Structural basis of DNA binding by the WhiB-like transcription factor WhiB3 in Mycobacterium tuberculosis
2023; Elsevier BV; Volume: 299; Issue: 6 Linguagem: Inglês
10.1016/j.jbc.2023.104777
ISSN1083-351X
AutoresTao Wan, Magdaléna Horová, Vimmy Khetrapal, Shanren Li, Camden Jones, Andrew Schacht, Xinghui Sun, Limei Zhang,
Tópico(s)RNA and protein synthesis mechanisms
ResumoMycobacterium tuberculosis (Mtb) WhiB3 is an iron–sulfur cluster-containing transcription factor belonging to a subclass of the WhiB-Like (Wbl) family that is widely distributed in the phylum Actinobacteria. WhiB3 plays a crucial role in the survival and pathogenesis of Mtb. It binds to the conserved region 4 of the principal sigma factor (σA4) in the RNA polymerase holoenzyme to regulate gene expression like other known Wbl proteins in Mtb. However, the structural basis of how WhiB3 coordinates with σA4 to bind DNA and regulate transcription is unclear. Here we determined crystal structures of the WhiB3:σA4 complex without and with DNA at 1.5 Å and 2.45 Å, respectively, to elucidate how WhiB3 interacts with DNA to regulate gene expression. These structures reveal that the WhiB3:σA4 complex shares a molecular interface similar to other structurally characterized Wbl proteins and also possesses a subclass-specific Arg-rich DNA-binding motif. We demonstrate that this newly defined Arg-rich motif is required for WhiB3 binding to DNA in vitro and transcriptional regulation in Mycobacterium smegmatis. Together, our study provides empirical evidence of how WhiB3 regulates gene expression in Mtb by partnering with σA4 and engaging with DNA via the subclass-specific structural motif, distinct from the modes of DNA interaction by WhiB1 and WhiB7. Mycobacterium tuberculosis (Mtb) WhiB3 is an iron–sulfur cluster-containing transcription factor belonging to a subclass of the WhiB-Like (Wbl) family that is widely distributed in the phylum Actinobacteria. WhiB3 plays a crucial role in the survival and pathogenesis of Mtb. It binds to the conserved region 4 of the principal sigma factor (σA4) in the RNA polymerase holoenzyme to regulate gene expression like other known Wbl proteins in Mtb. However, the structural basis of how WhiB3 coordinates with σA4 to bind DNA and regulate transcription is unclear. Here we determined crystal structures of the WhiB3:σA4 complex without and with DNA at 1.5 Å and 2.45 Å, respectively, to elucidate how WhiB3 interacts with DNA to regulate gene expression. These structures reveal that the WhiB3:σA4 complex shares a molecular interface similar to other structurally characterized Wbl proteins and also possesses a subclass-specific Arg-rich DNA-binding motif. We demonstrate that this newly defined Arg-rich motif is required for WhiB3 binding to DNA in vitro and transcriptional regulation in Mycobacterium smegmatis. Together, our study provides empirical evidence of how WhiB3 regulates gene expression in Mtb by partnering with σA4 and engaging with DNA via the subclass-specific structural motif, distinct from the modes of DNA interaction by WhiB1 and WhiB7. The causative agent of tuberculosis, Mycobacterium tuberculosis (Mtb), continues to affect millions of people annually with high mortality, and the devastation caused by this pathogen is exacerbated by the ongoing COVID-19 and HIV epidemic (1Pai M. Kasaeva T. Swaminathan S. Covid-19's devastating effect on tuberculosis care - a path to recovery.N. Engl. J. Med. 2022; 386: 1490-1493Google Scholar, 2Pawlowski A. Jansson M. Skold M. Rottenberg M.E. Kallenius G. Tuberculosis and HIV co-infection.PLoS Pathog. 