Stabilizing IκBα by “Consensus” Design

IkappaBalpha is the major regulator of transcription factor NF-kappaB function. The ankyrin repeat region of IkappaBalpha mediates specific interactions with NF-kappaB dimers, but ankyrin repeats 1, 5 and 6 display a highly dynamic character when not in complex with NF-kappaB. Using chemical denaturation, we show here that IkappaBalpha displays two folding transitions: a non-cooperative conversion under weak perturbation, and a major cooperative folding phase upon stronger insult. Taking advantage of a native Trp residue in ankyrin repeat (AR) 6 and engineered Trp residues in AR2, AR4 and AR5, we show that the cooperative transition involves AR2 and AR3, while the non-cooperative transition involves AR5 and AR6. The major structural transition can be affected by single amino acid substitutions converging to the "consensus" ankyrin repeat sequence, increasing the native state stability significantly. We further characterized the structural and dynamic properties of the native state ensemble of IkappaBalpha and the stabilized mutants by H/(2)H exchange mass spectrometry and NMR. The solution experiments were complemented with molecular dynamics simulations to elucidate the microscopic origins of the stabilizing effect of the consensus substitutions, which can be traced to the fast conformational dynamics of the folded ensemble.

[1]  Kristian M Müller,et al.  Structural perturbation and compensation by directed evolution at physiological temperature leads to thermostabilization of beta-lactamase. , 2005, Biochemistry.

[2]  D. Barford,et al.  Molecular recognition via coupled folding and binding in a TPR domain. , 2005, Journal of molecular biology.

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

[4]  S. Ghosh,et al.  X-ray Crystal Structure of an IκBβ·NF-κB p65 Homodimer Complex* , 2003, Journal of Biological Chemistry.

[5]  H. Dyson,et al.  Insights into the structure and dynamics of unfolded proteins from nuclear magnetic resonance. , 2002, Advances in protein chemistry.

[6]  Z. Peng,et al.  Consensus-derived structural determinants of the ankyrin repeat motif , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[7]  S. Nanduri,et al.  The structural basis of ankyrin-like repeat function as revealed by the solution structure of myotrophin. , 1998, Structure.

[8]  A. Baldwin,et al.  THE NF-κB AND IκB PROTEINS: New Discoveries and Insights , 1996 .

[9]  C. Pace.  Determination and analysis of urea and guanidine hydrochloride denaturation curves. , 1986, Methods in enzymology.

[10]  S. Smerdon,et al.  The ankyrin repeat: a diversity of interactions on a common structural framework. , 1999, Trends in biochemical sciences.

[11]  T. N. Bhat,et al.  The Protein Data Bank , 2000, Nucleic Acids Res..

[12]  E. Komives,et al.  Solvent exposed non-contacting amino acids play a critical role in NF-kappaB/IkappaBalpha complex formation. , 2002, Journal of molecular biology.

[13]  A. Fersht.  Structure and mechanism in protein science , 1998 .

[14]  B. Zhang,et al.  A minimum folding unit in the ankyrin repeat protein p16(INK4). , 2000, Journal of molecular biology.

[15]  C L Brooks,et al.  Taking a Walk on a Landscape , 2001, Science.

[16]  C. Pace,et al.  How to measure and predict the molar absorption coefficient of a protein , 1995, Protein science : a publication of the Protein Society.

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

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

[19]  G. R. Taylor,et al.  PCR: a practical approach. , 1991 .

[20]  L. Mosavi,et al.  Structure-based substitutions for increased solubility of a designed protein. , 2003, Protein engineering.

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

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

[23]  A. Fersht,et al.  Stability and folding of the tumour suppressor protein p16. , 1999, Journal of molecular biology.

[24]  G. Ghosh,et al.  The role of DNA in the mechanism of NFkappaB dimer formation: crystal structures of the dimerization domains of the p50 and p65 subunits. , 1997, Structure.

[25]  J. Onuchic,et al.  Funnels, pathways, and the energy landscape of protein folding: A synthesis , 1994, Proteins.

[26]  M. Tsai,et al.  Solution structure of the human oncogenic protein gankyrin containing seven ankyrin repeats and analysis of its structure--function relationship. , 2004, Biochemistry.

[27]  J. Mandell,et al.  Phosphorylation causes subtle changes in solvent accessibility at the interdomain interface of methylesterase CheB. , 2001, Journal of molecular biology.

[28]  E. Komives,et al.  Biophysical characterization of the free IkappaBalpha ankyrin repeat domain in solution. , 2004, Protein science : a publication of the Protein Society.

[29]  Doug Barrick,et al.  Limits of cooperativity in a structurally modular protein: response of the Notch ankyrin domain to analogous alanine substitutions in each repeat. , 2002, Journal of molecular biology.

[30]  Benjamin A. Shoemaker,et al.  Speeding molecular recognition by using the folding funnel: the fly-casting mechanism. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[31]  J. Lakowicz.  Principles of fluorescence spectroscopy , 1983 .

[32]  Michael Feig,et al.  MMTSB Tool Set: enhanced sampling and multiscale modeling methods for applications in structural biology. , 2004, Journal of molecular graphics & modelling.

