Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Jun;299(6):104777.
doi: 10.1016/j.jbc.2023.104777. Epub 2023 May 2.

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

Tao Wan  1 , Magdaléna Horová  1 , Vimmy Khetrapal  1 , Shanren Li  1 , Camden Jones  1 , Andrew Schacht  1 , Xinghui Sun  1 , LiMei Zhang  2
Affiliations

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

Tao Wan et al. J Biol Chem. 2023 Jun.

Abstract

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.

Keywords: Wbl family; WhiB3; X-ray crystallography; iron–sulfur cluster; transcription factor.

PubMed Disclaimer

Conflict of interest statement

Conflict of interest The authors declare no conflict of interest with the contents of this article.

Figures

Figure 1
Figure 1
Overview of the WhiB3TR:σA4-β-tipstructure.A, an optical image of the WhiB3TR:σA4-βtip crystals. The two forms of crystals, P43212 and R3, respectively, are shown in the same crystal drop. B, the trimeric structure of the WhiB3TR:σA4-βtip complexes in the R3 form. The trimer is centered on the His6-tags in the N terminus of σA4-βtip, which bind to Ni ions from the crystallization solution. The side chains of the His6-tag residues are shown in sticks, and the Ni atoms are shown in magenta spheres. C, cartoon representation of the WhiB3:σA4-βtipʹ complex in the R3 form, in which β-tip from the neighboring complex molecule (βtipʹ) is shown to reflect its interaction with σA4 and WhiB3. The four helices in WhiB3 and five helices in σA4 are labeled as hw1-4 and hs1-5, respectively. D, surface representation of the WhiB3TR: σA4-βtip complex, demonstrating the enclosed [4Fe-4S] cluster binding pocket in the complex. In all the structures, C atoms of WhiB3 are colored pale green with the N-terminus residues (aa 6–16) in salmon pink, σA4 in gray, and βtip in pink. The [4Fe-4S] clusters are shown in spheres, with Fe colored orange and S in yellow. [4Fe-4S] cluster, iron–sulfur cluster containing four iron and four sulfur ions; σA4-βtip, σA4 fused with βtip by an artificial linker; βtip, the C-terminal flap tip helix of the RNAP β-subunit; His6-tag, hexa histidine-tag; WhiB3TR, a truncated WhiB3 without C-terminal loop region (aa 91–102).
Figure 2
Figure 2
Molecular interface between WhiB3 σA4.A, a structural overlay of σA4-βtipʹ-bound WhiB3 (pale green, with the N-terminal region in salmon pink) and WhiB7 (PDB ID: 7KUG, gray). Only Wbl proteins are shown for clarity. B, close-up view of the hydrophilic network at the molecular interface of WhiB3 (pale green) and σA4 (gray). The βtipʹ residues interacting with σA4 in this region are shown in pink. C, SDS-PAGE analyses of the samples from the co-expression and affinity purification of tagless WhiB3 and His6-σAC170 (either wildtype [WT] or mutant as indicated). σA4-βtip, σA4 fused with βtip by an artificial linker.
Figure 3
Figure 3
Interactions between N-terminal WhiB3 and σA4-βtip.A, a hydrophobic interface among N-terminal residues (aa 6–11) of WhiB3 (pale green with N-terminal residues in salmon pink), σA4 (gray), and βtip (pink). βtip from the neighboring complex molecule (βtipʹ) is shown to reflect its interaction with σA4 and WhiB3 in the WhiB3TR:σA4-βtip complex. The hydrophobic residues at the molecular interface are shown in sticks. B, an overlay of σA4 in the WhiB3:σA4-βtip complex with the WhiB7-RNAP-DNA complex in the closed state (PDB ID: 7KIM, σA colored purple blue, the α-subunits in gray, the β-subunit in cyan, and DNA in orange). The WhiB3:σA4-βtip complex is shown in the cartoon representations, and the WhiB7–RNAP–DNA complex is shown in the surface representation. Only WhiB3 (pale green with the N-terminal WhiB3 in salmon red) in the WhiB3:σA4-βtip complex is shown for clarity. By comparison, the N-terminal WhiB7 points toward an opposite direction relative to the N terminus of WhiB3 and interacts with the βʹ-subunit of RNAP (Fig. S5). σA4-βtip, σA4 fused with βtip by an artificial linker; RNAP, RNA polymerase; WhiB3TR, a truncated WhiB3 without C-terminal loop region (aa 91–102).
Figure 4
Figure 4
