Jenifer K. Lum

Backbone-Driven Collapse in Unfolded Protein Chains

Collapse of unfolded protein chains is an early event in folding. It affects structural properties of intrinsically disordered proteins, which take a considerable fraction of the human proteome. Collapse is generally believed to be driven by hydrophobic forces imposed by the presence of nonpolar amino acid side chains. Contributions from backbone hydrogen bonds to protein folding and stability, however, are controversial. To date, the experimental dissection of side-chain and backbone contributions has not yet been achieved because both types of interactions are integral parts of protein structure. Here, we realized this goal by applying mutagenesis and chemical modification on a set of disordered peptides and proteins. We measured the protein dimensions and kinetics of intra-chain diffusion of modified polypeptides at the level of individual molecules using fluorescence correlation spectroscopy, thereby avoiding artifacts commonly caused by aggregation of unfolded protein material in bulk. We found no contributions from side chains to collapse but, instead, identified backbone interactions as a source sufficient to form globules of native-like dimensions. The presence of backbone hydrogen bonds decreased polypeptide water solubility dramatically and accelerated the nanosecond kinetics of loop closure, in agreement with recent predictions from computer simulation. The presence of side chains, instead, slowed loop closure and modulated the dimensions of intrinsically disordered domains. It appeared that the transient formation of backbone interactions facilitates the diffusive search for productive conformations at the early stage of folding and within intrinsically disordered proteins.

Long-Range Modulation of Chain Motions within the Intrinsically Disordered Transactivation Domain of Tumor Suppressor p53

The tumor suppressor p53 is a hub protein with a multitude of binding partners, many of which target its intrinsically disordered N-terminal domain, p53-TAD. Partners, such as the N-terminal domain of MDM2, induce formation of local structure and leave the remainder of the domain apparently disordered. We investigated segmental chain motions in p53-TAD using fluorescence quenching of an extrinsic label by tryptophan in combination with fluorescence correlation spectroscopy (PET-FCS). We studied the loop closure kinetics of four consecutive segments within p53-TAD and their response to protein binding and phosphorylation. The kinetics was multiexponential, showing that the conformational ensemble of the domain deviates from random coil, in agreement with previous findings from NMR spectroscopy. Phosphorylations or binding of MDM2 changed the pattern of intrachain kinetics. Unexpectedly, we found that upon binding and phosphorylation chain motions were altered not only within the targeted segments but also in remote regions. Long-range interactions can be induced in an intrinsically disordered domain by partner proteins that induce apparently only local structure or by post-translational modification.

S100 proteins interact with the N‐terminal domain of MDM2

MINT‐7905185, MINT‐7905347: s100A2 (uniprotkb:P29034) and p53 (uniprotkb:P04637) bind (MI:0407) by molecular sieving (MI:0071)

A High-Resolution Interaction Map of Three Transcriptional Activation Domains with a Key Coactivator from Photo-Cross-Linking and Multiplexed Mass Spectrometry

Transcriptional activators play a central role in gene regulation, stimulating the assembly of the transcriptional machinery at a promoter in a signal responsive manner.[1] Because of the significant role that mis-regulated or malfunctioning transcriptional activators play in human disease, there has been a strong effort towards identifying molecules that inhibit the ability of an activator to function via binding interactions with the transcriptional machinery.[2–4] One difficulty associated with this strategy is that distinct transcriptional activators often share binding partners within the transcriptional machinery, making the design or discovery of molecules specific for a particular transcription factor quite challenging.[5] For example, the activators p53, c-Myc and the viral oncoprotein E1A all interact with the coactivator and chromatin modifier TRRAP, yet it is not known if these activators utilize a shared binding surface for activation or if there are several distinct binding sites within the coactivator.[6–8] Covalent cross-linking has been successfully used to identify coactivator binding partners of activators, but high resolution analysis for binding site identification has not been reported;[9–15] a real challenge to high resolution analysis is the acid-rich composition of many eukaryotic activators, a characteristic that renders them difficult to analyze by standard cross-linking and mass spectrometric strategies.[16–20] Here we describe a multiplexed mass spectrometric strategy to produce for the first time a high-resolution map of the binding sites of transcriptional activators, focusing on three prototypical activators (Gal4, Gcn4, and VP16) in complex with the key coactivator Med15(Gal11) (Figure 1). These data in combination with genetic experiments demonstrate that while Gal4 and Gcn4 target the same binding surfaces within Med15, the VP16-derived activator utilizes a distinct binding surface. This finding suggests that some degree of inhibitor specificity for a given activator or activator class may indeed be achievable. Further, this general approach can be applied to a broader range of activator-coactivator binding partners to expand the high-resolution interaction map of transcriptional activators.

Transient-state Kinetic Analysis of Transcriptional Activator·DNA Complexes Interacting with a Key Coactivator

Several lines of evidence suggest that the prototypical amphipathic transcriptional activators Gal4, Gcn4, and VP16 interact with the key coactivator Med15 (Gal11) during transcription initiation despite little sequence homology. Recent cross-linking data further reveal that at least two of the activators utilize the same binding surface within Med15 for transcriptional activation. To determine whether these three activators use a shared binding mechanism for Med15 recruitment, we characterized the thermodynamics and kinetics of Med15·activator·DNA complex formation by fluorescence titration and stopped-flow techniques. Combination of each activator·DNA complex with Med15 produced biphasic time courses. This is consistent with a minimum two-step binding mechanism composed of a bimolecular association step limited by diffusion, followed by a conformational change in the Med15·activator·DNA complex. Furthermore, the equilibrium constant for the conformational change (K2) correlates with the ability of an activator to stimulate transcription. VP16, the most potent of the activators, has the largest K2 value, whereas Gcn4, the least potent, has the smallest value. This correlation is consistent with a model in which transcriptional activation is regulated at least in part by the rearrangement of the Med15·activator·DNA ternary complex. These results are the first detailed kinetic characterization of the transcriptional activation machinery and provide a framework for the future design of potent transcriptional activators.

Artificial Transcriptional Activation Domains

utilizing the same genomic content to generate unique gene expression profiles and many different cell types. Given the critical nature of this process, it is not surprising that misregulation at any step can result in aberrant cellular function. Often, this misregulation can be traced back to a malfunctioning transcriptional regulator. For example, in medulloblastoma, one of the most malignant pediatric cancers, the concentration of the transcriptional repressor REST/NRSF is abnormally high, resulting in the suppression of genes critical for proper differentiation of neuronal cells. 4] However, up-regulation of the REST/NRSF-controlled genes abrogates the tumorigenic potential of treated cells. 6] Thus there is growing interest in the identification of molecules that can selectively activate the expression of targeted genes, so-called artificial transcriptional activators (ATAs). ATAs are outstanding tools for increasing our understanding of the role of aberrant transcription patterns in disease and might provide a future basis for therapeutic intervention.

Disrupting Mediator with a Short Peptide Ligand

Although RNA polymerase II is the core enzyme that carries out transcription, it requires a large number of accessory proteins and protein complexes to regulate its activity. One of the most important of these complexes is Mediator, a group of 20–25 protein components that is highly conserved from Saccharomyces cerevisiae through to humans. The yeast Mediator is subdivided into three portions: a head region that contacts RNA polymerase II, a middle region, and a tail (Figure 1).