Structures of NF-κB p52 homodimer-DNA complexes rationalize binding mechanisms and transcription activation
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Evaluation Summary:
This manuscript provides a detailed structural and biophysical characterization of several complexes of the p52 homodimer of NF kB and different DNA binding sites. The main topic is the investigation of why the central base pair(s) have a strong influence on the transcriptional activity of the homodimer. The authors correlate structural changes with measurements of kinetic on and off rates to develop a model that explains the differences in activity. The paper is of interest to all working on understanding how transcriptional activity is regulated.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Abstract
The mammalian NF-κB p52:p52 homodimer together with its cofactor Bcl3 activates transcription of κB sites with a central G/C base pair (bp), while it is inactive toward κB sites with a central A/T bp. To understand the molecular basis for this unique property of p52, we have determined the crystal structures of recombinant human p52 protein in complex with a P-selectin(PSel)-κB DNA (5′-GGGGT G ACCCC-3′) (central bp is underlined) and variants changing the central bp to A/T or swapping the flanking bp. The structures reveal a nearly two-fold widened minor groove in the central region of the DNA as compared to all other currently available NF-κB-DNA complex structures, which have a central A/T bp. Microsecond molecular dynamics (MD) simulations of free DNAs and p52 bound complexes reveal that free DNAs exhibit distinct preferred conformations, and p52:p52 homodimer induces the least amount of DNA conformational changes when bound to the more transcriptionally active natural G/C-centric PSel-κB, but adopts closed conformation when bound to the mutant A/T and swap DNAs due to their narrowed minor grooves. Our binding assays further demonstrate that the fast kinetics favored by entropy is correlated with higher transcriptional activity. Overall, our studies have revealed a novel conformation for κB DNA in complex with NF-κB and pinpoint the importance of binding kinetics, dictated by DNA conformational and dynamic states, in controlling transcriptional activation for NF-κB.
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Evaluation Summary:
This manuscript provides a detailed structural and biophysical characterization of several complexes of the p52 homodimer of NF kB and different DNA binding sites. The main topic is the investigation of why the central base pair(s) have a strong influence on the transcriptional activity of the homodimer. The authors correlate structural changes with measurements of kinetic on and off rates to develop a model that explains the differences in activity. The paper is of interest to all working on understanding how transcriptional activity is regulated.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Meshcheryakov et al present a compelling story showing that upon binding to DNA, an NF-kB homodimer induces distinct conformations in DNA, regardless of the DNA sequence or their level of transcriptional activity. To understand how the protein achieves transcriptional selectivity for distinct sequences, authors use simulations, binding assays, and kinetic studies. They observe a correlation between kinetics and transcriptional activation, showing that binding to active DNA sequences is driven by entropy and occurs without a large deformation of the DNA. Conversely, binding to DNA sequences with lower transcriptional activity requires more significant alteration of DNA conformation and is largely enthalpic with slower kinetics. These studies uncover an important role of binding kinetics in DNA selectivity by …
Reviewer #1 (Public Review):
Meshcheryakov et al present a compelling story showing that upon binding to DNA, an NF-kB homodimer induces distinct conformations in DNA, regardless of the DNA sequence or their level of transcriptional activity. To understand how the protein achieves transcriptional selectivity for distinct sequences, authors use simulations, binding assays, and kinetic studies. They observe a correlation between kinetics and transcriptional activation, showing that binding to active DNA sequences is driven by entropy and occurs without a large deformation of the DNA. Conversely, binding to DNA sequences with lower transcriptional activity requires more significant alteration of DNA conformation and is largely enthalpic with slower kinetics. These studies uncover an important role of binding kinetics in DNA selectivity by transcription factors that should motivate many future studies to further explore and elucidate these mechanisms.
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Reviewer #2 (Public Review):
The overall conclusions of this work are: i) p52 homodimers are able to recognize kB sites with a G at the central position by naturally fitting the wider minor groove found in those sites, ii) binding affinity of p52 to kB sites is poorly correlated with transcriptional activity but the entropic contribution to binding is correlated, iii) higher on and off rates for p52/kB binding are associated with more transcriptionally active kB sites. The results are very interesting and address important questions that underlie mechanisms of transcriptional regulation.
