The regulation of transcription by RNA polymerase II is closely intertwined with the regulation of chromatin structure. A host of proteins required for the disassembly, reassembly, and modification of nucleosomes interacts with Pol II to aid its movement and counteract its disruptive effects on chromatin. The highly conserved Polymerase Associated Factor 1 Complex, Paf1C, travels with Pol II and exerts control over transcription elongation and chromatin structure, while broadly impacting the transcriptome in both single cell and multicellular eukaryotes. Recent studies have yielded exciting new insights into the mechanisms by which Paf1C regulates transcription elongation, epigenetic modifications, and post-transcriptional steps in eukaryotic gene expression. Importantly, these functional studies are now supported by an extensive foundation of high-resolution structural information, providing intimate views of Paf1C and its integration into the larger Pol II elongation complex. As a global regulatory factor operating at the interface between chromatin and transcription, the impact of Paf1C is broad and its influence reverberates into other domains of nuclear regulation, including genome stability, telomere maintenance, and DNA replication.

Chromatin remodelers act to regulate multiple cellular processes, such as transcription and DNA repair, by controlling access to genomic DNA. Four families of chromatin remodelers have been identified in yeast, each with non-redundant roles within the cell. There has been a recent surge in structural models of chromatin remodelers in complex with their nucleosomal substrate. These structural studies provide new insight into the mechanism of action for individual chromatin remodelers. In this review, we summarize available data for the structure and mechanism of action of the four chromatin remodeling complex families.

The highly conserved C-terminal domain (CTD) of the largest subunit of RNA polymerase II comprises a consensus heptad (Y1S2P3T4S5P6S7) repeated multiple times. Despite the simplicity of its sequence, the essential CTD domain orchestrates eukaryotic transcription and co-transcriptional processes, including transcription initiation, elongation, and termination, and mRNA processing. These distinct facets of the transcription cycle rely on specific post-translational modifications (PTM) of the CTD, in which five out of the seven residues in the heptad repeat are subject to phosphorylation. A hypothesis termed the "CTD code" has been proposed in which these PTMs and their combinations generate a sophisticated landscape for spatiotemporal recruitment of transcription regulators to Pol II. In this review, we summarize the recent experimental evidence understanding the biological role of the CTD, implicating a context-dependent theme that significantly enhances the ability of accurate transcription by RNA polymerase II. Furthermore, feedback communication between the CTD and histone modifications coordinates chromatin states with RNA polymerase II-mediated transcription, ensuring the effective and accurate conversion of information into cellular responses.

Regulation of transcription is a tightly choreographed process. The establishment of RNA polymerase II promoter proximal pausing soon after transcription initiation and the release of Pol II into productive elongation are key regulatory processes that occur in early elongation. We describe the techniques and tools that have become available for the study of promoter proximal pausing and their utility for future experiments. We then provide an overview of the factors and interactions that govern a multipartite pausing process and address emerging questions surrounding the mechanism of RNA polymerase II's subsequent advancement into the gene body. Finally, we address remaining controversies and future areas of study.

Pontin is a AAA+ ATPase protein that has functions in various biological contexts including gene transcription regulation, chromatin remodeling, DNA damage sensing and repair, as well as assembly of protein and ribonucleoprotein complexes. Pontin is known to regulate the transcription of several important signaling pathways, including Wnt signaling. However, its role in early embryonic signaling regulation remains unclear. Retinoic acid (RA) signaling plays a central role in vertebrate development. Using an in vivo biotin tagging technology, we mapped the genome-wide binding pattern of Pontin before and after RA-induced differentiation in the pluripotent embryo carcinoma cell line NTERA-2. Biotin ChIP-seq revealed significant changes in genome-wide Pontin binding sites upon RA stimulation. We also identified a substantial amount of overlapping binding peaks between Pontin and RARα, especially on all of the HOX gene loci (A-D clusters). Pontin knockdown experiments showed that its chromatin binding at the HOX gene clusters is required for RA-induced HOX gene expression. Furthermore, we performed Global Run-On sequencing (GRO-seq) to map de novo transcripts genome-wide and found that Pontin knockdown significantly diminished nascent HOX gene transcripts, indicating that Pontin regulates HOX gene expression at the transcriptional level. Finally, proteomic analysis demonstrated that Pontin associates with chromatin organization/remodeling complexes and various other functional complexes. Altogether, we have demonstrated that Pontin is a critical transcriptional co-activator for RA-induced HOX gene activation.

The general transcription factor TFIIH contains three ATP-dependent catalytic activities. TFIIH functions in nucleotide excision repair primarily as a DNA helicase and in Pol II transcription initiation as a dsDNA translocase and protein kinase. During initiation, the XPB/Ssl2 subunit of TFIIH couples ATP hydrolysis to dsDNA translocation facilitating promoter opening and the kinase module phosphorylates Pol II to facilitate the transition to elongation. These functions are conserved between metazoans and yeast; however, yeast TFIIH also drives transcription start-site scanning in which Pol II scans downstream DNA to locate productive start-sites. The ten-subunit holo-TFIIH from S. cerevisiae has a processive dsDNA translocase activity required for scanning and a structural role in scanning has been ascribed to the three-subunit TFIIH kinase module. Here, we assess the dsDNA translocase activity of ten-subunit holo- and core-TFIIH complexes (i.e. seven subunits, lacking the kinase module) from both S. cerevisiae and H. sapiens. We find that neither holo nor core human TFIIH exhibit processive translocation, consistent with the lack of start-site scanning in humans. Furthermore, in contrast to holo-TFIIH, the S. cerevisiae core-TFIIH also lacks processive translocation and its dsDNA-stimulated ATPase activity was reduced ~5-fold to a level comparable to the human complexes, potentially explaining the reported upstream shift in start-site observed in vitro in the absence of the S. cerevisiae kinase module. These results suggest that neither human nor S. cerevisiae core-TFIIH can translocate efficiently, and that the S. cerevisiae kinase module functions as a processivity factor to allow for robust transcription start-site scanning.

