Kristen M. Ramsey

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 (ΔG_RA and ΔG_MAML) vary among the four Notch paralogs, although variations for Notch2, 3, and 4 offset in the free energy of the ternary complex (ΔG_TC, where ΔG_TC = ΔG_RA + ΔG_MAML). 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 ΔG_TC, but that activation is further modified by CTR identity in a paralog‐specific way.

Summary: Notch signaling is mediated by four paralogous receptors with conserved architectures and overlapping yet non-redundant functions. Notch signaling generates a transcriptional activation complex (NTC) wherein the N-terminal RAM and ankyrin repeat (ANK) regions of the Notch intracellular domain (NICD) bind transcription factor CSL and recruit co-activator Mastermind-like (MAML). To better understand paralog-specific differences in Notch signaling, we analyzed the thermodynamics of binary and ternary NTCs for all four Notch paralogues and chimeric constructs. We find that RAMANK:CSL stability (ΔG _RA ) is primarily determined by the RAM region while MAML binding to preformed RAMANK:CSL complexes (ΔG _MAML ) is largely determined by the ANK region. We determined transcriptional activation data for the paralogous and chimeric NICDs and analyzed the data with an independent multiplicative model. This analysis shows ternary complex stability (ΔG _TC , where ΔG _TC = ΔG _RA +ΔG _MAML ) correlates well with transcriptional activations and provides insights into contributions of RAM, ANK and the C-terminal regions to Notch signaling.

The NFκB transcription factor family members RelA, p50, and cRel form homo- and heterodimers that are inhibited by IκBα, IκBβ, 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.

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 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 (HDXMS), 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 HDXMS to establish the presence of a seventh AR at the C‐terminus identified by only one of the three templates used for modeling.

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.

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 ¹H-¹³C heteronuclear multiple quantum coherence (HMQC) spectrum of ¹³C-Ile–labeled enzyme resembles the glucose-bound state. In β-type activation, glucose affinity is largely unchanged, proteolytic susceptibility of the loop is enhanced, and the ¹H-¹³C 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.