Binding site | EPFL Graph Search

In biochemistry and molecular biology, a binding site is a region on a macromolecule such as a protein that binds to another molecule with specificity. The binding partner of the macromolecule is often referred to as a ligand. Ligands may include other proteins (resulting in a protein-protein interaction), enzyme substrates, second messengers, hormones, or allosteric modulators. The binding event is often, but not always, accompanied by a conformational change that alters the protein's function. Binding to protein binding sites is most often reversible (transient and non-covalent), but can also be covalent reversible or irreversible. Function Binding of a ligand to a binding site on protein often triggers a change in conformation in the protein and results in altered cellular function. Hence binding site on protein are critical parts of signal transduction pathways. Types of ligands include neurotransmitters, toxins, neuropeptides, and steroid hormones. Binding sites in

Systematic analysis of biomolecular binding reactions using microfluidic systems

Amir Shahein

Transcription factor binding to a single binding site on DNA and its functional consequence in a promoter context are beginning to be relatively well understood. However, binding to clusters of sites has yet to be characterized in depth, and the functional relevance of binding site clusters remains uncertain. We employed a high-throughput biochemical method to characterize transcription factor binding to clusters varying across a range of affinities and configurations. We found that transcription factors can bind concurrently to overlapping sites, challenging the notion of binding exclusivity. Furthermore, compared to an individual high-affinity binding site, small clusters with binding sites an order of magnitude lower in affinity give rise to higher mean occupancies at physiologically-relevant transcription factor concentrations in vitro. To assess whether the observed in vitro occupancies translate to transcriptional activation in vivo, we tested low-affinity binding site clusters by inserting them into a synthetic minimal CYC1 and the native PHO5 S. cerevisiae promoter. In the minCYC1 promoter, clusters of low-affinity binding sites can generate transcriptional output comparable to a promoter containing three consensus binding sites. In the PHO5 promoter, replacing the native Pho4 binding sites with clusters of low-affinity binding sites recovered activation of these promoters as well. This systematic characterization demonstrates that clusters of low-affinity binding sites achieve substantial occupancies, and that this occupancy can drive expression in eukaryotic promoters. In the antibody discovery phase, before advancing a particular antibody sequence to clinical trials, a large number of candidates are characterized for properties of therapeutic interest. Above all, this includes their ability to bind to the target antigen. Standard processes currently in use to characterize antibody binding are either only semi-quantitative (ELISA), or low-throughput (SPR). We developed an experimental process to first retrieve DNA encoding promising synthetic antibody variants directly from the output pools of phage and ribosome display methods, followed by expressing these variants on a microfluidic chip for characterization in a fluorescence-based binding assay. We applied our method to characterize the binding of retrieved Sybody variants against the Spike trimer and RBD domain of the SARS-CoV-2 variant. After an initial proof-of-concept, we improved our method by removing bottlenecks, reducing costs, and incorporating a large degree of process automation to enable measuring binding affinities at scale.

EPFL2022

Systematic analysis of low-affinity transcription factor binding site clusters in vitro and in vivo establishes their functional relevance

Shiyu Cheng, Ivan Istomin, Maria Lopez Malo, Sebastian Maerkl, Evan James Olson, Amir Shahein

Binding to binding site clusters has yet to be characterized in depth, and the functional relevance of low-affinity clusters remains uncertain. We characterized transcription factor binding to low-affinity clusters in vitro and found that transcription factors can bind concurrently to overlapping sites, challenging the notion of binding exclusivity. Furthermore, small clusters with binding sites an order of magnitude lower in affinity give rise to high mean occupancies at physiologically-relevant transcription factor concentrations. To assess whether the observed in vitro occupancies translate to transcriptional activation in vivo, we tested low-affinity binding site clusters in a synthetic and native gene regulatory network in S. cerevisiae. In both systems, clusters of low-affinity binding sites generated transcriptional output comparable to single or even multiple consensus sites. This systematic characterization demonstrates that clusters of low-affinity binding sites achieve substantial occupancies, and that this occupancy can drive expression in eukaryotic promoters.

NATURE PORTFOLIO2022

RNA-Protein interactions at AU-rich elements

Ramona Pauchard-Batschulat

The expression of many proto-oncogenes, nuclear transcription factors and cytokines is regulated post-transcriptionally by specific interactions between distinct cis-elements within the mRNA and trans-acting factors that bind to these elements and thereby either promote or inhibit the rapid decay of the mRNA. This kind of regulation enables the cell to rapidly respond to changes in the cellular environment. These elements are usually located within the 3’UTR of the mRNA, rich in adenylate and uridylate and called AU-rich elements or AREs. To date, many of these trans-acting factors have been determined and among the best studied are the stabilizing HuR, the rather destabilizing AUF1 and the destabilizing KSRP and TTP. Previous studies have suggested that these ARE-BPs as well as yet unidentified factors might have overlapping target specificities and bind either in a concurrent, concomitant or maybe an even cooperative fashion to their targets to regulate mRNA half-life. However, the exact manner of how these factors might interact and how these interactions might be influenced, is largely unknown. Thus, my first project aimed to study the interactions between AUF1, KSRP and HuR in the context of the 3’UTRs of RANKL and Bcl-6 that were recently identified as AUF1 targets with a short half-life. By combining ARE-BP immunoprecipitations with qRT-PCR analysis of co-precipitated reporter RNAs, we were able to gain interesting insight into the RNA-protein interactions at AU-rich elements. However, this approach is very laborious and can study RNA-protein interactions only within a limited context. Yet, the pools of mRNA targets as well as binding factors are very multifaceted and likely depend on the cell type and condition as well as the cellular environment. Thus, a much broader approach is necessary to reveal the entire network of RNA-protein and protein-protein interactions at AU-rich elements. A promising technique is the combination of ARE-BP immunoprecipitation and high-throughput sequencing to analyze co-precipitated RNA. This approach allows for the extraction of a lot of important data from a single experiment as for example the virtually complete pool of target mRNAs under the studied conditions, the binding sites within these mRNAs as well as the frequency with which these sequences were detected. Based on these data, it would be possible to make suggestions about the binding specificities as well as potential, yet un-identified targets. If accumulating data sets for different cellular or environmental conditions, we might gain insight into the regulation of these RNA-protein interactions. In a similar way, the comparison of data sets for different ARE-binding proteins would give us further insight into the protein-protein interactions at and around AU-rich elements. Therefore, my second project focused on the development of a novel protocol that allows for the rapid, reliable and reproducible generation of high-throughput sequencing samples based on co-immunoprecipitated RNA fragments.

EPFL2013