Exclusivity and Compensation in NFκB Dimer Distributions and IκB Inhibition.

The NFκB transcription factor family members RelA, p50, and cRel form homo- and heterodimers that are inhibited by IκBα, IκBß, and IκBε. These NFκB family members have diverse biological functions, and their expression profiles differ, leading to different concentrations in different tissue types. Here we present definitive biophysical measurements of the NFκB dimer affinities and inhibitor affinities to better understand dimer exchange and how the presence of inhibitors may alter the equilibrium concentrations of NFκB dimers in the cellular context. Fluorescence anisotropy binding experiments were performed at low concentrations to mimic intracellular concentrations. We report binding affinities much stronger than those that had been previously reported by non-equilibrium gel shift and analytical ultracentrifugation assays. The results reveal a wide range of NFκB dimer affinities and a strong preference of each IκB for a small subset of NFκB dimers. Once the preferred IκB is bound, dimer exchange no longer occurs over a period of days. A mathematical model of the cellular distribution of these canonical NFκB transcription factors based on the revised binding affinities recapitulates intracellular observations and provides simple, precise explanations for observed cellular phenomena.

Dual allosteric activation mechanisms in monomeric human glucokinase.

Cooperativity in human glucokinase (GCK), the body's primary glucose sensor and a major determinant of glucose homeostatic diseases, is fundamentally different from textbook models of allostery because GCK is monomeric and contains only one glucose-binding site. Prior work has demonstrated that millisecond timescale order-disorder transitions within the enzyme's small domain govern cooperativity. Here, using limited proteolysis, we map the site of disorder in unliganded GCK to a 30-residue active-site loop that closes upon glucose binding. Positional randomization of the loop, coupled with genetic selection in a glucokinase-deficient bacterium, uncovers a hyperactive GCK variant with substantially reduced cooperativity. Biochemical and structural analysis of this loop variant and GCK variants associated with hyperinsulinemic hypoglycemia reveal two distinct mechanisms of enzyme activation. In α-type activation, glucose affinity is increased, the proteolytic susceptibility of the active site loop is suppressed and the (1)H-(13)C heteronuclear multiple quantum coherence (HMQC) spectrum of (13)C-Ile-labeled enzyme resembles the glucose-bound state. In ß-type activation, glucose affinity is largely unchanged, proteolytic susceptibility of the loop is enhanced, and the (1)H-(13)C HMQC spectrum reveals no perturbation in ensemble structure. Leveraging both activation mechanisms, we engineer a fully noncooperative GCK variant, whose functional properties are indistinguishable from other hexokinase isozymes, and which displays a 100-fold increase in catalytic efficiency over wild-type GCK. This work elucidates specific structural features responsible for generating allostery in a monomeric enzyme and suggests a general strategy for engineering cooperativity into proteins that lack the structural framework typical of traditional allosteric systems.

Binding of NFκB Appears to Twist the Ankyrin Repeat Domain of IκBα.

Total internal reflection fluorescence-based single-molecule Förster resonance energy transfer (FRET) measurements were previously carried out on the ankyrin repeat domain (ARD) of IκBα, the temporally regulated inhibitor of canonical NFκB signaling. Under native conditions, most of the IκBα molecules showed stable, high FRET signals consistent with distances between the fluorophores estimated from the crystal structures of the NFκB(RelA/p50)-IκBα complex. Similar high FRET efficiencies were found when the IκBα molecules were either free or in complex with NFκB(RelA/p50), and were interpreted as being consistent with the crystallographically observed ARD structure. An exception to this was observed when the donor and acceptor fluorophores were attached in AR3 (residue 166) and AR6 (residue 262). Surprisingly, the FRET efficiency was lower for the bound IκBα molecules (0.67) than for the free IκBα molecules (0.74), apparently indicating that binding of NFκB(RelA/p50) stretches the ARD of IκBα. Here, we conducted confocal-based single-molecule FRET studies to investigate this phenomenon in greater detail. The results not only recapitulated the apparent stretching of the ARD but also showed that the effect was more pronounced when the N-terminal domains (NTDs) of both RelA and p50 were present, even though the interface between NFκB(RelA/p50) and IκBα encompasses only the dimerization domains. We also performed mass spectrometry-detected amide hydrogen/deuterium exchange (HDXMS) experiments on IκBα as well as IκBα bound to dimerization-domain-only constructs or full-length NFκB(RelA/p50). Although we expected the stretched IκBα to have regions with increased exchange, instead the HDXMS experiments showed decreases in exchange in AR3 and AR6 that were more pronounced when the NFκB NTDs were present. Simulations of the interaction recapitulated the increased distance between residues 166 and 262, and also provide a plausible mechanism for a twisting of the IκBα ARD induced by interactions of the IκBα proline-glutamate-serine-threonine-rich sequence with positively charged residues in the RelA NTD.

Unraveling paralog-specific Notch signaling through thermodynamics of ternary complex formation and transcriptional activation of chimeric receptors.