Although we have made significant progress, we still possess a limited understanding of how genomic and epigenomic information directs gene expression programs through sequence-specific transcription factors (TFs). Extensive research has settled on three general classes of TF targets in metazoans: promoter accessibility via chromatin regulation (e.g., SAGA), assembly of the general transcription factors on promoter DNA (e.g., TFIID), and recruitment of RNA polymerase (Pol) II (e.g., Mediator) to establish a transcription pre-initiation complex (PIC). Here we discuss TFs and their targets. We also place this in the context of our current work with Saccharomyces (yeast), where we find that promoters typically lack an architecture that supports TF function. Moreover, yeast promoters that support TF binding also display interactions with cofactors like SAGA and Mediator, but not TFIID. It is unknown to what extent all genes in metazoans require TFs and their cofactors.

Packaging of the eukaryotic genome into chromatin places fundamental physical constraints on transcription. Clarifying how transcription operates within these constraints is essential to understand how eukaryotic gene expression programs are established and maintained. Here we review what is known about the mechanisms of transcription on chromatin templates. Current models indicate that transcription through chromatin is accomplished by the combination of an inherent nucleosome disrupting activity of RNA polymerase and the action of ATP-dependent chromatin remodeling motors. Collaboration between these two types of molecular motors is proposed to occur at all stages of transcription through diverse mechanisms. Further investigation of how these two motors combine their basic activities is essential to clarify the interdependent relationship between genome structure and transcription.

The BAF chromatin remodeling complex is critical for genome regulation. The central ATPase of BAF is either BRM or BRG1, both of which contain a C-terminal bromodomain, known to associate with acetylated lysines. We have recently demonstrated that in addition to acetyl-lysine binding, the BRG1/BRM bromodomain can associate with DNA through a lysine/arginine rich patch that is adjacent to the acetyl-lysine binding pocket. Flanking the bromodomain is an AT-hook separated by a short, proline-rich linker. We previously found that the AT-hook and bromodomain can associate with DNA in a multivalent manner. Here, we investigate the conservation of this composite module and find that the AT-hook, linker, and lysine/arginine rich bromodomain patch are ancient, conserved over ~1 billion years. We utilize extensive mutagenesis, NMR spectroscopy, and fluorescence anisotropy to dissect the contribution of each of these conserved elements in association of this module with DNA. Our results reveal a structural and functional coupling of the AT-hook and bromodomain mediated by the linker. The lysine/arginine rich patch on the bromodomain and the conserved elements of the AT-hook are critical for robust affinity for DNA, while the conserved elements of the linker are dispensable for overall DNA affinity but critical for maintaining the relative conformation of the AT-hook and bromodomain in binding to DNA. This supports that the coupled action of the AT-hook and bromodomain are important for BAF activity.

Regulation of transcription by RNA Polymerase II (RNAPII) is a rapidly evolving area of research. Technological developments in microscopy have revealed insight into the dynamics, structure, and localization of transcription components within single cells. A frequent observation in many studies is the appearance of 'spots' in cell nuclei associated with the transcription process. In this review we highlight studies that characterize the temporal and spatial characteristics of these spots, examine possible pitfalls in interpreting these kind of imaging data, and outline directions where single-cell imaging may advance in ways to further our understanding of transcription regulation.

Folding of RNA into secondary structures through intramolecular base pairing determines an RNA's three-dimensional architecture and associated function. Simple RNA structures like stem loops can provide specialized functions independent of coding capacity, such as protein binding, regulation of RNA processing and stability, stimulation or inhibition of translation. RNA catalysis is dependent on tertiary structures found in the ribosome, tRNAs and group I and II introns. While the extent to which non-coding RNAs contribute to cellular maintenance is generally appreciated, the fact that both non-coding and coding RNA can assume relevant structural states has only recently gained attention. In particular, the co-transcriptional folding of nascent RNA of all classes has the potential to regulate co-transcriptional processing, RNP (ribonucleoprotein particle) formation, and transcription itself. Riboswitches are established examples of co-transcriptionally folded coding RNAs that directly regulate transcription, mainly in prokaryotes. Here we discuss recent studies in both prokaryotes and eukaryotes showing that structure formation may carry a more widespread regulatory logic during RNA synthesis. Local structures forming close to the catalytic center of RNA polymerases have the potential to regulate transcription by reducing backtracking. In addition, stem loops or more complex structures may alter co-transcriptional RNA processing or its efficiency. Several examples of functional structures have been identified to date, and this review provides an overview of physiologically distinct processes where co-transcriptionally folded RNA plays a role. Experimental approaches such as single-molecule FRET and in vivo structural probing to further advance our insight into the significance of co-transcriptional structure formation are discussed.

The transcription elongation factor Spt5 is conserved from bacteria to humans. In eukaryotes, Spt5 forms a complex with Spt4 and regulates processive transcription elongation. Recent studies on transcription elongation suggest different mechanistic roles in yeast versus mammals. Higher eukaryotes utilize Spt4-Spt5 (DSIF) to regulate promoter-proximal pausing, a transcription-regulatory mechanism that connects initiation to productive elongation. DSIF is a versatile transcription factor and has been implicated in both gene-specific regulation and transcription through nucleosomes. Future studies will further elucidate the role of DSIF in transcriptional dynamics and disentangle its inhibitory and enhancing activities in transcription.

