the SARP domain activates transcription and how it is modulated by NOD and TPR domains. The structures of transcription initiation complexes (TICs) show that the SARP domain forms a side-by-side dimer to simultaneously engage the afs box overlapping the −35 element and the σ HrdB region 4 (R4), resembling a sigma adaptation mechanism. The SARP extensively interacts with the subunits of the RNA polymerase (RNAP) core enzyme including the β-flap tip helix (FTH), the β′ zinc-binding domain (ZBD), and the highly flexible C-terminal domain of the α subunit (αCTD). Transcription assays of full-length AfsR and truncated proteins reveal the inhibitory effect of NOD and TPR on SARP transcription activation, which can be eliminated by ATP binding. In vitro phosphorylation hardly affects transcription activation of AfsR, but counteracts the disinhibition of ATP binding. Overall, our results present a detailed molecular view of how AfsR serves to activate transcription.

Here, we report cryo-electron microscopy (cryo-EM) structures of the SARP-transcription initiation complex (TIC), elucidating a transcription-activating mechanism that could be generally used by SARP family activators. We find AfsR uses its SARP domain to engage the afs box and make extensive interactions with all RNAP subunits except ω subunit. We confirm the inhibitory effect of NOD and TPR on SARP transcription activation by in vitro assays. The inhibitory effect can be eliminated by ATP binding, but phosphorylation of AfsR counteracts the disinhibition by ATP. Overall, we present a detailed molecular view of how AfsR activates transcription.

Due to the presence of the NOD domain, AfsR is also classified as the signal transduction ATPases with numerous domains (STAND) family [ 25 ]. Representative examples of STAND include APAF1/CED4 that regulates apoptosis [ 26 ] and plant resistance proteins that respond to pathogens and stress [ 27 ], as well as bacterial transcriptional activator MalT in Escherichia coli [ 12 , 28 , 29 ]. STAND proteins are presumed to keep in a monomeric inactive state by arm-based NOD–sensor autoinhibitory interactions or inhibitor binding in the absence of cognate inducer molecules [ 26 , 30 – 32 ]. Activation involves a multistep process of inducer binding, autoinhibition release, nucleotide exchange, and conformational changes that switch the protein to an active state [ 33 ]. However, the mechanisms by which AfsR regulates itself and activates transcription are still obscure.

The RNAP holoenzyme consists of a dissociable σ subunit that establishes specific interactions with the −35 and −10 elements of the promoter to initiate transcription. The positioning of RNAP is primarily influenced by the contacts between σ region 4 (R4) and the −35 element, while the formation of an open complex is driven by the contacts between σ region 2 (R2) and the −10 element [ 16 , 17 ]. The afsS promoter comprises a suboptimal −10 element (CACTGT) and a poor −35 element (TTCAGC). This characteristic is commonly observed in streptomycete promoters [ 18 , 19 ], which complicates the identification of the promoters. The 22-bp afs box, centered at position −29.5 relative to the transcription start site (TSS) in the afsS promoter, overlaps with the spacer between the −10 and −35 elements. It is composed of two 11-bp direct repeats (DRs), with the upstream DR (−40 to −30) overlapping with the −35 element (−38 to −33) [ 20 , 21 ]. The catabolite activator protein (CAP), also known as the cAMP receptor protein (CRP), is an extensively studied transcription factor. The 22-bp CAP binding box (cap box) is centered at –61.5 in class I promoters and at –41.5 in class II promoters [ 22 – 24 ]. The binding position of AfsR suggests that it may possess a distinct activation mechanism.

Streptomyces coelicolor AfsR is a pleiotropic, global regulator of both primary and secondary metabolism belonging to the “large” SARP group, containing an SARP (Met1-Ala270) domain, a conserved approximately 35 kDa dubbed nucleotide-binding oligomerization domain (NOD) (Ala271 to Glu618), an arm domain (Arg619-Glu777) as well as a tentative tetratricopeptide repeats (TPRs) sensor domain (Asp778-Arg993) [ 9 ] ( Fig 1A ). AfsR is found for its vital role in antibiotic biosynthesis. AfsR null mutants show defects in afsS transcription, as well as production deficiencies in actinorhodin and undecylprodigiosin [ 10 ]. It is presumed that upon binding of AfsR to a 22-base pair (bp) binding box (afs box), RNA polymerase (RNAP) holoenzyme is recruited to the afsS promoter, allowing for transcriptional initiation [ 9 ]. AfsS serves as a master regulator of secondary metabolism and nutritional stress response [ 11 ]. AfsR-AfsS system is widely distributed among Streptomyces, and expressing AfsR in the heterologous hosts can awaken silent antibiotic production genes [ 12 ]. Additionally, AfsR and the master regulator PhoP bind to overlapping sequences within PhoR-PhoP regulon promoters, such as pstS, phoRP, and glnR, exerting crosstalking regulatory control over the response to phosphate and nitrogen scarcity [ 13 , 14 ] ( Fig 1B ). AfsR can be phosphorylated by several serine/threonine kinases including AfsK, AfsL, and PkaG, and achieves better DNA binding affinity [ 12 , 15 ]. The full-length AfsR, as well as truncations containing only the SARP domain or lacking the TPR domain, can bind to afs box and activate afsS transcription in vitro [ 9 ]. However, the NOD is essential for the functionality of AfsR in vivo [ 10 ].