Effect of the C-terminal WhiB3 on DNA binding.A and B, the EMSAs of the WhiB3:σA4-β-tip complex with the pks3 promoter, either with wildtype WhiB3 protein (A) or the truncated WhiB3 without the C-terminal restudies 91 to 102 (WhiB3TR, B). C, an overlay of WhiB3 (pale green) in the WhiB3:σA4-βtip with the σA4-bound WhiB1 (PDB ID: 6ONO, gray). Only Wbl proteins are shown for clarity. D, sequence alignment between Mtb WhiB3 and WhiB1. The putative C-terminal DNA binding motifs in the Wbl proteins and the conserved Arg-rich motif specific to the WhiB3 subclass are highlighted by dashed rectangles. The two invariant Arg residues (R38 and R42) in the conserved Arg-rich motif of WhiB3 and absent in WhiB1, are indicated by blue triangles (Fig. S1). R40 is a variant in the WhiB3 subclass. σA4-βtip, σA4 fused with βtip by an artificial linker; EMSA, electrophoretic mobility shift assay.
Figure 5
Figure 5
Comparison of the PwhiB7binding site between WhiB3:σA4-β-tipand WhiB7:σA4-β-tip.A, simulated-annealing composite omit map around the whiB7 promoter (PwhiB7) in the crystal structure of two adjacent WhiB3:σA4-βtip:PwhiB7 complex molecules, contoured at 1.0 σ. The gap in the electron density map between the two adjacent PwhiB7 DNA molecules is highlighted by a black dash circle, indicative of the correct assignment of the PwhiB7 DNA. B and C, a side-by-side comparison of the crystal structures of WhiB3:σA4-βtip:PwhiB7 and WhiB7:σA4-βtip:PwhiB7 (PDB ID: 7KUF), respectively. The Wbl residues (R38 of WhiB3; R83-G84-R85 of WhiB7) inserted into the minor groove of the DNA helix are labeled. D and E, cartoon illustrations of the different modes of PwhiB7 binding by WhiB3:σA4-βtip and WhiB7:σA4-βtip, respectively. The blue arrow indicate the direction of the transcription in WhiB7:σA4-βtip:PwhiB7. In all the structures, WhiB3 and WhiB7 are colored pale green, σA4-βtip in gray, and the PwhiB7 DNA in orange. σA4-βtip, σA4 fused with βtip by an artificial linker.
Figure 6
Figure 6
Identification of a conserved Arg-rich DNA binding motif in WhiB3.A, highlights of the hydrophilic interactions between the conserved Arg-rich motif of WhiB3 and DNA in the WhiB3:σA4-βtip:PwhiB7 complex. The AT-rich DNA sequence in PwhiB7 is colored cyan, with the rest of the DNA in orange, WhiB3 in pale green, and σA4-βtip in gray. The three Arg residues (i.e., R38, R40 and R42) in the conserved Arg-rich motif of Mtb WhiB3 are shown in stick representations, with the 2Fo-Fc density map contoured at 1.0 σ. B, EMSAs of the σA4-βtip-bound WhiB3 (wildtype or mutant as indicated) with the pks3 promoter DNA. All the three Arg residues are substituted by Ala in the WhiB3-3RtoA mutant. σA4-βtip, σA4 fused with βtip by an artificial linker; EMSA, electrophoretic mobility shift assay.
Figure 7
Figure 7
Identification of the essential DNA binding motif for the WhiB3-dependent transcription activation in Msm.A and B, are the RT-qPCR analyses of relative mRNA levels of MSMEG_4728 and whiB3, respectively, in the Msm wildtype (wt), the whiB3 deletion mutant (ΔwhiB3) alone or complemented with the wildtype whiB3 (cWhiB3), the whiB3-3RtoA mutant (cWhiB3-3RtoA) or the C-terminal truncated whiB3 (cWhiB3TR). The mRNA level in each sample was normalized to the level σA, and the fold of changes were calculated relative to wt. Data are representative of three biological replicates. Statistical significance is determined by Student’s t test and displayed as ∗p < 0.05 in the comparisons as indicated. The error bars represent mean ± SD. Msm, Mycobacterium smegmatis; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Pai M., Kasaeva T., Swaminathan S. Covid-19's devastating effect on tuberculosis care - a path to recovery. N. Engl. J. Med. 2022;386:1490–1493. - PubMed
    1. Pawlowski A., Jansson M., Skold M., Rottenberg M.E., Kallenius G. Tuberculosis and HIV co-infection. PLoS Pathog. 2012;8 - PMC - PubMed
    1. Davis 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;232:351–358. - PubMed
    1. Soliveri 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;146:333–343. - PubMed
    1. Gomez 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;97:8554–8559. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources