OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA , 114 ( 8 ) ISSN 0027-8424

Significance Stress-response transcription factors turn on hundreds of genes, and their activity must be turned off completely and quickly. The inhibitor protein IκBα turns off NFκB by forming a transient ternary complex with the NFκB–DNA complex and then promoting DNA dissociation (molecular stripping). Here we report a mutant IκBα that is impaired in its ability to strip NFκB from DNA. The mutant forms a more stable ternary complex, and biophysical characterization shows what IκBα looks like “in the act of stripping.” We also show in single-cell nuclear export assays that the decrease in the rate of DNA dissociation from the mutant ternary complex matches the decrease in the rate of nuclear NFκB export in cells. Stress-response transcription factors such as NFκB turn on hundreds of genes and must have a mechanism for rapid cessation of transcriptional activation. We recently showed that the inhibitor of NFκB signaling, IκBα, dramatically accelerates the dissociation of NFκB from transcription sites, a process we have called “stripping.” To test the role of the IκBα C-terminal PEST (rich in proline, glutamic acid, serine, and threonine residues) sequence in NFκB stripping, a mutant IκBα was generated in which five acidic PEST residues were mutated to their neutral analogs. This IκBα(5xPEST) mutant was impaired in stripping NFκB from DNA and formed a more stable intermediate ternary complex than that formed from IκBα(WT) because DNA dissociated more slowly. NMR and amide hydrogen–deuterium exchange mass spectrometry showed that the IκBα(5xPEST) appears to be “caught in the act of stripping” because it is not yet completely in the folded and NFκB-bound state. When the mutant was introduced into cells, the rate of postinduction IκBα-mediated export of NFκB from the nucleus decreased markedly.

[1]  A. Hoffmann,et al.  CK2 Is a C-Terminal IkappaB Kinase Responsible for NF-kappaB Activation during the UV Response. , 2003, Molecular cell.

[2]  Elizabeth A Komives,et al.  Folding kinetics of the cooperatively folded subdomain of the IκBα ankyrin repeat domain. , 2011, Journal of molecular biology.

[3]  L. Iakoucheva,et al.  Predicted disorder-to-order transition mutations in IκBα disrupt function. , 2014, Physical chemistry chemical physics : PCCP.

[4]  H. Dyson,et al.  Detection of a ternary complex of NF-κB and IκBα with DNA provides insights into how IκBα removes NF-κB from transcription sites , 2011, Proceedings of the National Academy of Sciences.

[5]  A. Hoffmann,et al.  The IkappaB-NF-kappaB signaling module: temporal control and selective gene activation. , 2002, Science.

[6]  Davit A Potoyan,et al.  Molecular stripping in the NF-κB/IκB/DNA genetic regulatory network , 2015, Proceedings of the National Academy of Sciences.

[7]  G. Ghosh,et al.  The crystal structure of the IkappaBalpha/NF-kappaB complex reveals mechanisms of NF-kappaB inactivation. , 1998, Cell.

[8]  B. Aggarwal,et al.  Nuclear factor-kappaB: its role in health and disease. , 2004, Journal of molecular medicine.

[9]  E. Komives,et al.  The IκBα/NF‐κB complex has two hot spots, one at either end of the interface , 2008, Protein science : a publication of the Protein Society.

[10]  A. Hoffmann,et al.  A Regulated, Ubiquitin-Independent Degron in IκBα. , 2015, Journal of molecular biology.

[11]  S. Joseph,et al.  Direct observation of a transient ternary complex during IκBα-mediated dissociation of NF-κB from DNA , 2013, Proceedings of the National Academy of Sciences.

[12]  S. Joseph,et al.  Binding of mRNA to the bacterial translation initiation complex. , 2007, Methods in enzymology.

[13]  G. Ghosh,et al.  The Crystal Structure of the IκBα/NF-κB Complex Reveals Mechanisms of NF-κB Inactivation , 1998, Cell.

[14]  S. Harrison,et al.  Structure of an IκBα/NF-κB Complex , 1998, Cell.

[15]  H. Dyson,et al.  Transfer of flexibility between ankyrin repeats in IkappaB* upon formation of the NF-kappaB complex. , 2008, Journal of molecular biology.