[33]  A. Plückthun,et al.  High-affinity binders selected from designed ankyrin repeat protein libraries , 2004, Nature Biotechnology.

[34]  Doug Barrick,et al.  An experimentally determined protein folding energy landscape. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[35]  P. Wolynes,et al.  The energy landscapes and motions of proteins. , 1991, Science.

[36]  N. A. Rodionova,et al.  Study of the “molten globule” intermediate state in protein folding by a hydrophobic fluorescent probe , 1991, Biopolymers.

[37]  G. Ghosh,et al.  IκBβ, but Not IκBα, Functions as a Classical Cytoplasmic Inhibitor of NF-κB Dimers by Masking Both NF-κB Nuclear Localization Sequences in Resting Cells* , 2001, The Journal of Biological Chemistry.

[38]  D. Barrick,et al.  Studies of the ankyrin repeats of the Drosophila melanogaster Notch receptor. 2. Solution stability and cooperativity of unfolding. , 2001, Biochemistry.

[39]  Andreas Plückthun,et al.  Designing repeat proteins: well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins. , 2003, Journal of molecular biology.

[40]  A. Plückthun,et al.  Folding of a designed simple ankyrin repeat protein , 2004, Protein science : a publication of the Protein Society.

[41]  E. Komives,et al.  Solvent Exposed Non-contacting Amino Acids Play a Critical Role in NF-κB/IκBα Complex Formation , 2002 .

[42]  S. Joo,et al.  An NF-kappaB-specific inhibitor, IkappaBalpha, binds to and inhibits cyclin-dependent kinase 4. , 2003, Biochemistry.

[43]  A. Baldwin,et al.  The NF-kappa B and I kappa B proteins: new discoveries and insights. , 1996, Annual review of immunology.

[44]  J. Mandell,et al.  Measurement of amide hydrogen exchange by MALDI-TOF mass spectrometry. , 1998, Analytical chemistry.

[45]  M. Karplus,et al.  CHARMM: A program for macromolecular energy, minimization, and dynamics calculations , 1983 .

[46]  Daniel C. Desrosiers,et al.  The ankyrin repeat as molecular architecture for protein recognition , 2004, Protein science : a publication of the Protein Society.

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

[48]  David Baltimore,et al.  NF-κB: Ten Years After , 1996, Cell.

[49]  A. Baldwin.  Series introduction: the transcription factor NF-kappaB and human disease. , 2001, The Journal of clinical investigation.

[50]  Alexander D. MacKerell,et al.  All-atom empirical potential for molecular modeling and dynamics studies of proteins. , 1998, The journal of physical chemistry. B.

[51]  Andreas Plückthun,et al.  Consensus Design of Repeat Proteins , 2004, Chembiochem : a European journal of chemical biology.

[52]  G. Ghosh,et al.  IKAPPABALPHA/NF-KAPPAB COMPLEX , 1999 .

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

[54]  Peer Bork,et al.  SMART 4.0: towards genomic data integration , 2004, Nucleic Acids Res..

[55]  E. Komives,et al.  Biophysical characterization of the free IκBα ankyrin repeat domain in solution , 2004 .

[56]  A. Baldwin.  Series Introduction: The transcription factor NF-κB and human disease , 2001 .

[57]  M. McPherson,et al.  PCR 2 : a practical approach , 2016 .

[58]  Sophie E Jackson,et al.  The folding and design of repeat proteins: reaching a consensus. , 2003, Current opinion in structural biology.

[59]  Peter G Wolynes,et al.  The energy landscape of modular repeat proteins: topology determines folding mechanism in the ankyrin family. , 2005, Journal of molecular biology.

[60]  Nuclear magnetic resonance assignment and secondary structure of an ankyrin‐like repeat‐bearing protein: Myotrophin , 1997, Protein science : a publication of the Protein Society.

[61]  A. Plückthun,et al.  Designed to be stable: Crystal structure of a consensus ankyrin repeat protein , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[62]  B. Zimm,et al.  Theory of the Phase Transition between Helix and Random Coil in Polypeptide Chains , 1959 .

[63]  Sophie E Jackson,et al.  A recurring theme in protein engineering: the design, stability and folding of repeat proteins. , 2005, Current opinion in structural biology.

[64]  David Baltimore,et al.  Genetic analysis of NF‐κB/Rel transcription factors defines functional specificities , 2003, The EMBO journal.

[65]  Tommi Kajander,et al.  A new folding paradigm for repeat proteins. , 2005, Journal of the American Chemical Society.

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

[67]  G. Ghosh,et al.  Preparation and Crystallization of Dynamic NF-κB·IκB Complexes* , 2000, The Journal of Biological Chemistry.

[68]  G. Ghosh,et al.  IkappaBbeta, but not IkappaBalpha, functions as a classical cytoplasmic inhibitor of NF-kappaB dimers by masking both NF-kappaB nuclear localization sequences in resting cells. , 2001, The Journal of biological chemistry.

[69]  Doug Barrick,et al.  Effect of multiple prolyl isomerization reactions on the stability and folding kinetics of the notch ankyrin domain: experiment and theory. , 2005, Journal of molecular biology.