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

Transcription factor binding to a single binding site 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. Further-more, 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

Receptor binding and escape from Beta antibody responses drive Omicron-B.1.1.529 evolution

On the 24th November 2021 the sequence of a new SARS CoV-2 viral isolate spreading rapidly in Southern Africa was announced. Omicron contains a total of 30 substitutions plus deletions and an insertion in Spike, far more than any previously reported variant. The mutations include those previously identified by In-vitro evolution to contribute to high-affinity binding to ACE2, including mutations Q498R and N501Y critical in forming additional interactions in the interface. Together with increased charge complementarity between the RBD and ACE2, these substantially increase affinity and potentially virus transmissibility through increased syncytia formation. Further mutations promote immune evasion. We have studied the binding of a large panel of potent monoclonal antibodies generated from early pandemic or Beta infected cases. Mutations in Omicron will likely compromise the binding of many of these and additionally, the binding of antibodies under commercial development, however residual binding should provide protection from severe disease.

Ecdysteroids

Ecdysteroid: member of a class of polyhydroxylated steroids found in invertebrate animals (zooecdysteroids; moulting hormones), plants (phytoecdysteroids) and fungi (mycoecdysteroids). Over 500 structural analogues are currently known. Biosynthetically, they derive from C27-, C28- or C29-sterols. The most frequently encountered analogue (in arthropods and plants) is 20-hydroxyecdysone (2β,3β,14α, 20R,22R,25-hexahydroxycholest-7-en-6-one). In arthropods, ecdysteroids occur universally and regulate development by inducing moulting and reproduction, where their action is mediated by high-affinity binding to an intracellular member of the class of nuclear receptor (NR) proteins (ecdysteroid receptor; EcR) dimerised with a second NR (USP/RxR). This receptor complex binds to specific DNA promoter sites and regulates gene expression. In plants, ecdysteroids are a class of secondary compounds, occurring in varying amounts in certain species, but not all in others. Phytoecdysteroids are believed to contribute to the reduction of invertebrate predation by acting as feeding deterrents or endocrine disruptors. Ecdysteroids also possess a wide range of positive pharmacological effects in mammals, where the mode of action involves moderate-affinity binding to plasma-membrane-bound receptors and not interaction with the classical NRs for vertebrate steroid hormones.

Multifaceted N-degron recognition and ubiquitylation by GID/CTLH E3 ligases

N-degron E3 ubiquitin ligases recognize specific residues at the N-termini of substrates. Although molecular details of N-degron recognition are known for several E3 ligases, the range of N-terminal motifs that can bind a given E3 substrate binding domain remains unclear. Here, studying the Gid4 and Gid10 substrate receptor subunits of yeast GID/human CTLH multiprotein E3 ligases, whose known substrates bear N-terminal prolines, we discovered capacity for high-affinity binding to diverse N-terminal sequences determined in part by context. Screening of phage displaying peptide libraries with exposed N-termini identified novel consensus motifs with non-Pro N-terminal residues distinctly binding Gid4 or Gid10 with high affinity. Structural data reveal that flexible loops in Gid4 and Gid10 conform to complementary folds of diverse interacting peptide sequences. Together with analysis of endogenous substrate degrons, the data show that degron identity, substrate domains harboring targeted lysines, and varying E3 ligase higher-order assemblies combinatorially determine efficiency of ubiquitylation and degradation.

Zinc Trafficking 1. Probing the Roles of Proteome, Metallothionein, and Glutathione

The cellular trafficking pathways that conduct zinc to its sites of binding in functional proteins remain largely unspecified. In this study, the hypothesis was investigated that non-specific proteomic binding sites serve as intermediates in zinc trafficking. Proteome from pig kidney LLC-PK1 cells contains a large concentration of such sites, displaying an average conditional stability constant of 1010-11, that are dependent on sulfhydryl ligands to achieve high affinity binding of zinc. As a result, the proteome competes effectively with induced metallothionein for Zn2+ upon exposure of cells to extracellular Zn2+ or during in vitro direct competition. The reaction of added Zn2+ bound to proteome with apo-carbonic anhydrase was examined as a potential model for intracellular zinc trafficking. The extent of this reaction was inversely dependent upon proteome concentration and under cellular conditions thought to be negligible. The rate of reaction was strictly first order in both Zn2+ and apo-carbonic anhydrase and also considered to be insignificant in cells. Adding the low molecular weight fraction of cell supernatant to the proteome markedly enhanced the speed of this reaction, a phenomenon dependent on the presence of glutathione. In agreement, inclusion of glutathione accelerated the reaction in a concentration-dependent manner. The implications of abundant high affinity binding sites for Zn2+ within the proteome are considered in relation to their interaction with glutathione in the efficient delivery of Zn2+ to functional binding sites and in the operation of fluorescent zinc sensors as a tool to observe zinc trafficking.