Surprising Features of Nuclear Receptor Interaction Networks Revealed by Live Cell Single Molecule Imaging.

Type 2 Nuclear Receptors (T2NRs) require heterodimerization with a common partner, the Retinoid X Receptor (RXR), to bind cognate DNA recognition sites in chromatin. Based on previous biochemical and over-expression studies, binding of T2NRs to chromatin is proposed to be regulated by competition for a limiting pool of the core RXR subunit. However, this mechanism has not yet been tested for endogenous proteins in live cells. Using single molecule tracking (SMT) and proximity-assisted photoactivation (PAPA), we monitored interactions between endogenously tagged retinoid X receptor (RXR) and retinoic acid receptor (RAR) in live cells. Unexpectedly, we find that higher expression of RAR, but not RXR increases heterodimerization and chromatin binding in U2OS cells. This surprising finding indicates the limiting factor is not RXR but likely its cadre of obligate dimer binding partners. SMT and PAPA thus provide a direct way to probe which components are functionally limiting within a complex TF interaction network providing new insights into mechanisms of gene regulation in vivo with implications for drug development targeting nuclear receptors.

Single-molecule tracking (SMT): a window into live-cell transcription biochemistry.

How molecules interact governs how they move. Single-molecule tracking (SMT) thus provides a unique window into the dynamic interactions of biomolecules within live cells. Using transcription regulation as a case study, we describe how SMT works, what it can tell us about molecular biology, and how it has changed our perspective on the inner workings of the nucleus. We also describe what SMT cannot yet tell us and how new technical advances seek to overcome its limitations. This ongoing progress will be imperative to address outstanding questions about how dynamic molecular machines function in live cells.

Recovering mixtures of fast-diffusing states from short single-particle trajectories.

Single-particle tracking (SPT) directly measures the dynamics of proteins in living cells and is a powerful tool to dissect molecular mechanisms of cellular regulation. Interpretation of SPT with fast-diffusing proteins in mammalian cells, however, is complicated by technical limitations imposed by fast image acquisition. These limitations include short trajectory length due to photobleaching and shallow depth of field, high localization error due to the low photon budget imposed by short integration times, and cell-to-cell variability. To address these issues, we investigated methods inspired by Bayesian nonparametrics to infer distributions of state parameters from SPT data with short trajectories, variable localization precision, and absence of prior knowledge about the number of underlying states. We discuss the advantages and disadvantages of these approaches relative to other frameworks for SPT analysis.

Promiscuous and Selective: How Intrinsically Disordered BH3 Proteins Interact with Their Pro-survival Partner MCL-1.

The BCL-2 family of proteins plays a central role in regulating cell survival and apoptosis. Disordered BH3-only proteins bind promiscuously to a number of different BCL-2 proteins, with binding affinities that vary by orders of magnitude. Here we investigate the basis for these differences in affinity. We show that eight different disordered BH3 proteins all bind to their BCL-2 partner (MCL-1) very rapidly, and that the differences in sequences result in different dissociation rates. Similarly, mutation of the binding surface of MCL-1 generally affects association kinetics in the same way for all BH3 peptides but has significantly different effects on the dissociation rates. Importantly, we infer that the evolution of homologous, competing interacting partners has resulted in complexes with significantly different lifetimes.

Phosphorylation of the IDP KID Modulates Affinity for KIX by Increasing the Lifetime of the Complex.

Intrinsically disordered proteins (IDPs) are known to undergo a range of posttranslational modifications, but by what mechanism do such modifications affect the binding of an IDP to its partner protein? We investigate this question using one such IDP, the kinase inducible domain (KID) of the transcription factor CREB, which interacts with the KIX domain of CREB-binding protein upon phosphorylation. As with many other IDPs, KID undergoes coupled folding and binding to form α-helical structure upon interacting with KIX. This single site phosphorylation plays an important role in the control of transcriptional activation in vivo. Here we show that, contrary to expectation, phosphorylation has no effect on association rates-unphosphorylated KID binds just as rapidly as pKID, the phosphorylated form-but rather, acts by increasing the lifetime of the complex. We propose that by controlling the lifetime of the bound complex of pKID:KIX via altering the dissociation rate, phosphorylation can facilitate effective control of transcription regulation.

pKID Binds to KIX via an Unstructured Transition State with Nonnative Interactions.

Understanding the detailed mechanism of interaction of intrinsically disordered proteins with their partners is crucial to comprehend their functions in signaling and transcription. Through its interaction with KIX, the disordered pKID region of CREB protein is central in the transcription of cAMP responsive genes, including those involved in long-term memory. Numerous simulation studies have investigated these interactions. Combined with experimental results, these can provide valuable and comprehensive understanding of the mechanisms involved. Here, we probe the transition state of this interaction experimentally through analyzing the kinetic effect of mutating both interface and solvent exposed residues in pKID. We show that very few specific interactions between pKID and KIX are required in the initial binding process. Only a small number of weak interactions are formed at the transition state, including nonnative interactions, and most of the folding occurs after the initial binding event. These properties are consistent with computational results and also the majority of experimental studies of intrinsically disordered protein coupled folding and binding in other protein systems, suggesting that these may be common features.

A critical role for the self-assembly of Amyloid-ß1-42 in neurodegeneration.

Amyloid ß1-42 (Aß1-42) plays a central role in Alzheimer's disease. The link between structure, assembly and neuronal toxicity of this peptide is of major current interest but still poorly defined. Here, we explored this relationship by rationally designing a variant form of Aß1-42 (vAß1-42) differing in only two amino acids. Unlike Aß1-42, we found that the variant does not self-assemble, nor is it toxic to neuronal cells. Moreover, while Aß1-42 oligomers impact on synaptic function, vAß1-42 does not. In a living animal model system we demonstrate that only Aß1-42 leads to memory deficits. Our findings underline a key role for peptide sequence in the ability to assemble and form toxic structures. Furthermore, our non-toxic variant satisfies an unmet demand for a closely related control peptide for Aß1-42 cellular studies of disease pathology, offering a new opportunity to decipher the mechanisms that accompany Aß1-42-induced toxicity leading to neurodegeneration.

Insights into Coupled Folding and Binding Mechanisms from Kinetic Studies.

Intrinsically disordered proteins (IDPs) are characterized by a lack of persistent structure. Since their identification more than a decade ago, many questions regarding their functional relevance and interaction mechanisms remain unanswered. Although most experiments have taken equilibrium and structural perspectives, fewer studies have investigated the kinetics of their interactions. Here we review and highlight the type of information that can be gained from kinetic studies. In particular, we show how kinetic studies of coupled folding and binding reactions, an important class of signaling event, are needed to determine mechanisms.