Genetics is at the basis of cancer initiation and evolution, but emerging evidence indicates that mutations are not sufficient to produce cancer, indicating a role for epigenetic contributions to the different stages of tumorigenesis. While the genetic tracks of cancer have been widely investigated, the epigenetic "drivers" remain a vague definition. Gene-environment interactions can produce gene-regulatory programs that dictate pathogenesis; this implies a reciprocal relationship where environmental factors contribute to genetic mechanisms of tumorigenesis (i.e. mutagenesis) and genetic factors influence the cellular response to extrinsic stress. In this review article, we attempt to summarise the most remarkable findings demonstrating a contribution of epigenetic factors as proper "drivers" of tumorigenesis. We also try to pose attention on the relevance of epigenetic mechanisms as downstream consequences of genes versus environment interaction.

Antibodies provide a comprehensive record of the encounters with threats and insults to the immune system. The ability to examine the repertoire of antibodies in serum and discover those that best represent "discriminating features" characteristic of various clinical situations, is potentially very useful. Recently, phage display technologies combined with Next-Generation Sequencing (NGS) produced a powerful experimental methodology, coined "Deep-Panning", in which the spectrum of serum antibodies is probed. In order to extract meaningful biological insights from the tens of millions of affinity-selected peptides generated by Deep-Panning, advanced bioinformatics algorithms are a must. In this study, we describe Motifier, a computational pipeline comprised of a set of algorithms that systematically generates discriminatory peptide motifs based on the affinity-selected peptides identified by Deep-Panning. These motifs are shown to effectively characterize antibody binding activities and through the implementation of machine-learning protocols are shown to accurately classify complex antibody mixtures representing various biological conditions.

Ricin toxin kills mammalian cells with notorious efficiency. The toxin's B subunit (RTB) is a Gal/GalNAc-specific lectin that attaches to cell surfaces and promotes retrograde transport of ricin's A subunit (RTA) to the trans Golgi network (TGN) and endoplasmic reticulum (ER). RTA is liberated from RTB in the ER and translocated into the cell cytoplasm, where it functions as a ribosome-inactivating protein. While antibodies against ricin's individual subunits have been reported, we now describe seven alpaca-derived, single-domain antibodies (VHHs) that span the RTA-RTB interface, including four Tier 1 VHHs with IC50 values <1 nM. Crystal structures of each VHH bound to native ricin holotoxin revealed three different binding modes, based on contact with RTA's F-G loop (mode 1), RTB's subdomain 2γ (mode 2) or both (mode 3). VHHs in modes 2 and 3 were highly effective at blocking ricin attachment to HeLa cells and immobilized asialofetuin, due to framework residues (FR3) that occupied the 2γ Gal/GalNAc-binding pocket and mimic ligand. The four Tier 1 VHHs also interfered with intracellular functions of RTB, as they neutralized ricin in a post-attachment cytotoxicity assay (e.g., the toxin was bound to cell surfaces before antibody addition) and reduced the efficiency of toxin transport to the TGN. We conclude that the RTA-RTB interface is a target of potent toxin-neutralizing antibodies that interfere with both extracellular and intracellular events in ricin's cytotoxic pathway.

MPV17 is an integral inner mitochondrial membrane protein, whose loss-of-function is linked to the hepatocerebral form of the mitochondrial-DNA-depletion syndrome, leading to a tissue-specific reduction of mitochondrial DNA and organ failure in infants. Several disease-causing mutations in MPV17 have been identified and earlier studies with reconstituted protein suggest that MPV17 forms a high conductivity channel in the membrane. However, the molecular and structural basis of the MPV17 functionality remain only poorly understood. In order to make MPV17 accessible to high-resolution structural studies, we here present an efficient protocol for its high-level production in E. coli and refolding into detergent micelles. Using biophysical and NMR methods, we show that refolded MPV17 in detergent micelles adopts a compact structure consisting of six membrane-embedded α-helices. Furthermore, we demonstrate that MPV17 forms oligomers in a lipid bilayer that are further stabilized by disulfide-bridges. In line with these findings, MPV17 could only be inserted into lipid nanodiscs of 8-12 nm in diameter if intrinsic cysteines were either removed by mutagenesis or blocked by chemical modification. Using this nanodisc reconstitution approach, we could show that disease-linked mutations in MPV17 abolish its oligomerization properties in the membrane. These data suggest that, induced by oxidative stress, MPV17 can alter its oligomeric state from a properly folded monomer to a disulfide-stabilized oligomeric pore which might be required for the transport of metabolic DNA precursors into the mitochondrial matrix to compensate for the damage caused by reactive oxygen species.

The nucleocapsid protein is one of four structural proteins encoded by SARS-CoV-2 and plays a central role in packaging viral RNA and manipulating the host cell machinery, yet its dynamic behavior and promiscuity in nucleotide binding has made standard structural methods to address its atomic-resolution details difficult. To begin addressing the SARS-CoV-2 nucleocapsid protein interactions with both RNA and the host cell along with its dynamic behavior, we have specifically focused on the folded N-terminal domain (NTD) and its flanking regions using nuclear magnetic resonance solution studies. Studies performed here reveal a large repertoire of interactions, which includes a temperature-dependent self-association mediated by the disordered flanking regions that also serve as binding sites for host cell cyclophilin-A while nucleotide binding is largely mediated by the central NTD core. NMR studies that include relaxation experiments have revealed the complicated dynamic nature of this viral protein. Specifically, while much of the N-terminal core domain exhibits micro-millisecond motions, a central β-hairpin shows elevated inherent flexibility on the pico-nanosecond timescale and the serine/arginine-rich region of residues 176-209 undergoes multiple exchange phenomena. Collectively, these studies have begun to reveal the complexities of the nucleocapsid protein dynamics and its preferred interaction sites with its biological targets.

