Structural basis of DNA binding by the WhiB-like transcription factor WhiB3 in Mycobacterium tuberculosis

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. 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 Scholar, M. A. Mycobacterium tuberculosis WhiB3 to in to modulate and 2016; Scholar). It is by Mtb to and in response to various acidic and starvation and by and in vitro M. A. Mycobacterium tuberculosis WhiB3 to in to modulate and 2016; Scholar, A. L. D. J. et tuberculosis WhiB3 to and via cluster and is essential for starvation Natl. Acad. Sci. U. S. A. Scholar, A. D. L. et tuberculosis WhiB3 by regulating virulence to modulate Pathog. Scholar, A. Mycobacterium tuberculosis a cluster protein that regulates and 2012; 16: Scholar, M. Bishai W.R. expression of Mycobacterium tuberculosis transcription in 2012; Scholar, Cumming B.M. L. et and essential for drug and virulence of Mycobacterium Rep. 2016; Scholar, B.M. K.H. et tuberculosis host at the to Pathog. Scholar, M. A. Mycobacterium tuberculosis WhiB3 and survival in response to and Med. Scholar, W.R. R.P. Mycobacterium tuberculosis WhiB3 interacts with to affect host survival but is dispensable for in Natl. Acad. Sci. U. S. A. Scholar). the WhiB3 is required for the of and proteins required for of in Streptomyces coelicolor 2000; Scholar, P. Bishai W.R. Chater K.F. that the Streptomyces developmental protein a of the binds a 2005; Scholar). with roles in regulating gene Mtb Wbl proteins to and of Wbl proteins widely distributed in Actinobacteria. conserved are found in Wbl binding conserved and a motif are in and the are the are is also to as the The of Wbl proteins in DNA binding Wbl proteins a cluster of in this region that is to in a 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, the Wbl proteins in Mtb, a defined DNA-binding motif, the in the binds to the of the in the WhiB3 also to a motif A. Mycobacterium tuberculosis a cluster protein that regulates and 2012; 16: Scholar). in the WhiB3 are conserved in the WhiB3 subclass and role in WhiB3 binding to DNA known is the of Wbl proteins to the motif. region in and the Wbl proteins and to the of Wbl Mtb Wbl and WhiB7, to regulate gene expression in the by binding to the conserved region 4 in the principal sigma factor (σA4) in the RNA polymerase holoenzyme W.R. R.P. Mycobacterium tuberculosis WhiB3 interacts with to affect host survival but is dispensable for in Natl. Acad. Sci. U. S. A. Scholar, J. G. M. P. A. et mycobacterial antibiotic resistance as a transcriptional activator by binding the sigma factor Scholar, et of a Wbl protein and for by M. Scholar). Mtb Wbl proteins also to bind to σA4 in a L. Z. Z. Hu Y. S. of transcriptional regulators in Mycobacterium 2016; Scholar). a study that the WhiB3 of Streptomyces also binds to region 4 of the principal sigma factor a of by Wbl proteins in Actinobacteria of the Streptomyces Wbl protein with the principal sigma factor depends on the Scholar). in the structural and of the Wbl proteins on how Wbl proteins with to regulate gene expression. Mtb WhiB1 is the first Wbl protein that structurally characterized at the first in the by and in the by et of a Wbl protein and for by M. Scholar, T. S. D.G. A. L. et basis of transcriptional regulation by the protein WhiB1 in M. Scholar). with the molecular and reveal an molecular interface in the complex by and a molecular of transcription regulation by WhiB1 in Actinobacteria T. S. D.G. A. L. et basis of transcriptional regulation by the protein WhiB1 in M. Scholar). the crystal of in complex with σA4 and and the of to in and by our group and by the T. M. D.G. S. the of WhiB-like proteins in Mycobacterium Scholar, M. basis of transcriptional by the Mycobacterium tuberculosis intrinsic transcription factor Scholar). The binding the of DNA a of possesses distinct structural from high and DNA the the in U. The of and intrinsic DNA Rev. of the structures the structural basis for how binding to the region the the DNA in the to the intrinsic of and with for transcriptional Together, an of how gene expression by DNA binding with σA4 via and the subclass-specific structural that to from WhiB1 T. M. D.G. S. the of WhiB-like proteins in Mycobacterium Scholar, M. basis of transcriptional by the Mycobacterium tuberculosis intrinsic transcription factor Scholar). WhiB3 is one of the Mtb Wbl proteins to for the