2012; 8e1002464Google Scholar). The survival and persistence of Mtb in the host depends on a complex regulatory system to rapidly sense and respond to various assaults launched by the host immune system, such as acidic, oxidative, and nutritional stress. The seven members of WhiB-like (Wbl) family proteins found in Mtb, namely WhiB1-7, are key components in this regulatory system. Wbl proteins are a group of small iron–sulfur cluster ([4Fe-4S])-bound proteins first discovered in Streptomyces and exclusive to Actinobacteria (3Davis N.K. Chater K.F. The Streptomyces coelicolor whiB gene encodes a small transcription factor-like protein dispensable for growth but essential for sporulation.Mol. Gen. Genet. 1992; 232: 351-358Google Scholar, 4Soliveri J.A. Gomez J. Bishai W.R. Chater K.F. Multiple paralogous genes related to the Streptomyces coelicolor developmental regulatory gene whiB are present in Streptomyces and other actinomycetes.Microbiology. 2000; 146: 333-343Google Scholar). Members of Wbl proteins play versatile and nonredundant roles in regulating biological processes and responding to various stresses Mtb encounters in the host, such as oxidative stress (WhiB1-7), cell division (WhiB2), acidic stress and nutritional starvation (WhiB3), virulence and reactivation (WhiB3, WhiB5, and WhiB6), and antibiotic resistance (WhiB2 and WhiB7) (5Gomez J.E. Bishai W.R. whmD is an essential mycobacterial gene required for proper septation and cell division.Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8554-8559Google Scholar, 6Morris R.P. Nguyen L. Gatfield J. Visconti K. Nguyen K. Schnappinger D. et al.Ancestral antibiotic resistance in Mycobacterium tuberculosis.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 12200-12205Google Scholar, 7Chen Z. Hu Y. Cumming B.M. Lu P. Feng L. Deng J. et al.Mycobacterial WhiB6 differentially regulates ESX-1 and the Dos regulon to modulate granuloma formation and virulence in Zebrafish.Cell Rep. 2016; 16: 2512-2524Google Scholar, 8Burian J. Ramon-Garcia S. Howes C.G. Thompson C.J. WhiB7, a transcriptional activator that coordinates physiology with intrinsic drug resistance in Mycobacterium tuberculosis.Expert Rev. Anti Infect. Ther. 2012; 10: 1037-1047Google Scholar). Among them, Mtb WhiB3 is one of the key global regulators involved in the early-stage response to acidic stress inside host macrophages (9Rohde K.H. Abramovitch R.B. Russell D.G. Mycobacterium tuberculosis invasion of macrophages: linking bacterial gene expression to environmental cues.Cell Host Microbe. 2007; 2: 352-364Google Scholar, 10Mehta M. Rajmani R.S. Singh A. Mycobacterium tuberculosis WhiB3 responds to vacuolar pH-induced changes in mycothiol redox potential to modulate phagosomal maturation and virulence.J. Biol. Chem. 2016; 291: 2888-2903Google Scholar). It is exploited by Mtb to maintain redox and metabolic homeostasis in response to various host-generated redox stress, acidic stress, and carbon starvation and induced by hypoxia and nitric oxide in vitro (10Mehta M. Rajmani R.S. Singh A. Mycobacterium tuberculosis WhiB3 responds to vacuolar pH-induced changes in mycothiol redox potential to modulate phagosomal maturation and virulence.J. Biol. Chem. 2016; 291: 2888-2903Google Scholar, 11Singh A. Guidry L. Narasimhulu K.V. Mai D. Trombley J. Redding K.E. et al.Mycobacterium tuberculosis WhiB3 responds to O2 and nitric oxide via its [4Fe-4S] cluster and is essential for nutrient starvation survival.Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 11562-11567Google Scholar, 12Singh A. Crossman D.K. Mai D. Guidry L. Voskuil M.I. Renfrow M.B. et al.Mycobacterium tuberculosis WhiB3 maintains redox homeostasis by regulating virulence lipid anabolism to modulate macrophage response.PLoS Pathog. 2009; 5e1000545Google Scholar, 13Saini V. Farhana A. Steyn A.J. Mycobacterium tuberculosis WhiB3: a novel iron-sulfur cluster protein that regulates redox homeostasis and virulence.Antioxid. Redox Signal. 