Bacterial NusG associates with RNA polymerase (RNAP) through its N-terminal domain, while the C-terminal domain (CTD) forms dynamic interactions with Rho, S10, NusB and NusA to affect transcription elongation. While virtually all bacteria encode for a core NusG, many also synthesize paralogs that transiently bind RNAP to alter expression of targeted genes. Yet, despite the importance of the genes they regulate, most of the subfamilies of NusG paralogs (e.g., UpxY, TaA, ActX and LoaP) have not been investigated in depth. Herein, we discover that LoaP requires a small RNA hairpin located within the 5' leader region of its targeted operons. LoaP binds the RNA element with nanomolar affinity and high specificity, in contrast to other NusG proteins, which have not been shown to exhibit RNA-binding activity. These data reveal a sequence feature that can be used to identify LoaP-regulated operons. This discovery also expands the repertoire of macromolecular interactions exhibited by the NusG CTD during transcription elongation to include an RNA ligand.

Glycans decorate the cell surface, secreted glycoproteins and glycolipids, and altered glycans are often found in cancers. Despite their high diagnostic and therapeutic potential, however, glycans are polar and flexible molecules that are quite challenging for the development and design of high-affinity binding antibodies. To understand the mechanisms by which glycan neoantigens are specifically recognized by antibodies, we analyze the biomolecular recognition of the tumor-associated carbohydrate antigen CA19-9 by two distinct antibodies using X-ray crystallography. Despite the potential plasticity of glycans and the very different antigen-binding surfaces presented by the antibodies, both structures reveal an essentially identical extended CA19-9 conformer, suggesting that this conformer's stability selects the antibodies. Starting from the bound structure of one of the antibodies, we use the AbLIFT computational algorithm to design a variant with seven core mutations in the variable domain's light-heavy chain interface that exhibits tenfold improved affinity for CA19-9. The results reveal strategies used by antibodies to specifically recognize glycan antigens and show how automated antibody-optimization methods may be used to enhance the clinical potential of existing antibodies.

Protein dynamics play a major role for the catalytic function of enzymes, the interaction of protein complexes or signal integration in regulatory proteins. In the context of multi-domain proteins involved in light-regulation of enzymatic effectors, the central role of conformational dynamics is well established. Light activation of sensory modules is followed by long-range signal transduction to different effectors; rather than domino-style structural rearrangements, a complex interplay of functional elements is required to maintain functionality. One family of such sensor-effector systems are red-light-regulated phytochromes that control diguanylate cyclases involved in cyclic-dimeric-GMP formation. Based on structural and functional studies of one prototypic family member, the central role of the coiled-coil sensor-effector linker was established. Interestingly, subfamilies with different linker lengths feature strongly varying biochemical characteristics. The dynamic interplay of the domains involved, however, is presently not understood. Here we show that the PHY domain dimer interface plays an essential role in signal integration, and that a functional coupling with the coiled-coil linker element is crucial. Chimaeras of two biochemically different family members highlight the phytochrome-spanning helical spine as an essential structural element involved in light-dependent upregulation of enzymatic turnover. However, isolated structural elements can frequently not be assigned to individual characteristics, which further emphasises the importance of global conformational dynamics. Our results provide insights into the intricate processes at play during light signal integration and transduction in these photosensory systems and thus provide additional guidelines for a more directed design of novel sensor-effector combinations with potential applications as optogenetic tools.

DNA glycosylases remove damaged or modified nucleobases by cleaving the N-glycosyl bond and the correct nucleotide is restored through subsequent base excision repair. In addition to excising threatening lesions, DNA glycosylases contribute to epigenetic regulation by mediating DNA demethylation and perform other important functions. However, the catalytic mechanism remains poorly defined for many glycosylases, including MBD4 (methyl-CpG binding domain IV), a member of the helix-hairpin-helix (HhH) superfamily. MBD4 excises thymine from G·T mispairs, suppressing mutations caused by deamination of 5-methylcytosine, and it removes uracil and modified uracils (e.g., 5-hydroxymethyluracil) mispaired with guanine. To investigate the mechanism of MBD4 we solved high-resolution structures of enzyme-DNA complexes at three stages of catalysis. Using a non-cleavable substrate analog, 2'-deoxy-pseudouridine, we determined the first structure of an enzyme-substrate complex for wild-type MBD4, which confirms interactions that mediate lesion recognition and suggests that a catalytic Asp, highly conserved in HhH enzymes, binds the putative nucleophilic water molecule and stabilizes the transition state. Observation that mutating the Asp (to Gly) reduces activity by 2700-fold indicates an important role in catalysis, but probably not one as the nucleophile in a double-displacement reaction, as previously suggested. Consistent with direct-displacement hydrolysis, a structure of the enzyme-product complex indicates a reaction leading to inversion of configuration. A structure with DNA containing 1-azadeoxyribose models a potential oxacarbenium-ion intermediate and suggests the Asp could facilitate migration of the electrophile towards the nucleophilic water. Finally, the structures provide detailed snapshots of the HhH motif, informing how these ubiquitous metal-binding elements mediate DNA binding.

Small heat shock proteins (sHsps) are a conserved class of ATP-independent chaperones which in stress conditions bind to unfolded protein substrates and prevent their irreversible aggregation. Substrates trapped in sHsps-containing aggregates are efficiently refolded into native structures by ATP-dependent Hsp70 and Hsp100 chaperones. Most γ-proteobacteria possess a single sHsp (IbpA), while in a subset of Enterobacterales, as a consequence of ibpA gene duplication event, a two-protein sHsp (IbpA and IbpB) system has evolved. IbpA and IbpB are functionally divergent. Purified IbpA, but not IbpB, stably interacts with aggregated substrates, yet both sHsps are required to be present at the substrate denaturation step for subsequent efficient Hsp70-Hsp100-dependent substrate refolding. IbpA and IbpB interact with each other, influence each other's expression levels and degradation rates. However, the crucial information on how these two sHsps interact and what is the basic building block required for proper sHsps functioning was missing. Here, based on NMR, mass spectrometry and crosslinking studies, we show that IbpA-IbpB heterodimer is a dominating functional unit of the two sHsp system in Enterobacterales. The principle of heterodimer formation is similar to one described for homodimers of single bacterial sHsps. β-hairpins formed by strands β5 and β7 of IbpA or IbpB crystallin domains associate with the other one's β-sandwich in the heterodimer structure. Relying on crosslinking and molecular dynamics studies, we also propose the orientation of two IbpA-IbpB heterodimers in a higher order tetrameric structure.

