Structural basis for defective membrane targeting of mutant enzyme in human VLCAD deficiency.

Very long-chain acyl-CoA dehydrogenase (VLCAD) is an inner mitochondrial membrane enzyme that catalyzes the first and rate-limiting step of long-chain fatty acid oxidation. Point mutations in human VLCAD can produce an inborn error of metabolism called VLCAD deficiency that can lead to severe pathophysiologic consequences, including cardiomyopathy, hypoglycemia, and rhabdomyolysis. Discrete mutations in a structurally-uncharacterized C-terminal domain region of VLCAD cause enzymatic deficiency by an incompletely defined mechanism. Here, we conducted a structure-function study, incorporating X-ray crystallography, hydrogen-deuterium exchange mass spectrometry, computational modeling, and biochemical analyses, to characterize a specific membrane interaction defect of full-length, human VLCAD bearing the clinically-observed mutations, A450P or L462P. By disrupting a predicted α-helical hairpin, these mutations either partially or completely impair direct interaction with the membrane itself. Thus, our data support a structural basis for VLCAD deficiency in patients with discrete mutations in an α-helical membrane-binding motif, resulting in pathologic enzyme mislocalization.

A multi-pronged approach targeting SARS-CoV-2 proteins using ultra-large virtual screening.

The unparalleled global effort to combat the continuing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic over the last year has resulted in promising prophylactic measures. However, a need still exists for cheap, effective therapeutics, and targeting multiple points in the viral life cycle could help tackle the current, as well as future, coronaviruses. Here, we leverage our recently developed, ultra-large-scale in silico screening platform, VirtualFlow, to search for inhibitors that target SARS-CoV-2. In this unprecedented structure-based virtual campaign, we screened roughly 1 billion molecules against each of 40 different target sites on 17 different potential viral and host targets. In addition to targeting the active sites of viral enzymes, we also targeted critical auxiliary sites such as functionally important protein-protein interactions.

A Multi-Pronged Approach Targeting SARS-CoV-2 Proteins Using Ultra-Large Virtual Screening.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), previously known as 2019 novel coronavirus (2019-nCoV), has spread rapidly across the globe, creating an unparalleled global health burden and spurring a deepening economic crisis. As of July 7th, 2020, almost seven months into the outbreak, there are no approved vaccines and few treatments available. Developing drugs that target multiple points in the viral life cycle could serve as a strategy to tackle the current as well as future coronavirus pandemics. Here we leverage the power of our recently developed in silico screening platform, VirtualFlow, to identify inhibitors that target SARS-CoV-2. VirtualFlow is able to efficiently harness the power of computing clusters and cloud-based computing platforms to carry out ultra-large scale virtual screens. In this unprecedented structure-based multi-target virtual screening campaign, we have used VirtualFlow to screen an average of approximately 1 billion molecules against each of 40 different target sites on 17 different potential viral and host targets in the cloud. In addition to targeting the active sites of viral enzymes, we also target critical auxiliary sites such as functionally important protein-protein interaction interfaces. This multi-target approach not only increases the likelihood of finding a potent inhibitor, but could also help identify a collection of anti-coronavirus drugs that would retain efficacy in the face of viral mutation. Drugs belonging to different regimen classes could be combined to develop possible combination therapies, and top hits that bind at highly conserved sites would be potential candidates for further development as coronavirus drugs. Here, we present the top 200 in silico hits for each target site. While in-house experimental validation of some of these compounds is currently underway, we want to make this array of potential inhibitor candidates available to researchers worldwide in consideration of the pressing need for fast-tracked drug development.

Targeting a helix-in-groove interaction between E1 and E2 blocks ubiquitin transfer.