2012; 16: 687-697Google Scholar, 14Larsson C. Luna B. Ammerman N.C. Maiga M. Agarwal N. Bishai W.R. Gene expression of Mycobacterium tuberculosis putative transcription factors whiB1-7 in redox environments.PLoS One. 2012; 7e37516Google Scholar, 15Saini V. Cumming B.M. Guidry L. Lamprecht D.A. Adamson J.H. Reddy V.P. et al.Ergothioneine maintains redox and bioenergetic homeostasis essential for drug susceptibility and virulence of Mycobacterium tuberculosis.Cell Rep. 2016; 14: 572-585Google Scholar, 16Cumming B.M. Rahman M.A. Lamprecht D.A. Rohde K.H. Saini V. Adamson J.H. et al.Mycobacterium tuberculosis arrests host cycle at the G1/S transition to establish long term infection.PLoS Pathog. 2017; 13e1006389Google Scholar, 17Mehta M. Singh A. Mycobacterium tuberculosis WhiB3 maintains redox homeostasis and survival in response to reactive oxygen and nitrogen species.Free Radic. Biol. Med. 2019; 131: 50-58Google Scholar, 18Steyn A.J. Collins D.M. Hondalus M.K. Jacobs Jr., W.R. Kawakami R.P. Bloom B.R. Mycobacterium tuberculosis WhiB3 interacts with RpoV to affect host survival but is dispensable for in vivo growth.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3147-3152Google Scholar). In Streptomyces, the WhiB3 ortholog, WhiD, is required for the late stage of sporulation (19Molle V. Palframan W.J. Findlay K.C. Buttner M.J. WhiD and WhiB, homologous proteins required for different stages of sporulation in Streptomyces coelicolor A3(2).J. Bacteriol. 2000; 182: 1286-1295Google Scholar, 20Jakimowicz P. Cheesman M.R. Bishai W.R. Chater K.F. Thomson A.J. Buttner M.J. Evidence that the Streptomyces developmental protein WhiD, a member of the WhiB family, binds a [4Fe-4S] cluster.J. Biol. Chem. 2005; 280: 8309-8315Google Scholar). Correlated with their diverse roles in regulating gene expression, Mtb Wbl proteins share only 30 to 50% sequence identity and represent five different subclasses of Wbl proteins widely distributed in Actinobacteria. Two conserved motifs are found in all Wbl proteins—a [4Fe-4S]-cluster binding domain containing four conserved Cys and a "G[I/V]W[G/A]G" motif (the invariant residues are highlighted in bold fonts and the preferred residues are underlined; the same notations are used below), which is also referred to as the β turn (Fig. S1). The C terminus of Wbl proteins has been implicated in DNA binding since many Wbl proteins feature a cluster of basic residues in this region that is predicted to be in a helix-turn-helix fold (4Soliveri J.A. Gomez J. Bishai W.R. Chater K.F. Multiple paralogous genes related to the Streptomyces coelicolor developmental regulatory gene whiB are present in Streptomyces and other actinomycetes.Microbiology. 2000; 146: 333-343Google Scholar). However, among the Wbl proteins in Mtb, only WhiB7 has a defined DNA-binding motif, the AT-hook ("RGRP"), in the C terminus. WhiB7 AT-hook preferably binds to the AT-rich sequence upstream of the −35 element in the target promoters. WhiB3 was also suggested to possess a C-terminal AT-hook–like motif (13Saini V. Farhana A. Steyn A.J. Mycobacterium tuberculosis WhiB3: a novel iron-sulfur cluster protein that regulates redox homeostasis and virulence.Antioxid. Redox Signal. 2012; 16: 687-697Google Scholar). But these basic residues in the C-terminal WhiB3 are not conserved in the WhiB3 subclass (Fig. S1), and their role in WhiB3 binding to DNA has not been verified. Even less known is the N terminus of Wbl proteins prior to the Fe–S cluster–binding motif. This region varies substantially in length and sequence among the Wbl proteins (Fig. S1) and lacks information regarding its significance to the function of Wbl proteins. Several Mtb Wbl proteins, including WhiB1, WhiB3, and WhiB7, have been shown to regulate gene expression in the [4Fe-4S]-bound (holo-) form by binding to the conserved region 4 in the σ70-family principal sigma factor σA (σA4) in the RNA polymerase (RNAP) holoenzyme (18Steyn A.J. Collins D.M. Hondalus M.K. Jacobs Jr., W.R. Kawakami R.P. Bloom B.R. Mycobacterium tuberculosis WhiB3 interacts with RpoV to affect host survival but is dispensable for in vivo growth.