(A) Domain structures of SARPs. The N-terminal ODB together with the following BTA is referred as an SARP domain. S. coelicolor AfsR is a large SARP with additional NOD and TPR domains. (B) Hypothetical scheme for the regulation of S. coelicolor AfsR. (C) Fluorescence polarization assays of the SARP with afs box. Error bars represent mean ± SEM of n = 3 experiments. (D) Transcription assays with increasing concentrations (62.5 nM, 125 nM, 250 nM, 500 nM, 750 nM, 1,000 nM) of the SARP. CK represents the control group without the addition of the SARP. Data are presented as mean ± SEM from 3 independent assays. (E) Assembly of the SARP-TIC. The protein compositions in the dotted line boxed fractions are shown in the SDS-PAGE. The original gel image can be found in S1 Raw Images. (F) The afsS promoter fragment used for the SARP-TIC assembly. The −35 element, −10 element, the TSS, and the 6-bp noncomplementary bubble are denoted. The afs box is colored orange and contains DRup (the upstream direct repeat) and DRdown (the downstream direct repeat). The top (non-template, NT) strand and bottom (template, T) strand are colored light green and dark green, respectively. (G) Two views of cryo-EM map. The map was generated by merging the consensus map of the full SARP-TIC and the focused map of the SARP region in Chimera X. (H) Cartoon representation of the SARP-TIC structure. The subunits are colored as in the color scheme. The data underlying C, D, and E are provided in S1 Data . BTA, bacterial transcriptional activation; cryo-EM, cryo-electron microscopy; NOD, nucleotide-binding oligomerization domain; ODB, OmpR-type DNA-binding; RNAP, RNA polymerase; SARP, Streptomyces antibiotic regulatory protein; TIC, transcription initiation complex; TPR, tetratricopeptide repeat; TSS, transcription start site.

Streptomycetes are multicellular bacteria with a complex developmental cycle [ 1 ]. They produce numerous bioactive natural products, including many antibiotics with important applications in medicine and agriculture. This capability makes streptomycetes one of the most important industrial microbial genera. Complex regulatory systems tightly govern the natural product biosynthesis of streptomycetes, involving both global and pathway-specific regulators. Streptomyces antibiotic regulatory protein (SARP) family regulators include many of the pathway-specific regulators and some global regulators. They are prominent initiators and activators of natural product biosynthesis and have mainly been found in actinomycetes [ 2 ]. Recently reported members of the SARP family include ChlF2 from Streptomyces antibioticus and MilR3 from Streptomyces bingchenggensis. ChlF2 activates the production of chlorothricin, known for its anti-inflammatory properties, and MilR3 stimulates the synthesis of an excellent insecticide milbemycin [ 3 , 4 ]. SARPs feature an N-terminal OmpR-type DNA-binding (ODB) domain and a C-terminal bacterial transcriptional activation (BTA) domain, collectively referred to as the SARP domain. The ODB domain is characterized by a winged helix-turn-helix (HTH) comprising 3 α-helices and 2 antiparallel β-sheets [ 5 ], whereas the BTA domain is all-helical [ 6 ]. In contrast to “small” SARPs that only contain an SARP domain, “large” SARPs possess extra domains at their C-terminals that are hypothesized to modulate the activity of the SARP domain ( Fig 1A ). “Small” SARPs are more frequently observed and extensively studied among actinomycetes, compared to their “large” counterparts [ 7 ]. SARPs bind to the target promoters having common features. They contain direct repeats, the 3′ repeat of which is located 8 bp from the −10 element, and each repeat is separated from the adjacent repeat by 11 bp or 22 bp, corresponding to 1 or 2 complete turns of the DNA helix, respectively [ 8 ].

