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The LexA–RecA* structure reveals a cryptic lock-and-key mechanism for SOS activation

Nature Structural & Molecular Biology (2024)Cite this article

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Abstract

The bacterial SOS response plays a key role in adaptation to DNA damage, including genomic stress caused by antibiotics. SOS induction begins when activated RecA*, an oligomeric nucleoprotein filament that forms on single-stranded DNA, binds to and stimulates autoproteolysis of the repressor LexA. Here, we present the structure of the complete Escherichia coli SOS signal complex, constituting full-length LexA bound to RecA*. We uncover an extensive interface unexpectedly including the LexA DNA-binding domain, providing a new molecular rationale for ordered SOS gene induction. We further find that the interface involves three RecA subunits, with a single residue in the central engaged subunit acting as a molecular key, inserting into an allosteric binding pocket to induce LexA cleavage. Given the pro-mutagenic nature of SOS activation, our structural and mechanistic insights provide a foundation for developing new therapeutics to slow the evolution of antibiotic resistance.

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Fig. 1: Structure of the SOS signal complex.
Fig. 2: The role of the RecA L2 loops in complex formation.
Fig. 3: The role of the LexA NTD in RecA*-dependent proteolysis.
Fig. 4: The hyperactive LexA variant autopopulates the allosteric pocket in a similar manner to RecA*-bound LexA.
Fig. 5: RecA F203 variants modulate binding and dictate LexA cleavage efficiency.
Fig. 6: A single active subunit with F203 in RecA* is sufficient for LexA cleavage.

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Data availability

The cryo-EM maps and associated atomic model for this study have been deposited to the Electron Microscopy Data Bank (EMD-41579) and Protein Data Bank (8TRG). The earlier maps and model used as a comparison in this work are available on the Electron Microscopy Data Bank (EMD-34152) or the Protein Data Bank (1JHE, 3JSP, 8GMS). Source data are provided with this paper. All other data necessary to evaluate the claims in the paper is present in the text or Supplementary Information. Plasmids for LexA or RecA variants are available upon request.

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Acknowledgements

This work was supported by the National Institutes of Health (grant no. R01-GM127593 to R.M.K. and E.J.P.). R.M.K. holds an Investigators in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund. The National Institutes of Health also provided training grants (grant nos. T32-AI141393 for M.B.C. and T32-GM133398 for C.M.H. and R.M.P.) and mass spectrometry instrumentation support (grant no. S10-OD030460). Structural data collection was performed with the help of the Institute of Structural Biology, the Electron Microscopy Resource Laboratory and the Beckman Center for Cryo-EM at the University of Pennsylvania (RRID: SCR_022375). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Authors and Affiliations

  1. Graduate Group in Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, USA

    Michael B. Cory, Christina M. Hurley & Peter J. Carman

  2. Department of Chemistry, University of Pennsylvania, Philadelphia, PA, USA

    Allen Li, Ryann M. Perez, Yarra Venkatesh, Xinning Li & E. James Petersson

  3. Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, USA

    Ruth A. Pumroy, Kushol Gupta, E. James Petersson & Rahul M. Kohli

  4. Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Zachary M. Hostetler & Rahul M. Kohli

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Contributions

M.B.C., E.J.P. and R.M.K. conceived of the experiments. M.B.C. designed the overall experimental plan. M.B.C., A.L., C.M.H., Z.M.H. and Y.V. designed and executed biochemical experiments. M.B.C., A.L., P.J.C., R.A.P. and K.G. designed structural biology experiments, collected associated data and performed analysis. R.M.P. and X.L. performed computational modeling experiments. M.B.C., E.J.P. and R.M.K. wrote the manuscript. All authors were involved in editing and reviewing.

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Correspondence to E. James Petersson or Rahul M. Kohli.

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Nature Structural & Molecular Biology thanks Michael Cox, Edward Egelman and Yu Feng for their contribution to the peer review of this work. Dimitris Typas was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Cryo-EM analysis pipeline.

The flow of data from the collected and filtered micrographs through the final local refinement is shown. Each labeled step includes relevant information for the partitioning of data at each junction. For each refinement and reconstruction step, the FSC curve generated by CryoSPARC is shown.

Extended Data Fig. 2 Characteristics of the EM density and model.

a) Final sharpened map colored by the estimated local resolution using Relion. The entire complex is shown at top, with the relevant sub-complex components at the bottom with RecA* and LexA labeled and colored relative to grayed out other components. b) Orientation distribution of the final particle stack as determined by cryoEF. Orientation efficiency, Eod is given below. c) Closeup of the ATP binding pocket at the interface of two RecA protomers within the filament, showing the coordinated Mg2+ ion in green. Density from the two independent half maps and the corresponding full map at a contour level of 0.203 and 0.172 respectively shown in mesh. d) Closeup of the bound ssDNA within the filament. Density from the two independent half maps and the corresponding full map at a contour level of 0.203 and 0.172 respectively shown in mesh.

Extended Data Fig. 3 Comparison to prior models.

a) Global comparison of this current model of full-length LexA bound to RecA* (8TRG, blue and pink) to prior published model of a RecA* dimer bound to the CTD-only LexA (8GMS, gold and sea green). Global RMSD is between Cα atoms of residues present in both models, RMSD of the L2 pocket is all-atom of the residues shown by sticks. b) Overlay of the current model of the SOS complex with native, full-length LexA into the cryoEM density from Gao et al (Ref. 32). The displayed density is that derived from 8GMS, and is colored to match the model colors from A (LexA in gold, RecA* in sea green), including the density from a second LexA CTD (dark orange). The ribbon structures show our fit model, colored accordingly (8TRG, blue and pink). The overlay demonstrates that full-length LexA containing the NTD is not compatible with the symmetrically decorated filament previously studied with CTD-only LexA. c) Molecular dynamics was performed to build potential poses for the missing NTD from the unbound LexA subunit. Five distinctive poses were selected in this overlay, represented by different colors in ribbons, with the cryo-EM density-derived model shown as a surface.