Intrinsically disordered proteins (IDPs) are key components of regulatory networks that control crucial aspects of cell decision making. The intrinsically disordered transactivation domain (TAD) of tumor suppressor p53 mediates its interactions with multiple regulatory pathways to control the p53 homeostasis during the cellular response to genotoxic stress. Many cancer-associated mutations have been discovered in p53-TAD, but their structural and functional consequences are poorly understood. Here, by combining atomistic simulations, NMR spectroscopy, and binding assays, we demonstrate that cancer-associated mutations can significantly perturb the balance of p53 interactions with key activation and degradation regulators. Importantly, the four mutations studied in this work do not all directly disrupt the known interaction interfaces. Instead, at least three of these mutations likely modulate the disordered state of p53-TAD to perturb its interactions with regulators. Specifically, NMR and simulation analysis together suggest that these mutations can modulate the level of conformational expansion as well as rigidity of the disordered state. Our work suggests that the disordered conformational ensemble of p53-TAD can serve as a central conduit in regulating the response to various cellular stimuli at the protein-protein interaction level. Understanding how the disordered state of IDPs may be modulated by regulatory signals and/or disease associated perturbations will be essential in the studies on the role of IDPs in biology and diseases.

Members of the αv family of integrins regulate activation of transforming growth factor beta (TGFβ) and are directly involved in pro-tumorigenic phenotypes. Thus, αv integrins may be therapeutic targets for fibrosis and cancer, yet the isolation of selective inhibitors is currently a challenge. We generated synthetic antibodies selective for αv integrins by phage display selections on cell lines that displayed integrin heterodimers. We identified antibodies that targeted two distinct epitopes on cell-surface αv integrins and partially inhibited cell adhesion mediated by interactions between integrins and the latency-associated peptide, part of the pro-form of TGFβ. Using the isolated antibody paratope sequences we engineered a bispecific antibody capable of binding to both epitopes simultaneously; this antibody potently and completely inhibited cell adhesion mediated by integrins αvβ1, αvβ3 and αvβ5. In addition, the bispecific antibody inhibited proliferation and migration of lung carcinoma lines, where the highest and lowest potencies observed correlated with integrin-αv cell surface expression levels. Taken together, our results demonstrate that phage display selections with live cells can yield high quality anti-integrin antibodies, which we used as biparatopic building blocks to construct a bispecific antibody that strongly inhibited integrin function and may be a therapeutic candidate for cancer and fibrosis.

Our current knowledge of protein folding is primarily based on experimental data obtained from isolated domains. In fact, because of their complexity, multidomain proteins have been elusive to the experimental characterization. Thus, the folding of a domain in isolation is generally assumed to resemble what should be observed for more complex structural architectures. Here we compared the folding mechanism of a protein domain in isolation and in the context of its supramodular multidomain structure. By carrying out an extensive mutational analysis we illustrate that while the early events of folding are malleable and influenced by the absence/presence of the neighboring structures, the late events appear to be more robust. These effects may be explained by analyzing the local frustration patterns of the domain, providing critical support for the funneled energy landscape theory of protein folding, and highlighting the role of protein frustration in sculpting the early events of the reaction.

Stalled DNA replication forks can result in incompletely replicated genomes and cell death. DNA replication restart pathways have evolved to deal with repair of stalled forks and E. coli Rep helicase functions in this capacity. Rep and an accessory protein, PriC, assemble at a stalled replication fork to facilitate loading of other replication proteins. A Rep monomer is a rapid and processive single stranded (ss) DNA translocase but needs to be activated to function as a helicase. Activation of Rep in vitro requires self-assembly to form a dimer, removal of its auto-inhibitory 2B sub-domain, or interactions with an accessory protein. Rep helicase activity has been shown to be stimulated by PriC, although the mechanism of activation is not clear. Using stopped flow kinetics, analytical sedimentation and single molecule fluorescence methods, we show that a PriC dimer activates the Rep monomer helicase and can also stimulate the Rep dimer helicase. We show that PriC can self-assemble to form dimers and tetramers and that Rep and PriC interact in the absence of DNA. We further show that PriC serves as a Rep processivity factor, presumably co-translocating with Rep during DNA unwinding. Activation is specific for Rep since PriC does not activate the UvrD helicase. Interaction of PriC with the C-terminal acidic tip of the ssDNA binding protein, SSB, eliminates Rep activation by stabilizing the PriC monomer. This suggests a likely mechanism for Rep activation by PriC at a stalled replication fork.

Biogenic polyamines are natural aliphatic polycations formed from amino acids by biochemical pathways that are highly conserved from bacteria to humans. Their cellular concentrations are carefully regulated and dysregulation causes severe cell growth defects. Polyamines have high affinity for nucleic acids and are known to interact with mRNA, tRNA and rRNA to stimulate the translational machinery, but the exact molecular mechanism(s) for this stimulus is still unknown. Here we exploit that Escherichia coli is viable in the absence of polyamines, including the universally conserved putrescine and spermidine. Using global macromolecule labelling approaches we find that ribosome efficiency is reduced by 50-70% in the absence of polyamines and this reduction is caused by slow translation elongation speed. The low efficiency causes rRNA and multiple tRNA species to be overproduced in the absence of polyamines, suggesting an impact on the feedback regulation of stable RNA transcription. Importantly, we find that polyamine deficiency affects both tRNA levels and tRNA modification patterns. Specifically, a large fraction of tRNAhis, tRNAtyr and tRNAasn lack the queuosine modification in the anticodon "wobble" base, which can be reversed by addition of polyamines to the growth medium. In conclusion, we demonstrate that polyamines are needed for modification of specific tRNA, possibly by facilitating the interaction with modification enzymes.