The authors provide data to support their conclusions, but in some cases, the data and interpretations are not entirely convincing. The structural data support a wider minor groove for the PSel kB site, but it is not clear how p52 recognizes this …
Reviewer #2 (Public Review):
The overall conclusions of this work are: i) p52 homodimers are able to recognize kB sites with a G at the central position by naturally fitting the wider minor groove found in those sites, ii) binding affinity of p52 to kB sites is poorly correlated with transcriptional activity but the entropic contribution to binding is correlated, iii) higher on and off rates for p52/kB binding are associated with more transcriptionally active kB sites. The results are very interesting and address important questions that underlie mechanisms of transcriptional regulation.
The authors provide data to support their conclusions, but in some cases, the data and interpretations are not entirely convincing. The structural data support a wider minor groove for the PSel kB site, but it is not clear how p52 recognizes this difference. The loss of cross-strand hydrogen bonds near the center of the site is noted, but the addition of a canonical Arg-Gua interaction is shown but not discussed. A higher level of asymmetry in the dimer-DNA interface is described, but it is not clear how this relates to the central 3 bp in question.
The colorimetric studies are fascinating. A range of binding enthalpies from -5 to -15 kcal/mol are reported for the four kB sites studied and a range of -5 to +5 kcal/mol for the enthalpic contribution to binding (TΔS). The more favorable entropic free energy of binding is associated with kB sites that are more transcriptionally active. It is unclear what the source of these wide variations might be; the authors do not attempt to explain the results in light of the structural models.
Binding kinetic studies are also revealing and interesting. The more transcriptionally active kB sites (e.g., PSel kB) have faster on-rates and faster off-rates. The least active kB sites (e.g., MHC) have lower on-rates and lower off-rates. The authors suggest that increased resident time of p52/Bcl3 on the promoter is associated with lower transcriptional activity; a possible reason is that longer times allow the recruitment of co-repressors. The binding affinities derived from BLI experiments are more than 50-fold lower than those measured by ITC. Assuming that the ITC values are correct, this raises questions about what aspects of the kinetic measurements are valid and which might not be.
Overall, I think this is an interesting and important study, but the connections between the structural studies and the binding experiments need to be strengthened and there are several questions/issues about the individual experiments and interpretations that need to be resolved.
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Reviewer #3 (Public Review):
Understanding how transcriptional activity is regulated is a very important topic. The authors provide a detailed structural and biophysical characterization of several complexes of the p52 homodimer of NF kB and different DNA binding sites. The focus is on the central base pair as well as the flanking base pairs on both sides. By x-ray crystallography the authors show that the minor grove of the central base pairs is widened with the G:C base pairs compared to the A:T base pairs. Using MD simulations of the DNA the authors show that DNA molecules can adopt different conformations and the binding of the p52 homodimer induces the least conformational changes in the naturally occurring sequence. The authors go further and correlate these conformational changes with binding affinity and with kinetic kon and …
Reviewer #3 (Public Review):
Understanding how transcriptional activity is regulated is a very important topic. The authors provide a detailed structural and biophysical characterization of several complexes of the p52 homodimer of NF kB and different DNA binding sites. The focus is on the central base pair as well as the flanking base pairs on both sides. By x-ray crystallography the authors show that the minor grove of the central base pairs is widened with the G:C base pairs compared to the A:T base pairs. Using MD simulations of the DNA the authors show that DNA molecules can adopt different conformations and the binding of the p52 homodimer induces the least conformational changes in the naturally occurring sequence. The authors go further and correlate these conformational changes with binding affinity and with kinetic kon and koff measurements. Overall, there is little correlation between affinity and transcriptional activity. The only correlation they observe is between fast on and off kinetics and higher transcriptional activity. This result is surprising and does not immediately provide a mechanistic interpretation of how high transcriptional activity is achieved. The authors try to provide an explanation via binding of co-repressors but the fundamental biophysical investigations for such a model are missing at the moment.
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