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3147-3152Google Scholar, 21Burian J. Yim G. Hsing M. Axerio-Cilies P. Cherkasov A. Spiegelman G.B. et al.The mycobacterial antibiotic resistance determinant WhiB7 acts as a transcriptional activator by binding the primary sigma factor SigA (RpoV).Nucleic Acids Res. 2013; 41: 10062-10076Google Scholar, 22Kudhair B.K. Hounslow A.M. Rolfe M.D. Crack J.C. Hunt D.M. Buxton R.S. et al.Structure of a Wbl protein and implications for NO sensing by M. tuberculosis.Nat. Commun. 2017; 8: 2280Google Scholar). Other Mtb Wbl proteins except WhiB5 have also been reported to bind to σA4 in a [4Fe-4S]-dependent manner (23Feng L. Chen Z. Wang Z. Hu Y. Chen S. Genome-wide characterization of monomeric transcriptional regulators in Mycobacterium tuberculosis.Microbiology (Reading). 2016; 162: 889-897Google Scholar). Moreover, a recent study has shown that the WhiB3 ortholog of Streptomyces venezuelae also binds to region 4 of the principal sigma factor σHrdB, suggesting a shared mechanism of action by Wbl proteins in Actinobacteria (24Stewart M.Y.Y. Bush M.J. Crack J.C. Buttner M.J. Le Brun N.E. Interaction of the Streptomyces Wbl protein WhiD with the principal sigma factor σHrdB depends on the WhiD [4Fe-4S] cluster.J. Biol. Chem. 2020; 295: 9752-9765Google Scholar). Recent advances in the structural and biochemical characterization of the Wbl proteins have shed light on how Wbl proteins partner with σA to regulate gene expression. Mtb WhiB1 is the first Wbl protein that has been structurally characterized at the atomic level, first in the free holo-form by nuclear magnetic resonance and subsequently in the σA4-bound form by X-ray crystallography (22Kudhair B.K. Hounslow A.M. Rolfe M.D. Crack J.C. Hunt D.M. Buxton R.S. et al.Structure of a Wbl protein and implications for NO sensing by M. tuberculosis.Nat. Commun. 2017; 8: 2280Google Scholar, 25Wan T. Li S. Beltran D.G. Schacht A. Zhang L. Becker D.F. et al.Structural basis of non-canonical transcriptional regulation by the σA-bound iron-sulfur protein WhiB1 in M. tuberculosis.Nucleic Acids Res. 2020; 48: 501-516Google Scholar). Together with the molecular and biochemical analyses, these studies reveal an unexpected molecular interface in the WhiB1:σA4 complex dominated by hydrophobic interactions and support a new molecular mechanism of transcription regulation by WhiB1 in Actinobacteria (25Wan T. Li S. Beltran D.G. Schacht A. Zhang L. Becker D.F. et al.Structural basis of non-canonical transcriptional regulation by the σA-bound iron-sulfur protein WhiB1 in M. tuberculosis.Nucleic Acids Res. 2020; 48: 501-516Google Scholar). Subsequently, the crystal structure of WhiB7 in complex with σA4 and its own promoter (PwhiB7) and the single-particle cryo-electron microscopy (cryo-EM) structure of WhiB7 bond to σA in RNAP and PwhiB7 were reported by our group and by the Campbell group, respectively (26Wan T. Horova M. Beltran D.G. Li S. Wong H.X. Zhang L.M. Structural insights into the functional divergence of WhiB-like proteins in Mycobacterium tuberculosis.Mol. Cell. 2021; 81: 2887-2900.e5Google Scholar, 27Lilic M. Darst S.A. Campbell E.A. Structural basis of transcriptional activation by the Mycobacterium tuberculosis intrinsic antibiotic-resistance transcription factor WhiB7.Mol. Cell. 2021; 81: 2875-2886.e5Google Scholar). The WhiB7 AT-hook binding site has the characteristics of A-track DNA (i.e., a short run of consecutive four or more adenine-thymine base pairs), which possesses distinct structural properties from canonical B-form DNA, including narrow minor grooves, high propeller twists, and DNA bending toward the AT-rich minor groove (see the review in (28Haran T.E. Mohanty U. The unique structure of A-tracts and intrinsic DNA bending.Q. Rev. Biophys. 