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As transcription factors (TFs) play a major role in gene regulation, we studied their binding motifs (positional weight matrices, PWMs) and binding sites (TFBSs) in the human genome, and how TFs bind DNA motifs, including the involvement of binding co-factors. Using the chromatin immunoprecipitation sequencing data recently released by ENCODE (Encyclopedia of DNA Elements), we obtained new PWMs for 196 TFs and revised PWMs for 119 TFs. From these and the PWMs previously obtained for 235 TFs, we inferred the canonical PWMs for 500 TFs, including 243 new PWMs. Analysis revealed that most TFBSs are in introns (42.6%) and intergenic regions (31.6%), with only 11.3% in promoters. However, the TFBS density is considerably higher in promoters, showing a bell-shaped distribution of TFBSs with a peak at the transcription start site. Many TFBSs lie close to CTCF (CCCTC-binding factor) binding sites. Tethered binding is far more frequent than co-binding, with the latter often requiring co-factors.

Most general anaesthetics and classical benzodiazepine drugs act through positive modulation of γ-aminobutyric acid type A (GABA A ) receptors to dampen neuronal activity in the brain 1 – 5 . However, direct structural information on the mechanisms of general anaesthetics at their physiological receptor sites is lacking. Here we present cryo-electron microscopy structures of GABA A receptors bound to intravenous anaesthetics, benzodiazepines and inhibitory modulators. These structures were solved in a lipidic environment and are complemented by electrophysiology and molecular dynamics simulations. Structures of GABA A receptors in complex with the anaesthetics phenobarbital, etomidate and propofol reveal both distinct and common transmembrane binding sites, which are shared in part by the benzodiazepine drug diazepam. Structures in which GABA A receptors are bound by benzodiazepine-site ligands identify an additional membrane binding site for diazepam and suggest an allosteric mechanism for anaesthetic reversal by flumazenil. This study provides a foundation for understanding how pharmacologically diverse and clinically essential drugs act through overlapping and distinct mechanisms to potentiate inhibitory signalling in the brain. Cryo-electron microscopy structures of GABA A receptors bound to intravenous anaesthetics and benzodiazepines reveal both common and distinct transmembrane binding sites, and show that the mechanisms of action of anaesthetics partially overlap with those of benzodiazepines.

Molecular communication in biology is mediated by protein interactions. According to the current paradigm, the specificity and affinity required for these interactions are encoded in the precise complementarity of binding interfaces. Even proteins that are disordered under physiological conditions or that contain large unstructured regions commonly interact with well-structured binding sites on other biomolecules. Here we demonstrate the existence of an unexpected interaction mechanism: the two intrinsically disordered human proteins histone H1 and its nuclear chaperone prothymosin-α associate in a complex with picomolar affinity, but fully retain their structural disorder, long-range flexibility and highly dynamic character. On the basis of closely integrated experiments and molecular simulations, we show that the interaction can be explained by the large opposite net charge of the two proteins, without requiring defined binding sites or interactions between specific individual residues. Proteome-wide sequence analysis suggests that this interaction mechanism may be abundant in eukaryotes. A high-affinity complex of histone H1 and prothymosin-α reveals an unexpected interaction mechanism, where the large opposite net charge enables the two proteins to remain highly disordered even in the complex. High-affinity complexes of disordered proteins Disordered protein regions have been increasingly implicated in high-affinity protein–protein interactions. However, once the resulting protein complexes have formed, at least one interacting protein partner has been found to be stably folded. Using a suite of independent biophysical approaches, Ben Schuler and colleagues reveal a ultrahigh-affinity (picomolar) complex between two proteins (histone H1 and its nuclear chaperone prothymosin-α) that both remain fully disordered when bound to each other. High-affinity binding results from numerous, dynamic and non-specific electrostatic interactions along the extended chains of the highly charged polypeptides. This structural feature is prevalent among signalling molecules in eukaryotes, including in humans.

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is initiated by virus binding to the ACE2 cell-surface receptors 1 – 4 , followed by fusion of the virus and cell membranes to release the virus genome into the cell. Both receptor binding and membrane fusion activities are mediated by the virus spike glycoprotein 5 – 7 . As with other class-I membrane-fusion proteins, the spike protein is post-translationally cleaved, in this case by furin, into the S1 and S2 components that remain associated after cleavage 8 – 10 . Fusion activation after receptor binding is proposed to involve the exposure of a second proteolytic site (S2′), cleavage of which is required for the release of the fusion peptide 11 , 12 . Here we analyse the binding of ACE2 to the furin-cleaved form of the SARS-CoV-2 spike protein using cryo-electron microscopy. We classify ten different molecular species, including the unbound, closed spike trimer, the fully open ACE2-bound trimer and dissociated monomeric S1 bound to ACE2. The ten structures describe ACE2-binding events that destabilize the spike trimer, progressively opening up, and out, the individual S1 components. The opening process reduces S1 contacts and unshields the trimeric S2 core, priming the protein for fusion activation and dissociation of ACE2-bound S1 monomers. The structures also reveal refolding of an S1 subdomain after ACE2 binding that disrupts interactions with S2, which involves Asp614 13 – 15 and leads to the destabilization of the structure of S2 proximal to the secondary (S2′) cleavage site. Cryo-electron microscopy structures of consecutive binding events of ACE2 in complex with the spike protein of SARS-CoV-2 reveal the mechanisms of receptor binding by the spike protein and activation for membrane fusion by the spike protein of SARS-CoV-2.