2. Results

SARP engages DNA and RNAP to activate transcription Despite abundant genetical and biochemical studies on SARPs, the molecular basis of SARP-based transcription activation remains unknown. The isolated SARP domain of AfsR is able to bind afs box as evidenced by fluorescence polarization assays (Fig 1C), and mutating the upstream DR (M1) or the downstream DR (M2) attenuates this specific interaction (S1 Fig), in agreement with the previous report [9]. In vitro MangoIII-based transcription assays demonstrated the SARP domain could activate the transcription of afsS promoter (Fig 1D), while it has no significant effect on a control actII-4 promoter comprising a consensus −10 and −35 element but lacking the afs box (S1C Fig). Consistent with the DNA binding results, when the DRs were mutated, the SARP domain lost much of its ability to activate transcription, especially with mutation at the downstream DR (S1C Fig). To investigate the molecular basis of SARP-based transcription activation, we combined the isolated SARP domain, a DNA scaffold, RbpA, CarD, and RNAP σHrdB-holoenzyme to assembly the initiation complex (Fig 1E). The DNA scaffold is engineered from the afsS promoter, consisting of a 44-bp (−50 to −7) upstream promoter double-stranded DNA (dsDNA) which contains the 22-bp afs box and the consensus –10 element, a 6-bp (−6 to −1) non-complementary transcription bubble, and a 15-bp (+1 to +15) downstream promoter dsDNA (Fig 1F). RbpA and CarD, 2 RNAP-binding proteins discovered in actinobacteria, can stabilize the TIC and have been found in many TIC structures of Mycobacterium tuberculosis [34–36]. Through in vitro transcription assays, we observed that SARP maintained its role as a transcriptional activator in the presence of RbpA and CarD (S2 Fig). The final cryo-EM map of the SARP-TIC was reconstructed using a total of 95,223 single particles and refined to a nominal resolution of 3.35 Å, with approximately 3 Å at the center of RNAP and approximately 6 Å at the peripheral SARP (S3 Fig and S1 Table). Local refinement focused on the SARP region generated a 3.77-Å-resolution map. In the cryo-EM maps, the densities allowed unambiguous docking of 2 SARP protomers, a 61-bp promoter DNA (−46 to +15), a σHrdB subunit except disordered region 1.1, 5 subunits of RNAP core (α 2 ββ′ω), RbpA, and CarD. In addition, the cryo-EM density map shows a clear signal for an αCTD (Fig 1G). The 2 SARP protomers simultaneously engage the afs box and RNAP σHrdB-holoenzyme. SARP-TIC is an open complex containing a transcription bubble of 13 nt. The overall structure of S. coelicolor RNAP in SARP-TIC closely resembles that in our recently reported S. coelicolor RNAPσHrdB-Zur-DNA structure (PDB ID: 7X75, rmsd of 0.334 Å for 3,002 aligned Cα atoms) [37], and other actinobacteria RNAP structures including M. tuberculosis RNAP-promoter open complex (PDB ID: 6VVY, rmsd of 1.133 Å for 2,745 aligned Cα atoms) [38] and Mycobacterium smegmatis RNAP TIC (PDB ID: 5VI5, rmsd of 1.048 Å for 2,621 aligned Cα atoms) [39] (S4 Fig). As shown in Fig 1H, σHrdB R4 is positioned on top of 2 SARP protomers, instead of binding in the major groove of the −35 element as observed in the reported structures [37–39], suggesting a sigma adaptation mechanism like M. smegmatis PafBC [40]. CarD binds the β-subunit and interacts with the upstream double-stranded/single-stranded (ds/ss) junction of the transcription bubble [41], while RbpA enhances interactions between β′, σHrdB, and promoter DNA [42].