2009; 42: 41-81Google Scholar)). Analysis of the 3D structures reveals the structural basis for how WhiB7 binding to the AT-rich region opens the minor groove, reversely bends the DNA in the direction opposite to the expected intrinsic bending of A-track DNAs, and orchestrates with σA for transcriptional regulation. Together, these studies provide an atomic view of how WhiB7 activates gene expression by coordinating DNA binding with σA4 via its AT-hook and unravel the WhiB7 subclass-specific structural features that enable WhiB7 to function differently from WhiB1 (26Wan T. Horova M. Beltran D.G. Li S. Wong H.X. Zhang L.M. Structural insights into the functional divergence of WhiB-like proteins in Mycobacterium tuberculosis.Mol. Cell. 2021; 81: 2887-2900.e5Google Scholar, 27Lilic M. Darst S.A. Campbell E.A. Structural basis of transcriptional activation by the Mycobacterium tuberculosis intrinsic antibiotic-resistance transcription factor WhiB7.Mol. Cell. 2021; 81: 2875-2886.e5Google Scholar). WhiB3 is one of the most extensively investigated Mtb Wbl proteins owing to its importance for the pathogenesis of Mtb (10Mehta M. Rajmani R.S. Singh A. Mycobacterium tuberculosis WhiB3 responds to vacuolar pH-induced changes in mycothiol redox potential to modulate phagosomal maturation and virulence.J. Biol. Chem. 2016; 291: 2888-2903Google Scholar, 11Singh A. Guidry L. Narasimhulu K.V. Mai D. Trombley J. Redding K.E. et al.Mycobacterium tuberculosis WhiB3 responds to O2 and nitric oxide via its [4Fe-4S] cluster and is essential for nutrient starvation survival.Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 11562-11567Google Scholar, 12Singh A. Crossman D.K. Mai D. Guidry L. Voskuil M.I. Renfrow M.B. et al.Mycobacterium tuberculosis WhiB3 maintains redox homeostasis by regulating virulence lipid anabolism to modulate macrophage response.PLoS Pathog. 2009; 5e1000545Google Scholar, 13Saini V. Farhana A. Steyn A.J. Mycobacterium tuberculosis WhiB3: a novel iron-sulfur cluster protein that regulates redox homeostasis and virulence.Antioxid. Redox Signal. 2012; 16: 687-697Google Scholar, 14Larsson C. Luna B. Ammerman N.C. Maiga M. Agarwal N. Bishai W.R. Gene expression of Mycobacterium tuberculosis putative transcription factors whiB1-7 in redox environments.PLoS One. 2012; 7e37516Google Scholar, 15Saini V. Cumming B.M. Guidry L. Lamprecht D.A. Adamson J.H. Reddy V.P. et al.Ergothioneine maintains redox and bioenergetic homeostasis essential for drug susceptibility and virulence of Mycobacterium tuberculosis.Cell Rep. 2016; 14: 572-585Google Scholar, 16Cumming B.M. Rahman M.A. Lamprecht D.A. Rohde K.H. Saini V. Adamson J.H. et al.Mycobacterium tuberculosis arrests host cycle at the G1/S transition to establish long term infection.PLoS Pathog. 2017; 13e1006389Google Scholar, 17Mehta M. Singh A. Mycobacterium tuberculosis WhiB3 maintains redox homeostasis and survival in response to reactive oxygen and nitrogen species.Free Radic. Biol. Med. 2019; 131: 50-58Google Scholar, 18Steyn A.J. Collins D.M. Hondalus M.K. Jacobs Jr., W.R. Kawakami R.P. Bloom B.R. Mycobacterium tuberculosis WhiB3 interacts with RpoV to affect host survival but is dispensable for in vivo growth.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3147-3152Google Scholar, 29You D. Xu Y. Yin B.C. Ye B.C. Nitrogen regulator GlnR Controls redox sensing and lipids anabolism by directly activating the whiB3 in Mycobacterium smegmatis.Front. Microbiol. 