Extended Data Fig. 4 Discrimination between operator-bound LexA and free LexA by RecA*.

a) SDS-PAGE gel of autoproteolysis of fluorescent LexA-CF variant at pH 7.5 in the absence of operator or in the presence of either 20 bp or 40 bp consensus operator (single replicate). b) Fluorescence anisotropy of LexA-δ with various in vitro binding partners. Each data point represents a single replicate. The various contributing species to the observed anisotropic signal are given to the right. c) Equilibrium endpoint anisotropy titration of either Ec or Mtb LexA with FAM-labeled 40-mer consensus operator. Data shows a single replicate and the solid line is a fit to a quadratic equation, using a fixed [operator] of 1 nM. d) SDS-PAGE analysis of RecA*-dependent cleavage of E. coli LexA with either an Ec or Mtb inter-domain linker when incubated with consensus operator (single replicate). e) Structural overlay of our modeled SOS complex (8TRG, blue and pink) with the crystal structure of the DNA-bound LexA dimer (3JSP, yellow). Steric clashes are colored in red. Lower left panel shows a close-up view of one of the modeled bound LexA NTD alongside the corresponding DNA-bound NTD, demonstrating the distinctive orientations of the NTDs in the two different structures. The relative numbering of each alpha helix in the NTD is numbered according to the topological diagram on the left side of the panel.

Source data

Extended Data Fig. 5 Alkaline autoproteolysis rates of each tested LexA variant.

Data were fit to a single exponential decay (solid line) with 95% confidence intervals shown (shaded region). The best-fit value for the decay rate is shown on each graph. Data represents the mean from three replicates, with error bars denoting standard deviation.

Source data

Extended Data Fig. 6 Analysis of potential charge-charge interactions between RecA* and LexA.

a–c) Three different sections within the interaction interface that provide potential charge-charge interactions. Each of the RecA protomers within the three consecutive RecA units providing a majority of the contacts are highlighted and labeled in shades of pink to purple. The bound LexA monomer is shown in blue. Insets highlight the distances between interacting residues in green and show the sharpened map density as a wire-mesh surface. The panels highlight A) the ‘CTD Patch’ of interaction residues between RecA* and LexA. B) the ‘L2 Stabilizing Patch’ interaction residues, and C) the ‘NTD Patch’ interaction residues. d) Top: Representative SDS-PAGE analysis of RecA*-dependent cleavage of CTD patch residues in isolation or in combination. Bottom: Quantified LexA cleavage of CTD mutants expressed as a percentage of WT LexA rate (normalized to 100% shown by the dotted line; derived from n = 10 independent WT cleavage replicates). Each bar represents the mean from replicates (grey circles; n = 7 independent cleavage experiments for single mutants and n = 4 for QM) and error bars denote standard deviation. Below the graph, the posterior likelihoods of being either less than WT or greater than QMCTD are given via pairwise Bayesian comparisons of sample means, assuming unequal variance between samples. e) Top: Representative SDS-PAGE analysis of RecA*-dependent cleavage of L2-stabilizing patch residues. Bottom: Quantified LexA cleavage of L2-stabilizing mutants in isolation or in combination, expressed as a percentage of WT LexA rate (normalized to 100% shown by the dotted line; derived from n = 10 independent WT cleavage replicates). Each bar represents the mean from replicates (grey circles; n = 7 independent cleavage experiments for single mutants and n = 3 for QM) and error bars denote standard deviation. Below the graph, the posterior likelihoods of being either less than WT or greater than DML2 are given via pairwise Bayesian comparisons of sample means, assuming unequal variance between samples.

Source data

Extended Data Fig. 7 Allosteric binding pocket on LexA and species variation.

a) Surface (left) and cartoon (right) representations of the SOS complex model, as shown in Fig. 2. RecA F203 (pink) is bound to LexA (blue) within the hydrophobic pocket formed by the highlighted LexA residues (purple). The map density is shown on the right as a mesh surface. b) Sequence alignment of LexA and RecA proteins from select different species, showing the LexA hydrophobic pocket residues (left) and a subsection of the RecA L2 loop (right). F203 is highlighted in pink.

Extended Data Fig. 8 Biochemical analysis of RecA3x mutant filamentation and LexA binding.

a) RecA3x mutant filamentation expressed as a percentage of RecA3x WT anisotropy (normalized to 100% shown by the dotted line; derived from n = 3 independent RecA3x filamentation experiments) in the FAM-ssDNA binding assay. Data show the means of three replicates, with error bars denoting standard deviation. b) RecA3x mutant binding to LexA-δ expressed as a percentage of RecA3x WT anisotropy (normalized to 100% shown by the dotted line; derived from n = 3 independent RecA3x binding experiments) in the LexA binding assay. Data show the means of three replicates, with error bars denoting standard deviation. All samples had >0.999 posterior likelihood (via pairwise Bayesian comparisons of sample means, assuming unequal variance between samples) of being less than WT, and the posterior likelihoods of each successive mutant being less than the prior mutant is shown.

Extended Data Fig. 9 LexA and RecA constructs.

Shown is an SDS-PAGE gel including various LexA constructs evaluated in this study with the molecular weights of the ladder labeled at left (single replicate).

Source data

Supplementary information

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Cory, M.B., Li, A., Hurley, C.M. et al. The LexA–RecA* structure reveals a cryptic lock-and-key mechanism for SOS activation. Nat Struct Mol Biol (2024). https://doi.org/10.1038/s41594-024-01317-3

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