A side-by-side SARP dimer interacts with afs box The SARP-TIC structure elucidates the binding interactions between SARP protomers and 2 major grooves of the afs box, with each protomer making contacts with 1 DR (Fig 2A). The contacts span the first 8 conserved base pairs. In contrast, the last 3 base pairs do not make any contact with SARP (Fig 2B). Overall conformations of the 2 protomers are essentially the same with an overall rmsd of 0.8 Å (S5 Fig). A dimer interface of approximately 1,120 Å2 is formed between the 2 SARP protomers, revealing a unique side-by-side arrangement (Fig 2C). Since SARP is monomeric in solution, the dimerization of SARP occurs upon DNA binding. As a representative example of SARP family transcription activators, the SARP folds into an N-terminal ODB domain (residues 25–111) and a C-terminal BTA domain (residues 120–270) (Fig 2D). The ODB domain consists of 3 α helices packed against 2 antiparallel β sheets. Two helices, α2 and α3, and the seven-residue connecting loop constitute an HTH DNA-binding motif, followed by a β hairpin that contacts the strand between the helices α1 and α2. The BTA domain comprises 7 α-helices, of which the first 3 ones stand on the first β sheet and the first 2 helices of the N-terminal ODB domain, burying an interfacial area of approximately 860 Å2. The overall structure of SARP protomers closely resembles EmbR (PDB ID: 2FEZ, rmsd of 2.8 Å for 247 aligned Cα atoms) [6], a transcriptional regulator of M. tuberculosis, except that EmbR comprises an additional C-terminal forkhead-associated (FHA) domain (S6 Fig). PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 2. Interactions of SARP with afs box. (A) Two SARP protomers bind to the afs box (−19 to −40) side-by-side with each protomer contacting 1 DR. The density map is shown as transparent surface. T -30 (nt) is the last nucleotide of DRup. Upstream and downstream SARP are colored blue and orange, respectively. (B) Conserved sequences corresponding to the 11-nt DR of the afs box generated by MEME. (C) The dimer interface between 2 SARP protomers. (D) The domain organization indicated in the downstream SARP protomer. (E) Detailed interactions of the upstream protomer with the DNA backbone. Hydrogen bonds and salt bridges are shown as yellow and red dashed lines, respectively. (F) Contacts of SARP with specific nucleotides. The residues R92 and T88 make hydrogen bonds (shown as yellow dashed lines) with O4 of T -38 (nt) and N7 of G -36 (t), respectively. (G) Sequence alignments of SARP regulators from different Streptomyces strains, highlighting the residues interacting with DNA (green). Only ODB domains were compared. These proteins include AfsR (P25941), ActII-4 (P46106), RedD (P16922) from S. coelicolor, PimR (Q70DY8) from S. natalensis, PolY (ABX24502.1), PolR (ABX24503.1) from S. asoensis, SanG (Q5IW77) from S. ansochromogenes, ChlF2 (Q0R4N4) from S. antibioticus, MilR3 (D7BZQ7) from S. bingchenggensis, CcaR (P97060) from S. clavuligerus, DnrI (P25047) from S. peucetius, MtmR (Q194R8) from S. argillaceus, and TlyS (M4ML56) from S. fradiae. The black boxes highlight the positions conserved. DR, direct repeat; ODB, OmpR-type DNA-binding; SARP, Streptomyces antibiotic regulatory protein. https://doi.org/10.1371/journal.pbio.3002528.g002 The 2 SARP protomers establish a total contact surface of approximately 1,300 Å2 with the dsDNA, resulting in a 20° bend of the helical axis of the upstream dsDNA at T -30 (nt) (-30T on the non-template strand) (S7 Fig). The DNA bending at the midpoint between the 2 binding sites suggests that both protomers equally contribute to curve the DNA. The DNA contacts made by the upstream SARP protomer were described in the following section (Fig 2E). The N-terminal end of helix α1, the C-terminal end of helix α2, and the HTH loop make extensive contacts with the backbone phosphate groups from C- 40 to T -37 of the nt-strand, involving both hydrogen bonds (S46 and Q48) and van der Waals interactions (W74, P79, S80, and Q81). The recognition helix α3 penetrates the DNA major groove and is almost perpendicular to the DNA axis. R87, R94, and K95 form salt bridges with the backbone phosphate groups from T −35 to G -33 of the t-strand. The side chain of S91 makes a hydrogen bond with the phosphate group of T −35 (t). In addition to these nonspecific contacts with backbone phosphate groups, the recognition helix α3 also establishes specific DNA contacts (Fig 2F). The R92 makes hydrogen bonds with the O4 atom of T -38 (nt) via its guanidinium group. The side chain of T88 makes a hydrogen bond with N7 of G -36 (t). Consistent with the structural observations, previous EMSAs show that changing the T- 38 (nt) of the upstream DR or the corresponding T- 27 (nt) of the downstream DR to adenosine prevents the binding of AfsR [21]. However, changing the G -36 (t) to adenosine does not impair the binding of AfsR since A -25 (t) is observed at the corresponding position of the downstream DR [21]. The 2 ODB domains make similar contacts with DNA, inserting the helix α3 of the HTH into the major groove of the DR (S8 Fig). Most residues involved in DNA interactions are highly conserved in both “small” and “large” SARP homologues across various Streptomyces strains (Fig 2G), including Q48, W74, P79, T88, R94, and K95.

[END]
---
[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002528

Published and (C) by PLOS One
Content appears here under this condition or license: Creative Commons - Attribution BY 4.0.

via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/

You are viewing proxied material from magical.fish. The copyright of proxied material belongs to its original authors. Any comments or complaints in relation to proxied material should be directed to the original authors of the content concerned. Please see the disclaimer for more details.