pathogenesis of Mtb M. A. Mycobacterium tuberculosis WhiB3 to in to modulate and 2016; Scholar, A. L. D. J. et tuberculosis WhiB3 to and via cluster and is essential for starvation Natl. Acad. Sci. U. S. A. Scholar, A. D. L. et tuberculosis WhiB3 by regulating virulence to modulate Pathog. Scholar, A. Mycobacterium tuberculosis a cluster protein that regulates and 2012; 16: Scholar, M. Bishai W.R. expression of Mycobacterium tuberculosis transcription in 2012; Scholar, Cumming B.M. L. et and essential for drug and virulence of Mycobacterium Rep. 2016; Scholar, B.M. K.H. et tuberculosis host at the to Pathog. Scholar, M. A. Mycobacterium tuberculosis WhiB3 and survival in response to and Med. Scholar, W.R. R.P. Mycobacterium tuberculosis WhiB3 interacts with to affect host survival but is dispensable for in Natl. Acad. Sci. U. S. A. Scholar, D. Y. and by the in Mycobacterium 10: Scholar). However, the DNA-binding motif the DNA-binding of WhiB3 determined to It how WhiB3 binds to σA4 and DNA for transcriptional we crystal structures of the WhiB3 and in complex with DNA at 1.5 Å and 2.45 Å, Together, the from our and an essential DNA-binding motif in WhiB3 and on how WhiB3 coordinates with σA4 and interacts with DNA for transcriptional structural we how WhiB3 from WhiB1 and in Mtb by binding to the on σA4 and the subclass-specific structural motif for DNA in the a protein for the of the WhiB3 by σA4 with the via an to the interaction and the in the holoenzyme T. M. D.G. S. the of WhiB-like proteins in Mycobacterium Scholar, A. J. M. M. The of a transcription how is to J. Scholar, M. et of the bacterial RNA polymerase sigma Scholar). 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Scholar). the complex DNA binding the structural the structural basis that for the in DNA binding WhiB3 and The WhiB3 and WhiB1 are with an of in the structural the structural of to the DNA-binding motif is in the and an of and one is found in the of WhiB1 and WhiB3 and However, a of the WhiB3 subclass-specific an Arg-rich motif to in Mtb the that the interface with DNA and and The role of this motif for WhiB3 binding to DNA reveal the structural basis of the complex binding to we of with the DNA of of to the regulation of WhiB3 M. A. Mycobacterium tuberculosis WhiB3 to in to modulate and 2016; Scholar, A. D. L. et tuberculosis WhiB3 by regulating virulence to modulate Pathog. Scholar, B.M. K.H. et tuberculosis host at the to Pathog. Scholar, L. S. A. et in Mycobacterium tuberculosis expression and WhiB6 as a ESX-1 Scholar). of the from the to for WhiB3 possesses a motif, of we and that the complex binds to the DNA We the complex with that in the study of the complex T. M. D.G. 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D.G. S. the of WhiB-like proteins in Mycobacterium is from in the complex However, that σA4 binds to the of the and in the is in the we the of the from the our structural that in the conserved Arg-rich motif play a role in WhiB3 binding to the the in is of the Arg-rich motif DNA binding in the of by an WhiB3 binding to the with the effect of the the other and with the structural and as The WhiB3 are required for DNA binding in the are in the It is that with DNA and in the in the crystal with the structural and our polymerase study that the conserved Arg-rich motif is required for the transcriptional regulation in Mycobacterium the study D. Y. and by the in Mycobacterium 10: our that WhiB3 regulates the expression of in encodes a The of the of in the by the WhiB3 but by the the essential role of the conserved Arg-rich motif for transcriptional of the WhiB3 a effect on the expression of to the that the region is essential for the transcriptional with the high of this region in the WhiB3 subclass and the DNA binding in the complex structural study provides an of how WhiB3 interacts with σA4 and of the WhiB3:σA4 complex with the WhiB1 and that the Wbl proteins a similar molecular interface with and the subclass-specific structural the structural basis for DNA binding by by molecular and we a conserved Arg-rich DNA binding motif the in the WhiB3 subclass and the of the for DNA binding and transcription by Mtb It is to that WhiB3 subclass such as in Streptomyces and to the DNA binding motif of Mtb the of the newly Arg-rich motif for DNA binding by the WhiB3 subclass the of the conserved Arg-rich DNA-binding motif in WhiB3 