2019; 10: 74Google Scholar). However, neither the DNA-binding motif nor the DNA-binding preference of WhiB3 has been determined to date. It remains enigmatic how WhiB3 binds to σA4 and DNA for transcriptional regulation. Here, we report crystal structures of the σA4-bound WhiB3 alone and in complex with DNA at 1.5 Å and 2.45 Å, respectively. Together, the results from our structural, biochemical, and functional analyses uncover an essential DNA-binding motif in WhiB3 and shed light on how WhiB3 coordinates with σA4 and interacts with DNA for transcriptional regulation. By structural comparison, we provide insights into how WhiB3 functions differently from WhiB1 and WhiB7 in Mtb by binding to the same site on σA4 and utilizing the subclass-specific structural motif for DNA binding. As described in the previous studies, a chimeric protein denoted σA4-βtip was used for the crystallographic characterization of the σA4-bound WhiB3 by fusing σA4 with the RNAP β-subunit flap tip helix (βtip) via an artificial linker to mimic the interaction between σA and the β subunit in the RNAP holoenzyme (see Experimental procedures) (26Wan T. Horova M. Beltran D.G. Li S. Wong H.X. Zhang L.M. Structural insights into the functional divergence of WhiB-like proteins in Mycobacterium tuberculosis.Mol. Cell. 2021; 81: 2887-2900.e5Google Scholar, 30Blanco A.G. Canals A. Bernues J. Sola M. Coll M. The structure of a transcription activation subcomplex reveals how σ70 is recruited to PhoB promoters.EMBO J. 2011; 30: 3776-3785Google Scholar, 31Campbell E.A. Muzzin O. Chlenov M. Sun J.L. Olson C.A. Weinman O. et al.Structure of the bacterial RNA polymerase promoter specificity sigma subunit.Mol. Cell. 2002; 9: 527-539Google Scholar). We also generated a truncated WhiB3, denoted WhiB3TR, without the C-terminal residues (aa 91–102) containing the putative DNA binding motif to improve the protein stability and crystallizability. Phasing was performed using single-wavelength anomalous diffraction (SAD) of the iron–sulfur cluster containing four iron and four sulfur ions [4Fe-4S] cluster in the WhiB3:σA4-βtip complex (see Experimental procedures, Table 1 and Fig. S2). Two crystal forms were observed from the same crystallization drop, with the larger crystals in the P43212 form and the small ones in the R3 form (Fig. 1A). The final model of WhiB3:σA4-βtip was refined to 1.35 Å in the P43212 form and 1.5 Å in the R3 form.Table 1Data collection and refinement statisticsProteinsWhiB3TR:σA4-βtip P43212 (phasing)WhiB3TR:σA4-βtip P43212WhiB3TR:σA4-βtip R3WhiB3FL:σA4-βtip:PwhiB7Data collectionaThe highest resolution shell statistics are shown in parentheses. Space groupP43212P43212R3P212121 Cell dimensionsa, b, c (Å)122.2, 122.2, 115.1121.8, 121.8, 114.3120.8, 120.8, 31.850.7, 72.9, 95.4α, β, γ (°)90.0, 90.0, 90.090.0, 90.0, 90.090.0, 90.0, 120.090.0, 90.0, 90.0 Wavelength (Å)1.72200.97950.97950.9787 Resolution (Å)50–1.96 (1.99–1.96)50–1.35 (1.37–1.35)50–1.50 (1.53–1.50)50–2.45 (2.49–2.45) Rmerge0.099 (1.094)0.082 (3.02)0.077 (2.16)0.128 (1.344) I/σΙ48.1 (3.7)48.2 (1.2)45.4 (1.6)25.4 (1.2) Completeness (%)100.0 (100.0)99.9 (100.0)99.8 (99.7)99.4 (94.6) MultiplicitybFor the data used for SAD phasing, the anomalous multiplicity was shown.49.8 (48.8)25.3 (24.7)18.1 (14.2)15.3 (6.9) No. unique reflections62,861 (3092)187,184 (18,429)27,420 (2696)13,547 (617) CC1/2(%)99.1 (95.3)99.9 (65.9)100.0 (74.1)98.7 (71.3)Refinement Resolution (Å)50–1.35 (1.40–1.35)50–1.50 (1.56–1.50)50–2.45 (2.53–2.45) No. molecules per asymmetric unit311 Rwork/Rfree0.152/0.1740.183/0.2120.211/0.243 Included residue No.WhiB315–89; 15–89; 14–89cThe three sets of the residues are for each of the three complexes in the asymmetric unit.6–9016–90σA4446–528446–528450–525βtip815–826815–827815–826His-tag1–61–6- No. atoms539816891974Macromolecules469615551958Ligand1802913Water5221053 