Cardiolipin (CL) is a mitochondrial anionic lipid that plays important roles in the regulation and signaling of mitochondrial apoptosis. CL peroxidation catalyzed by the assembly of CL-cytochrome c (cyt c) complexes at the inner mitochondrial membrane is a critical checkpoint. The structural changes in the protein, associated with peroxidase activation by CL and different anionic lipids, are not known at a molecular level. To better understand these peripheral protein-lipid interactions, we compare how phosphatidylglycerol (PG) and CL lipids trigger cyt c peroxidase activation, and correlate functional differences to structural and motional changes in membrane-associated cyt c. Structural and motional studies of the bound protein are enabled by magic angle spinning solid state NMR spectroscopy, while lipid peroxidase activity is assayed by mass spectrometry. PG binding results in a surface-bound state that preserves a nativelike fold, which nonetheless allows for significant peroxidase activity, though at a lower level than binding its native substrate CL. Lipid-specific differences in peroxidase activation are found to correlate to corresponding differences in lipid-induced protein mobility, affecting specific protein segments. The dynamics of omega loops C and D are upregulated by CL binding, in a way that is remarkably controlled by the protein:lipid stoichiometry. In contrast to complete chemical denaturation, membrane-induced protein destabilization reflects a destabilization of select cyt c foldons, while the energetically most stable helices are preserved. Our studies illuminate the interplay of protein and lipid dynamics in the creation of lipid peroxidase-active proteolipid complexes implicated in early stages of mitochondrial apoptosis.

Mycobacterium tuberculosis is responsible for more than 1.6 million deaths each year. One potential antibacterial target in M. tuberculosis is filamentous temperature sensitive protein Z (FtsZ), which is the bacterial homologue of mammalian tubulin, a validated cancer target. M. tuberculosis FtsZ function is essential, with its inhibition leading to arrest of cell division, elongation of the bacterial cell and eventual cell death. However, the development of potent inhibitors against FtsZ has been a challenge owing to the lack of structural information. Here we report multiple crystal structures of M. tuberculosis FtsZ in complex with a coumarin analogue. The 4-hydroxycoumarin binds exclusively to two novel cryptic pockets in nucleotide-free FtsZ, but not to the binary FtsZ-GTP or GDP complexes. Our findings provide a detailed understanding of the molecular basis for cryptic pocket formation, controlled by the conformational flexibility of the H7 helix, and thus reveal an important structural and mechanistic rationale for coumarin antibacterial activity.

Rho-dependent termination of transcription (RDTT) is a critical regulatory mechanism specific to bacteria. In a subset of species including most Actinobacteria and Bacteroidetes, the Rho factor contains a large, poorly conserved N-terminal insertion domain (NID) of cryptic function. To date, only two NID-bearing Rho factors from high G + C Actinobacteria have been thoroughly characterized. Both can trigger RDTT at promoter-proximal sites or with structurally constrained transcripts that are unsuitable for the archetypal, NID-less Rho factor of Escherichia coli (EcRho). Here, we provide the first biochemical characterization of a NID-bearing Rho factor from a low G + C bacterium. We show that Bacteroides fragilis Rho (BfRho) is a bona fide RNA-dependent NTPase motor able to unwind long RNA:DNA duplexes and to disrupt transcription complexes. The large NID (~40% of total mass) strongly increases BfRho affinity for RNA, is strictly required for RDTT, but does not promote RDTT at promoter-proximal sites or with a structurally constrained transcript. Furthermore, the NID does not preclude modulation of RDTT by transcription factors NusA and NusG or by the Rho inhibitor bicyclomycin. Although the NID contains a prion-like Q/N-rich motif, it does not spontaneously trigger formation of β-amyloids. Thus, despite its unusually large RNA binding domain, BfRho behaves more like the NID-less EcRho than NID-bearing counterparts from high G + C Actinobacteria. Our data highlight the evolutionary plasticity of Rho's N-terminal region and illustrate how RDTT is adapted to distinct genomic contents.

We describe an enhancement of traditional genomics-based approaches to improve the success of structure determination of membrane proteins. Following a broad screen of sequence space to identify initial expression-positive targets, we employ a second step to select orthologs with closely related sequences to these hits. We demonstrate that a greater percentage of these latter targets express well and are stable in detergent, increasing the likelihood of identifying candidates that will ultimately yield structural information.

Comment in J Mol Biol. 2021 Jul 23;433(15):167052.

Our understanding of what determines ligand affinity of proteins is poor, even with high-resolution structures available. Both the non-covalent ligand-protein interactions and the relative free energies of available conformations contribute to the affinity of a protein for a ligand. Distant, non-binding site residues can influence the ligand affinity by altering the free energy difference between a ligand-free and ligand-bound conformation. Our hypothesis is that when different ligands induce distinct ligand-bound conformations, it should be possible to tweak their affinities by changing the free energies of the available conformations. We tested this idea for the maltose-binding protein (MBP) from Escherichia coli. We used single-molecule Förster resonance energy transfer (smFRET) to distinguish several unique ligand-bound conformations of MBP. We engineered mutations, distant from the binding site, to affect the stabilities of different ligand-bound conformations. We show that ligand affinity can indeed be altered in a conformation-dependent manner. Our studies provide a framework for the tuning of ligand affinity, apart from modifying binding site residues.