Notch signaling in humans is mediated by four paralogous receptors that share conserved architectures and possess overlapping, yet non-redundant functions. The receptors share a canonical activation pathway wherein upon extracellular ligand binding, the Notch intracellular domain (NICD) is cleaved from the membrane and translocates to the nucleus where its N-terminal RBP-j-associated molecule (RAM) region and ankyrin repeat (ANK) domain bind transcription factor CSL and recruit co-activator Mastermind-like-1 (MAML1) to activate transcription. However, different paralogs can lead to distinct outcomes. To better understand paralog-specific differences in Notch signaling, we performed a thermodynamic analysis of the Notch transcriptional activation complexes for all four Notch paralogs using isothermal titration calorimetry. Using chimeric constructs, we find that the RAM region is the primary determinant of stability of binary RAMANK:CSL complexes, and that the ANK regions are largely the determinants of MAML1 binding to pre-formed RAMANK:CSL complexes. Free energies of these binding reactions (ΔGRA and ΔGMAML) vary among the four Notch paralogs, although variations for Notch2, 3, and 4 offset in the free energy of the ternary complex (ΔGTC, where ΔGTC = ΔGRA + ΔGMAML). To probe how these affinity differences affect Notch signaling, we performed transcriptional activation assays with the paralogous and chimeric NICDs, and analyzed the results with an independent multiplicative model that quantifies contributions of the paralogous RAM, ANK, and C-terminal regions (CTR) to activation. This analysis shows that transcription activation correlates with ΔGTC, but that activation is further modified by CTR identity in a paralog-specific way.

Structural features of the Notch ankyrin domain-Deltex WWE2 domain heterodimer determined by NMR spectroscopy and functional implications.

The Notch signaling pathway, an important cell fate determination pathway, is modulated by the ubiquitin ligase Deltex. Here, we investigate the structural basis for Deltex-Notch interaction. We used nuclear magnetic resonance (NMR) spectroscopy to assign the backbone of the Drosophila Deltex WWE2 domain and mapped the binding site of the Notch ankyrin (ANK) domain to the N-terminal WWEA motif. Using cultured Drosophila S2R+ cells, we find that point substitutions within the ANK-binding surface of Deltex disrupt Deltex-mediated enhancement of Notch transcriptional activation and disrupt ANK binding in cells and in vitro. Likewise, ANK substitutions that disrupt Notch-Deltex heterodimer formation in vitro block disrupt Deltex-mediated stimulation of Notch transcription activation and diminish interaction with full-length Deltex in cells. Surprisingly, the Deltex-Notch intracellular domain (NICD) interaction is not disrupted by deletion of the Deltex WWE2 domain, suggesting a secondary Notch-Deltex interaction. These results show the importance of the WWEA:ANK interaction in enhancing Notch signaling.

Prediction of the presence of a seventh ankyrin repeat in IκBε from homology modeling combined with hydrogen-deuterium exchange mass spectrometry (HDX-MS).

The ankyrin repeat (AR) structure is a common protein-protein interaction motif and ankyrin repeat proteins comprise a vast family across a large array of different taxa. Natural AR proteins adopt a conserved fold comprised of several repeats with the N- and C-terminal repeats generally being of more divergent sequences. Obtaining experimental crystal structures for natural ankyrin repeat domains (ARD) can be difficult and often requires complexation with a binding partner. Homology modeling is an attractive method for creating a model of AR proteins due to the highly conserved fold; however, modeling the divergent N- and C-terminal "capping" repeats remains a challenge. We show here that amide hydrogen/deuterium exchange mass spectrometry (HDX-MS), which reports on the presence of secondary structural elements and "foldedness," can aid in the refinement and selection of AR protein homology models when multiple templates are identified with variations between them localizing to these terminal repeats. We report a homology model for the AR protein IκBε from three different templates and use HDX-MS to establish the presence of a seventh AR at the C-terminus identified by only one of the three templates used for modeling.

RNAs interact with BRD4 to promote enhanced chromatin engagement and transcription activation.

The bromodomain and extra-terminal motif (BET) protein BRD4 binds to acetylated histones at enhancers and promoters via its bromodomains (BDs) to regulate transcriptional elongation. In human colorectal cancer cells, we found that BRD4 was recruited to enhancers that were co-occupied by mutant p53 and supported the synthesis of enhancer-directed transcripts (eRNAs) in response to chronic immune signaling. BRD4 selectively associated with eRNAs that were produced from BRD4-bound enhancers. Using biochemical and biophysical methods, we found that BRD4 BDs function cooperatively as docking sites for eRNAs and that the BDs of BRD2, BRD3, BRDT, BRG1, and BRD7 directly interact with eRNAs. BRD4-eRNA interactions increased BRD4 binding to acetylated histones in vitro and augmented BRD4 enhancer recruitment and transcriptional cofactor activities. Our results suggest a mechanism by which eRNAs are directly involved in gene regulation by modulating enhancer interactions and transcriptional functions of BRD4.

DNA and IκBα Both Induce Long-Range Conformational Changes in NFκB.

We recently discovered that IκBα enhances the rate of release of nuclear factor kappa B (NFκB) from DNA target sites in a process we have termed molecular stripping. Coarse-grained molecular dynamics simulations of the stripping pathway revealed two mechanisms for the enhanced release rate: the negatively charged PEST region of IκBα electrostatically repels the DNA, and the binding of IκBα appears to twist the NFκB heterodimer so that the DNA can no longer bind. Here, we report amide hydrogen/deuterium exchange data that reveal long-range allosteric changes in the NFκB (RelA-p50) heterodimer induced by DNA or IκBα binding. The data suggest that the two Ig-like subdomains of each Rel-homology region, which are connected by a flexible linker in the heterodimer, communicate in such a way that when DNA binds to the N-terminal DNA-binding domains, the nuclear localization signal becomes more highly exchanging. Conversely, when IκBα binds to the dimerization domains, amide exchange throughout the DNA-binding domains is decreased as if the entire domain is becoming globally stabilized. The results help understand how the subtle mechanism of molecular stripping actually occurs.