B-factors (Å2)30.141.380.7Macromolecules28.441.276.6Ligand38.338.879.3Water42.543.966.2 r.m.s deviationsBond lengths (Å)0.0120.010.018Bond angles (°)1.681.292.29 Ramachandran statistics Favored regions (%)98.1497.8991.77Allowed regions (%)1.301.588.23Outliers (%)0.560.530PDB code8CWT8CWR8CYFa The highest resolution shell statistics are shown in parentheses.b For the data used for SAD phasing, the anomalous multiplicity was shown.c The three sets of the residues are for each of the three complexes in the asymmetric unit. Open table in a new tab Like the other two σA4-bound Wbl proteins, the WhiB3:σA4-βtip complex exists as a single complex in solution determined by size-exclusion chromatography (25Wan T. Li S. Beltran D.G. Schacht A. Zhang L. Becker D.F. et al.Structural basis of non-canonical transcriptional regulation by the σA-bound iron-sulfur protein WhiB1 in M. tuberculosis.Nucleic Acids Res. 2020; 48: 501-516Google Scholar, 26Wan T. Horova M. Beltran D.G. Li S. Wong H.X. Zhang L.M. Structural insights into the functional divergence of WhiB-like proteins in Mycobacterium tuberculosis.Mol. Cell. 2021; 81: 2887-2900.e5Google Scholar). However, in the crystals, the His6-tags in the N-terminal σA4-βtip of three WhiB3:σA4-βtip complexes are "glued" together by multiple nickel ions from the crystallization solution, resulting in a trimer of the WhiB3:σA4-βtip complexes in both crystal forms (Figs. 1B and S2A). Three WhiB3:σA4-βtip complexes in the trimer form are found in the asymmetric unit of the P43212 structure. The R3 structure contains one WhiB3:σA4-βtip complex per asymmetric unit, as the trimer axis coincides with the crystallographic threefold axis. Correlated with the trimerization in the crystal form, we observed two structural re-arrangement in the WhiB3-bound σA4-βtip compared to that bound to WhiB7. First, the N-terminal residues (aa 446–456) of σA4 in the WhiB3:σA4-βtip trimer form a β-hairpin with the His6-tag residues, instead of being part of helix hs1 of σA4 as expected (Figs. 1B and S2B) (26Wan T. Horova M. Beltran D.G. Li S. Wong H.X. Zhang L.M. Structural insights into the functional divergence of WhiB-like proteins in Mycobacterium tuberculosis.Mol. Cell. 2021; 81: 2887-2900.e5Google Scholar, 31Campbell E.A. Muzzin O. Chlenov M. Sun J.L. Olson C.A. Weinman O. et al.Structure of the bacterial RNA polymerase promoter specificity sigma subunit.Mol. Cell. 2002; 9: 527-539Google Scholar). Because the residues 446 to 456 of σA4 are far from the WhiB3 binding site, we do not anticipate this structural change affects the mode of WhiB3 binding to σA4. Second, βtip in the WhiB3:σA4-βtip complex, which was expected to form intramolecular contacts with σA4 in the σA4-βtip chimera, sticks into a neighboring protomer of the trimer and forms intermolecular interactions with σA4 in a second WhiB3:σA4-βtip complex (Figs. 1C and S2B). The resulting σA4-βtipʹ, however, resembles σA4-βtip observed in the WhiB7:σA4-βtip complex and the RNAP holoenzyme (Fig. S3C) (26Wan T. Horova M. Beltran D.G. Li S. Wong H.X. Zhang L.M. Structural insights into the functional divergence of WhiB-like proteins in Mycobacterium tuberculosis.Mol. Cell. 2021; 81: 2887-2900.e5Google Scholar, 31Campbell E.A. Muzzin O. Chlenov M. Sun J.L. Olson C.A. Weinman O. et al.Structure of the bacterial RNA polymerase promoter specificity sigma subunit.Mol. Cell. 2002; 9: 527-539Google Scholar), and thus it is used for the following structural analysis. The crystal structures of the WhiB3:σA4-βtip complex in the P43212 and R3 forms are essentially identical except for the N-terminal loop region, with an average Cα root-mean-square deviation of 0.27 Å for WhiB3 and 0.42 Å for σA4, respectively (Fig. S3, A and B). The R3 