Modeling the impact of amino acid mutations on protein-protein interaction plays a crucial role in protein engineering and drug design. In this study, we develop GeoPPI, a novel structure-based deep-learning framework to predict the change of binding affinity upon mutations. Based on the three-dimensional structure of a protein, GeoPPI first learns a geometric representation that encodes topology features of the protein structure via a self-supervised learning scheme. These representations are then used as features for training gradient-boosting trees to predict the changes of protein-protein binding affinity upon mutations. We find that GeoPPI is able to learn meaningful features that characterize interactions between atoms in protein structures. In addition, through extensive experiments, we show that GeoPPI achieves new state-of-the-art performance in predicting the binding affinity changes upon both single- and multi-point mutations on six benchmark datasets. Moreover, we show that GeoPPI can accurately estimate the difference of binding affinities between a few recently identified SARS-CoV-2 antibodies and the receptor-binding domain (RBD) of the S protein. These results demonstrate the potential of GeoPPI as a powerful and useful computational tool in protein design and engineering. Our code and datasets are available at: https://github.com/Liuxg16/GeoPPI .

Although organelles compartmentalize eukaryotic cells, they can communicate and integrate their activities by connecting at membrane contact sites (MCSs). The roles of MCSs in biology are becoming increasingly clear, with MCSs now known to function in intracellular signalling, lipid metabolism, membrane dynamics, organelle biogenesis and the cellular stress response.

RNA-binding proteins are key regulators of gene expression, yet only a small fraction have been functionally characterized. Here we report a systematic analysis of the RNA motifs recognized by RNA-binding proteins, encompassing 205 distinct genes from 24 diverse eukaryotes. The sequence specificities of RNA-binding proteins display deep evolutionary conservation, and the recognition preferences for a large fraction of metazoan RNA-binding proteins can thus be inferred from their RNA-binding domain sequence. The motifs that we identify in vitro correlate well with in vivo RNA-binding data. Moreover, we can associate them with distinct functional roles in diverse types of post-transcriptional regulation, enabling new insights into the functions of RNA-binding proteins both in normal physiology and in human disease. These data provide an unprecedented overview of RNA-binding proteins and their targets, and constitute an invaluable resource for determining post-transcriptional regulatory mechanisms in eukaryotes. This study reports a global analysis of binding sites for over 200 RNA-binding proteins (RBPs) from 24 species; conserved RNA-binding motifs are identified, and their analysis allows prediction of interaction sites based on the sequence of the RNA-binding domain alone. RNA-binding protein targets The sequence and context of RNA that dictate the interaction of RNA-binding proteins with their targets have tended to be studied on a protein-by-protein basis. A study by Timothy Hughes and colleagues now reports a global analysis of binding sites for more than 200 RNA-binding proteins from 24 eukaryote species. Conserved RNA-binding motifs are identified, and their analysis allows for the prediction of interaction sites on the basis of the RNA-binding domain sequence alone. The motifs also are found to reflect each molecule's function, which will aid in understanding the roles of previously uncharacterized examples.

We describe Cleavage Under Targets and Release Using Nuclease (CUT&RUN), a chromatin profiling strategy in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing. Unlike Chromatin Immunoprecipitation (ChIP), which fragments and solubilizes total chromatin, CUT&RUN is performed in situ, allowing for both quantitative high-resolution chromatin mapping and probing of the local chromatin environment. When applied to yeast and human nuclei, CUT&RUN yielded precise transcription factor profiles while avoiding crosslinking and solubilization issues. CUT&RUN is simple to perform and is inherently robust, with extremely low backgrounds requiring only ~1/10th the sequencing depth as ChIP, making CUT&RUN especially cost-effective for transcription factor and chromatin profiling. When used in conjunction with native ChIP-seq and applied to human CTCF, CUT&RUN mapped directional long range contact sites at high resolution. We conclude that in situ mapping of protein-DNA interactions by CUT&RUN is an attractive alternative to ChIP-seq. The DNA in a person’s skin cell will contain the same genes as the DNA in their muscle or brain cells. However, these cells have different identities because different genes are active in skin, muscle and brain cells. Proteins called transcription factors dictate the patterns of gene activation in the different kinds of cells by binding to DNA and switching nearby genes on or off. Transcription factors interact with other proteins such as histones that help to package DNA into a structure known as chromatin. Together, transcription factors, histones and other chromatin-associated proteins determine whether or not nearby genes are active. Sometimes transcription factors and other chromatin-associated proteins bind to the wrong sites on DNA; this situation can lead to diseases in humans, such as cancer. This is one of the many reasons why researchers are interested in working out where specific DNA-binding proteins are located in different situations. A technique called chromatin immunoprecipitation (or ChIP for short) can be used to achieve this goal, yet despite being one of the most widely used techniques in molecular biology, ChIP is hampered by numerous problems. As such, many researchers are keen to find alternative approaches. Skene and Henikoff have now developed a new method, called CUT&RUN (which is short for “Cleavage Under Targets & Release Using Nuclease”) to map specific interactions between protein and DNA in a way that overcomes some of the problems with ChIP. In CUT&RUN, unlike in ChIP, the DNA in the starting cells does not need to be broken up first; this means that protein-DNA interactions are more likely to be maintained in their natural state. With CUT&RUN, as in ChIP, a specific antibody identifies the protein of interest. But in CUT&RUN, this antibody binds to the target protein in intact cells and cuts out the DNA that the protein is bound to, releasing the DNA fragment from the cell. This new strategy allows the DNA fragments to be sequenced and identified more efficiently than is currently possible with ChIP. Skene and Henikoff showed that their new method could more accurately identify where transcription factors bind to DNA from yeast and human cells. CUT&RUN also identified a specific histone that is rarely found in yeast chromatin and the technique can be used with a small number of starting cells. Given the advantages that CUT&RUN offers over ChIP, Skene and Henikoff anticipate that the method will be viewed as a cost-effective and versatile alternative to ChIP. In future, the method could be automated so that multiple analyses can be performed at once.