[16]  Terence Hwa,et al.  Transcriptional regulation by the numbers: models. , 2005, Current opinion in genetics & development.

[17]  Elizabeth A. Komives,et al.  Regions of IκBα that are critical for its inhibition of NF-κB·DNA interaction fold upon binding to NF-κB , 2006, Proceedings of the National Academy of Sciences.

[18]  John R Engen,et al.  High-speed and high-resolution UPLC separation at zero degrees Celsius. , 2008, Analytical chemistry.

[19]  Davit A. Potoyan,et al.  PEST Control of Molecular Stripping of NFκB from DNA Transcription Sites. , 2016, The journal of physical chemistry. B.

[20]  Elizabeth A Komives,et al.  Long-range effects and functional consequences of stabilizing mutations in the ankyrin repeat domain of IκBα. , 2013, Journal of molecular biology.

[21]  P. Wolynes,et al.  Binding of NFκB Appears to Twist the Ankyrin Repeat Domain of IκBα. , 2016, Biophysical journal.

[22]  D. Baltimore,et al.  I kappa B: a specific inhibitor of the NF-kappa B transcription factor. , 1988, Science.

[23]  David Baltimore,et al.  IkappaBbeta acts to inhibit and activate gene expression during the inflammatory response. , 2010 .

[24]  Diego U. Ferreiro,et al.  Molecular Mechanisms of System Control of NF-κB Signaling by IκBα , 2009 .

[25]  H. Dyson,et al.  Interaction of the IkappaBalpha C-terminal PEST sequence with NF-kappaB: insights into the inhibition of NF-kappaB DNA binding by IkappaBalpha. , 2009, Journal of molecular biology.

[26]  Alexander Hoffmann,et al.  Anatomy of a negative feedback loop: the case of IκBα , 2015, Journal of The Royal Society Interface.

[27]  J. Mccammon,et al.  Functional Dynamics of the Folded Ankyrin Repeats of IκBα Revealed by Nuclear Magnetic Resonance† , 2009, Biochemistry.

[28]  J. Ragoussis,et al.  Principles of dimer-specific gene regulation revealed by a comprehensive characterization of NF-κB family DNA binding , 2011, Nature Immunology.

[29]  David Baltimore,et al.  IκBβ acts to both inhibit and activate gene expression at different stages of the inflammatory response , 2010, Nature.

[30]  E. Komives,et al.  Thermodynamics reveal that helix four in the NLS of NF-kappaB p65 anchors IkappaBalpha, forming a very stable complex. , 2006, Journal of molecular biology.

[31]  E. Komives,et al.  Structural characterization of the ternary complex that mediates termination of NF-κB signaling by IκBα , 2016, Proceedings of the National Academy of Sciences.

[32]  A. Hoffmann,et al.  The I (cid:1) B –NF-(cid:1) B Signaling Module: Temporal Control and Selective Gene Activation , 2022 .

[33]  M J May,et al.  NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses. , 1998, Annual review of immunology.

[34]  E. Komives,et al.  Thermodynamics Reveal that Helix Four in the NLS of NF-κB p65 Anchors IκBα, Forming a Very Stable Complex , 2006 .

[35]  A. Hoffmann,et al.  CK2 Is a C-Terminal IκB Kinase Responsible for NF-κB Activation during the UV Response , 2003 .

[36]  Melinda M. Mulvihill,et al.  Protein interactions among Fe65, the low-density lipoprotein receptor-related protein, and the amyloid precursor protein. , 2011, Biochemistry.

[37]  A. Hoffmann,et al.  Circuitry of nuclear factor kappaB signaling. , 2006, Immunological reviews.

[38]  E. Komives,et al.  Flexible Regions within IκBα Create the Ubiquitin-independent Degradation Signal* , 2010, The Journal of Biological Chemistry.

[39]  S. Harrison,et al.  Structure of an IkappaBalpha/NF-kappaB complex. , 1998, Cell.

[40]  Elizabeth A Komives,et al.  Regions of IkappaBalpha that are critical for its inhibition of NF-kappaB.DNA interaction fold upon binding to NF-kappaB. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[41]  Bharat B. Aggarwal,et al.  Nuclear factor-κB: its role in health and disease , 2004, Journal of Molecular Medicine.