The heterotetrameric bacterial KdpFABC transmembrane protein complex is an ion channel-pump hybrid that consumes ATP to import K+ against its transmembrane chemical potential gradient in low external K+ environments. The KdpB ion-pump subunit of KdpFABC is a P-type ATPase, and catalyses ATP hydrolysis. Under high external K+ conditions, K+ can diffuse into the cells through passive ion channels. KdpFABC must therefore be inhibited in high K+ conditions to conserve cellular ATP. Inhibition is thought to occur via unusual phosphorylation of residue Ser162 of the TGES motif of the cytoplasmic A domain. It is proposed that phosphorylation most likely traps KdpB in an inactive E1-P like conformation, but the molecular mechanism of phosphorylation-mediated inhibition remains unknown. Here, we employ molecular dynamics (MD) simulations of the dephosphorylated and phosphorylated versions of KdpFABC to demonstrate that phosphorylated KdpB is trapped in a conformation where the ion-binding site is hydrated by an intracellular pathway between transmembrane helices M1 and M2 which opens in response to the rearrangement of cytoplasmic domains resulting from phosphorylation. Cytoplasmic access of water to the ion-binding site is accompanied by a remarkable loss of secondary structure of the KdpB N-terminus and disruption of a key salt bridge between Glu87 in the A domain and Arg212 in the P domain. Our results provide the molecular basis of a unique mechanism of regulation amongst P-type ATPases, and suggest that the N-terminus has a significant role to play in the conformational cycle and regulation of KdpFABC.

Extracellular vesicles (EVs) are nano-sized, membrane-enclosed vesicles released by cells for intercellular communication. EVs are involved in pathological processes and miRNAs in EVs have gained interest as easily accessible biomolecules in liquid biopsies for diagnostic purposes. To validate potential miRNA biomarker, transcriptome analyses must be carried out to detect suitable reference miRNAs. miREV is a database with over 400 miRNA sequencing data sets and helps the researcher to find suitable reference miRNAs for their individual experimental setup. The researcher can put together a specific sample set in miREV, which is similar to his own experimental concept in order to find the most suitable references. This allows to run validation experiments without having to carry out a complex and costly transcriptome analysis priorly. Additional read count tables of each generated sample set are downloadable for further analysis. miREV is freely available at https://www.physio.wzw.tum.de/mirev/.

Rapidly spreading new variants of SARS-CoV-2 carry multiple mutations in the viral spike protein which attaches to the angiotensin converting enzyme 2 (ACE2) receptor on host cells. Among these mutations are amino acid changes N501Y (lineage B.1.1.7, first identified in the UK), and the combination N501Y, E484K, K417N (B.1.351, first identified in South Africa), all located at the interface on the receptor binding domain (RBD). We experimentally establish that RBD containing the N501Y mutation results in 7-fold stronger binding to the hACE2 receptor than wild type RBD. The E484K mutation only slightly enhances the affinity for the receptor, while K417N attenuates affinity. As a result, RBD from B.1.351 containing all three mutations binds 3-fold stronger to hACE2 than wild type RBD but 2-fold weaker than N501Y. However, the recently emerging double mutant E484K/N501Y binds even stronger than N501Y. The independent evolution of lineages containing mutations with different effects on receptor binding affinity, viral transmission and immune evasion underscores the importance of global viral genome surveillance and functional characterization.

The COVID-19 pandemic has triggered concerns about the emergence of more infectious and pathogenic viral strains. As a public health measure, efficient screening methods are needed to determine the functional effects of new sequence variants. Here we show that structural modeling of SARS-CoV-2 Spike protein binding to the human ACE2 receptor, the first step in host-cell entry, predicts many novel variant combinations with enhanced binding affinities. By focusing on natural variants at the Spike-hACE2 interface and assessing over 700 mutant complexes, our analysis reveals that high-affinity Spike mutations (including N440K, S443A, G476S, E484R, G502P) tend to cluster near known human ACE2 recognition sites (K31 and K353). These Spike regions are structurally flexible, allowing certain mutations to optimize interface interaction energies. Although most human ACE2 variants tend to weaken binding affinity, they can interact with Spike mutations to generate high-affinity double mutant complexes, suggesting variation in individual susceptibility to infection. Applying structural analysis to highly transmissible variants, we find that circulating point mutations S477N, E484K and N501Y form high-affinity complexes (~40% more than wild-type). By combining predicted affinities and available antibody escape data, we show that fast-spreading viral variants exploit combinatorial mutations possessing both enhanced affinity and antibody resistance, including S477N/E484K, E484K/N501Y and K417T/E484K/N501Y. Thus, three-dimensional modeling of the Spike/hACE2 complex predicts changes in structure and binding affinity that correlate with transmissibility and therefore can help inform future intervention strategies.

The nucleosome comprises two histone dimers of H2A-H2B and one histone tetramer of (H3-H4)2, wrapped around by ~145 bp of DNA. Detailed core structures of nucleosomes have been established by X-ray and cryo-EM, however, histone tails have not been visualized. Here, we have examined the dynamic structures of the H2A and H2B tails in 145-bp and 193-bp nucleosomes using NMR, and have compared them with those of the H2A and H2B tail peptides unbound and bound to DNA. Whereas the H2A C-tail adopts a single but different conformation in both nucleosomes, the N-tails of H2A and H2B adopt two distinct conformations in each nucleosome. To clarify these conformations, we conducted molecular dynamics (MD) simulations, which suggest that the H2A N-tail can locate stably in either the major or minor grooves of nucleosomal DNA. While the H2B N-tail, which sticks out between two DNA gyres in the nucleosome, was considered to adopt two different orientations, one toward the entry/exit side and one on the opposite side. Then, the H2A N-tail minor groove conformation was obtained in the H2B opposite side and the H2B N-tail interacts with DNA similarly in both sides, though more varied conformations are obtained in the entry/exit side. Collectively, the NMR findings and MD simulations suggest that the minor groove conformer of the H2A N-tail is likely to contact DNA more strongly than the major groove conformer, and the H2A N-tail reduces contact with DNA in the major groove when the H2B N-tail is located in the entry/exit side.