structure was used for the following structural analysis of the WhiB3:σA4-βtip complex because of the better-defined electron density of the N-terminal residues (aa 6–13) in WhiB3 (Fig. S3, D and E). The overall architecture of the WhiB3:σA4-βtip complex is comparable to the previously reported WhiB1:σA4 and WhiB7:σA4-βtip complexes. In all three cases, the molecular interface between the Wbl protein and σA4 is hinged at the [4Fe-4S] cluster (Fig. 1C) (25Wan T. Li S. Beltran D.G. Schacht A. Zhang L. Becker D.F. et al.Structural basis of non-canonical transcriptional regulation by the σA-bound iron-sulfur protein WhiB1 in M. tuberculosis.Nucleic Acids Res. 2020; 48: 501-516Google Scholar, 26Wan T. Horova M. Beltran D.G. Li S. Wong H.X. Zhang L.M. Structural insights into the functional divergence of WhiB-like proteins in Mycobacterium tuberculosis.Mol. Cell. 2021; 81: 2887-2900.e5Google Scholar). The 4Fe-4S cluster binding pocket in WhiB3:σA4-βtip is enclosed (Fig. 1D), similar to that of WhiB1:σA4 and in contrast to the solvent-accessible cluster in the case of WhiB7:σA4-βtip. Complex formation between WhiB3 and σA4 is driven by the conserved aromatic residues near the [4Fe-4S] cluster binding pocket (Figs. 2A and S1B), as previously observed in the cases of WhiB1 and WhiB7 (25Wan T. Li S. Beltran D.G. Schacht A. Zhang L. Becker D.F. et al.Structural basis of non-canonical transcriptional regulation by the σA-bound iron-sulfur protein WhiB1 in M. tuberculosis.Nucleic Acids Res. 2020; 48: 501-516Google Scholar, 26Wan T. Horova M. Beltran D.G. Li S. Wong H.X. Zhang L.M. Structural insights into the functional divergence of WhiB-like proteins in Mycobacterium tuberculosis.Mol. Cell. 2021; 81: 2887-2900.e5Google Scholar). A single Ala substitution of F31, F32, or W76 in WhiB3 (corresponding to F17, F18, and W60 in WhiB1, respectively) or H516 in σA4 completely abolishes the complex formation in the pull-down assays (Fig. 2C). To our initial surprise, a W17A mutation in WhiB3 does not abolish σA4 binding in the pull-down assay (Figs. 2C and S4). W17 is invariant in the WhiB3 subclass, corresponding to the invariant W3 in WhiB1. It has been shown to play a crucial role in Fe–S cluster stability and complex formation in WhiB1:σA4, while the absence of a W3 counterpart in WhiB7 leads to a solvent-accessible Fe–S cluster with increased O2 sensitivity in the WhiB7:σA4 complex (25Wan T. Li S. Beltran D.G. Schacht A. Zhang L. Becker D.F. et al.Structural basis of non-canonical transcriptional regulation by the σA-bound iron-sulfur protein WhiB1 in M. tuberculosis.Nucleic Acids Res. 2020; 48: 501-516Google Scholar, 26Wan T. Horova M. Beltran D.G. Li S. Wong H.X. Zhang L.M. Structural insights into the functional divergence of WhiB-like proteins in Mycobacterium tuberculosis.Mol. Cell. 2021; 81: 2887-2900.e5Google Scholar). Subsequent sequence analysis reveals an additional conserved Trp in the WhiB3 subclass, W15, which is close to W17 and the Fe–S cluster binding pocket (Figs. 2A and S1B) and thus may compensate for the loss of W17 in the WhiB3 W17A mutant based on our pull-down assay. As shown in Figure. 2C and Fig. S4, although a single W15A mutation does not affect σA4 binding, the double mutation of W15A and W17 A in WhiB3 completely abolishes the interaction in the pull-down assays. The existence of redundant Trp residues in the N-terminal WhiB3 highlights their importance in the complex formation, consistent with the observations from our studies on WhiB1 and WhiB7 (25Wan T. Li S. Beltran D.G. Schacht A. Zhang L. Becker D.F. et al.Structural basis of non-canonical transcriptional regulation by the σA-bound iron-sulfur protein WhiB1 in M. tuberculosis.Nucleic Acids Res. 2020; 48: 501-516Google
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