provides a for the in DNA-binding WhiB3:σA4 and and on the DNA binding of It is known that the is conserved in mycobacterial in T. G. et of transcriptional an in Mycobacterium Rep. Scholar, J. P. M. et and small proteins are of the mycobacterial Genet. Scholar, M. The complex of mycobacterial Scholar). σA4 bind to DNA in the without the DNA binding motif of a Wbl protein in the T. S. D.G. A. L. et basis of transcriptional regulation by the protein WhiB1 in M. Scholar, T. M. D.G. S. the of WhiB-like proteins in Mycobacterium Scholar). this the Wbl in Actinobacteria but in by to as to for the DNA and of σA4 in the transcriptional regulation in response to environmental the of DNA binding by WhiB3 and are the the in DNA binding motif. The motif of in and the of the like a The of interaction with DNA provides the structural basis for how binding the and the DNA to σA4 binding to the T. M. D.G. S. the of WhiB-like proteins in Mycobacterium Scholar). a WhiB3 is the DNA and interacts with DNA via the group like the DNA in the and WhiB3 with the in the like WhiB7. The that σA4 is from the binding to WhiB3 that WhiB3 the the The from the also the evidence for the high and of WhiB3 in this study and the A. D. L. et tuberculosis WhiB3 by regulating virulence to modulate Pathog. Scholar). These the of how WhiB3 regulates gene expression. on the of Mtb in Streptomyces and that coordinates with for DNA binding and transcriptional bind to DNA P. Y. interacts with to a regulatory effect on cell division Scholar, G. that is a transcription factor that regulon with to developmental cell division in 2016; Scholar, M. et basis of of cell division by the transcription and Natl. Acad. Sci. U. S. A. Scholar). WhiB3 also this for engaging with DNA and is in with the that the is required for WhiB3 binding to DNA in the in DNA binding in our structural and is dispensable for transcription in It is to that the of the region WhiB3 binding to DNA in the and to in the to the on WhiB1 that the and are also dispensable for transcriptional are required for DNA binding by to essential T. S. D.G. A. L. et basis of transcriptional regulation by the protein WhiB1 in M. Scholar, J. of the Mycobacterium tuberculosis transcription factor 2012; Scholar, et tuberculosis WhiB1 is an essential DNA-binding protein with a J. Scholar). study is to also the for DNA binding and It is that the of Wbl proteins involved in σA4 binding are However, the structural basis the in the Wbl and molecular of WhiB3 crucial evidence for how WhiB3 from other characterized Wbl proteins WhiB1 and WhiB7) via the conserved Arg-rich motif in the and regulates gene expression in with of structural is for the of pathogenesis and persistence in Mtb and a for for the of the of the in and at and the related without and with a gene in this study in on with and the with at for and for and for and for The bacterial and in this study are in The genes Mtb WhiB3 and the of σA4 the from Mtb DNA from group at the of by and and to WhiB3 and with a The and for the of the proteins with for and as in the related the from by for the expression of a WhiB3 without the in the for and T. M. D.G. S. the of WhiB-like proteins in Mycobacterium the the protein for and the for the expression of the protein in the the by DNA for protein expression. and of the proteins of from for structural and as in our study of T. M. D.G. S. the of WhiB-like proteins in Mycobacterium Scholar). of by and by the proteins in and in of the proteins an The at of proteins roles for Scholar, J. et the of the cluster of WhiB-like to the of the cluster in the protein a Wbl by the the role of the in WhiB3 and σA4 for the complex the a Mtb WhiB3 and a respectively, for protein and by as T. M. D.G. S. the of WhiB-like proteins in Mycobacterium Scholar). The protein by and to the binding of Mtb WhiB3 and in complex with to the of the gene as T. S. D.G. A. L. et basis of transcriptional regulation by the protein WhiB1 in M. Scholar, T. M. D.G. S. the of WhiB-like proteins in Mycobacterium Scholar). The of the of the the region in the A. D. L. et tuberculosis WhiB3 by regulating virulence to modulate Pathog. from Mtb DNA the of the binding with Mtb WhiB3 in the of and at for the on and to The DNA the to the The in as and of gene in Mycobacterium Rep. 2016; Scholar). The by and by DNA the in the the DNA the for expression of from the G.