Sensory photoreceptors enable organisms to adjust their physiology, behavior, and development in response to light, generally with spatiotemporal acuity and reversibility. These traits underlie the use of photoreceptors as genetically encoded actuators to alter by light the state and properties of heterologous organisms. Subsumed as optogenetics, pertinent approaches enable regulating diverse cellular processes, not least gene expression. Here, we controlled the widely used Tet repressor by coupling to light-oxygen-voltage (LOV) modules that either homodimerize or dissociate under blue light. Repression could thus be elevated or relieved, and consequently protein expression was modulated by light. Strikingly, the homodimeric RsLOV module from Rhodobacter sphaeroides not only dissociated under light but intrinsically reacted to temperature. The limited light responses of wild-type RsLOV at 37 °C were enhanced in two variants that exhibited closely similar photochemistry and structure. One variant improved the weak homodimerization affinity of 40 µM by two-fold and thus also bestowed light sensitivity on a receptor tyrosine kinase. Certain photoreceptors, exemplified by RsLOV, can evidently moonlight as temperature sensors which immediately bears on their application in optogenetics and biotechnology. Properly accounted for, the temperature sensitivity can be leveraged for the construction of signal-responsive cellular circuits.

In order to form functional filaments, human septins must assemble into hetero-oligomeric rod-like particles which polymerize end-to-end. The rules governing the assembly of these particles and the subsequent filaments are incompletely understood. Although crystallographic approaches have been successful in studying the separate components of the system, there has been difficulty in obtaining high resolution structures of the full particle. Here we report a first cryo-EM structure for a hexameric rod composed of human septins 2, 6 and 7 with a global resolution of ~3.6 Å and a local resolution of between ~3.0 Å and ~5.0 Å. By fitting the previously determined high-resolution crystal structures of the component subunits into the cryo-EM map, we are able to provide an essentially complete model for the particle. This exposes SEPT2 NC-interfaces at the termini of the hexamer and leaves internal cavities between the SEPT6-SEPT7 pairs. The floor of the cavity is formed by the two α0 helices including their polybasic regions. These are locked into place between the two subunits by interactions made with the α5 and α6 helices of the neighbouring monomer together with its polyacidic region. The cavity may serve to provide space allowing the subunits to move with respect to one another. The elongated particle shows a tendency to bend at its centre where two copies of SEPT7 form a homodimeric G-interface. Such bending is almost certainly related to the ability of septin filaments to recognize and even induce membrane curvature.

Ion conduction is an essential function for electrical activity in all organisms. The non-selective ion channel NaK was previously shown to adopt two stable conformations of the selectivity filter. Here, we present solid-state NMR measurements of NaK demonstrating a population shift between these conformations induced by changing the ions in the sample while the overall structure of NaK is not affected. We show that two K+-selective mutants (NaK2K and NaK2K-Y66F) suffer a complete loss of selectivity filter stability under Na+ conditions, but do not collapse into a defined structure. Widespread chemical shift perturbations are seen between the Na+ and K+ states of the K+-selective mutants in the region of the pore helix indicating structural changes. We conclude that the stronger link between the selectivity filter and the pore helix in the K+-selective mutants, compared to the non-selective wild-type NaK channel, reduces the ion-dependent conformational flexibility of the selectivity filter.

Long-term memory storage is modulated by the prion nature of CPEB3 forming the molecular basis for the maintenance of synaptic facilitation. Here we report that the first prion sub-domain PRD1 of mouse CPEB3 can autonomously form amyloid fibrils in vitro and punctate-like structures in vivo. A ninety-four amino acid sequence within the PRD1 domain, PRD1-core, displays high propensity towards aggregation and associated amyloid characteristics. PRD1-core is characterized using electron microscopy, X-ray diffraction, and solution-state NMR deuterium exchange experiments. Secondary structure elements deduced from solid-state NMR reveal a β-rich core comprising of forty amino acids at the N-terminus of PRD1-core. The synthesized twenty-three amino acid long peptide containing the longest rigid segment (E124-H145) of the PRD1-core rapidly self-aggregates and forms fibrils, indicating a limited aggregation-prone region that could potentially activate the aggregation of the full-length protein. This study provides the first step in identifying the structural trigger for the CPEB3 aggregation process.

In Gram-negative bacteria, periplasmic domains in inner membrane proteins are cotranslationally translocated across the inner membrane through the SecYEG translocon. To what degree such domains also start to fold cotranslationally is generally difficult to determine using currently available methods. Here, we apply Force Profile Analysis (FPA) - a method where a translational arrest peptide is used to detect folding-induced forces acting on the nascent polypeptide - to follow the cotranslational translocation and folding of the large periplasmic domain of the E. coli inner membrane protease LepB in vivo. Membrane insertion of LepB's two N-terminal transmembrane helices is initiated when their respective N-terminal ends reach 45-50 residues away from the peptidyl transferase center (PTC) in the ribosome. The main folding transition in the periplasmic domain involves all but the ~15 most C-terminal residues of the protein and happens when the C-terminal end of the folded part is ~70 residues away from the PTC; a smaller putative folding intermediate is also detected. This implies that wildtype LepB folds post-translationally in vivo, and shows that FPA can be used to study both co- and post-translational protein folding in the periplasm.

Being essential for oxidative protein folding in the mitochondrial intermembrane space, the mitochondrial disulfide relay relies on the electron transfer (ET) from the sulfhydryl oxidase Erv1 to cytochrome c (Cc). Using solution NMR spectroscopy, we demonstrate that while the yeast Cc-Erv1 system is functionally active, no observable binding of the protein partners takes place. The transient interaction between Erv1 and Cc can be rationalized by molecular modeling, suggesting that a large surface area of Erv1 can sustain a fast ET to Cc via a collision-type mechanism, without the need for a canonical protein complex formation. We suggest that, by preventing the direct ET to molecular oxygen (O2), the collision-type Cc-Erv1 interaction plays a role in protecting the organism against reactive oxygen species.