The ubiquitin-proteasome system (UPS) is a highly regulated protein disposal process critical to cell survival. Inhibiting the pathway induces proteotoxic stress and can be an effective cancer treatment. The therapeutic window observed upon proteasomal blockade has motivated multiple UPS-targeting strategies, including preventing ubiquitination altogether. E1 initiates the cascade by transferring ubiquitin to E2 enzymes. A small molecule that engages the E1 ATP-binding site and derivatizes ubiquitin disrupts enzymatic activity and kills cancer cells. However, binding-site mutations cause resistance, motivating alternative approaches to block this promising target. We identified an interaction between the E2 N-terminal alpha-1 helix and a pocket within the E1 ubiquitin-fold domain as a potentially druggable site. Stapled peptides modeled after the E2 alpha-1 helix bound to the E1 groove, induced a consequential conformational change and inhibited E1 ubiquitin thiotransfer, disrupting E2 ubiquitin charging and ubiquitination of cellular proteins. Thus, we provide a blueprint for a distinct E1-targeting strategy to treat cancer.

Homogeneous Oligomers of Pro-apoptotic BAX Reveal Structural Determinants of Mitochondrial Membrane Permeabilization.

BAX is a pro-apoptotic protein that transforms from a cytosolic monomer into a toxic oligomer that permeabilizes the mitochondrial outer membrane. How BAX monomers assemble into a higher-order conformation, and the structural determinants essential to membrane permeabilization, remain a mechanistic mystery. A key hurdle has been the inability to generate a homogeneous BAX oligomer (BAXO) for analysis. Here, we report the production and characterization of a full-length BAXO that recapitulates physiologic BAX activation. Multidisciplinary studies revealed striking conformational consequences of oligomerization and insight into the macromolecular structure of oligomeric BAX. Importantly, BAXO enabled the assignment of specific roles to particular residues and α helices that mediate individual steps of the BAX activation pathway, including unexpected functionalities of BAX α6 and α9 in driving membrane disruption. Our results provide the first glimpse of a full-length and functional BAXO, revealing structural requirements for the elusive execution phase of mitochondrial apoptosis.

Design of stapled antimicrobial peptides that are stable, nontoxic and kill antibiotic-resistant bacteria in mice.

The clinical translation of cationic α-helical antimicrobial peptides (AMPs) has been hindered by structural instability, proteolytic degradation and in vivo toxicity from nonspecific membrane lysis. Although analyses of hydrophobic content and charge distribution have informed the design of synthetic AMPs with increased potency and reduced in vitro hemolysis, nonspecific membrane toxicity in vivo continues to impede AMP drug development. Here, we analyzed a 58-member library of stapled AMPs (StAMPs) based on magainin II and applied the insights from structure-function-toxicity measurements to devise an algorithm for the design of stable, protease-resistant, potent and nontoxic StAMP prototypes. We show that a lead double-stapled StAMP named Mag(i+4)1,15(A9K,B21A,N22K,S23K) can kill multidrug-resistant Gram-negative pathogens, such as colistin-resistant Acinetobacter baumannii in a mouse peritonitis-sepsis model, without observed hemolysis or renal injury in murine toxicity studies. Inputting the amino acid sequences alone, we further generated membrane-selective StAMPs of pleurocidin, CAP18 and esculentin, highlighting the generalizability of our design platform.

Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells.

Functional antibody delivery in living cells would enable the labelling and manipulation of intracellular antigens, which constitutes a long-thought goal in cell biology and medicine. Here we present a modular strategy to create functional cell-permeable nanobodies capable of targeted labelling and manipulation of intracellular antigens in living cells. The cell-permeable nanobodies are formed by the site-specific attachment of intracellularly stable (or cleavable) cyclic arginine-rich cell-penetrating peptides to camelid-derived single-chain VHH antibody fragments. We used this strategy for the non-endocytic delivery of two recombinant nanobodies into living cells, which enabled the relocalization of the polymerase clamp PCNA (proliferating cell nuclear antigen) and tumour suppressor p53 to the nucleolus, and thereby allowed the detection of protein-protein interactions that involve these two proteins in living cells. Furthermore, cell-permeable nanobodies permitted the co-transport of therapeutically relevant proteins, such as Mecp2, into the cells. This technology constitutes a major step in the labelling, delivery and targeted manipulation of intracellular antigens. Ultimately, this approach opens the door towards immunostaining in living cells and the expansion of immunotherapies to intracellular antigen targets.

Binding of MBD proteins to DNA blocks Tet1 function thereby modulating transcriptional noise.

Aberrant DNA methylation is a hallmark of various human disorders, indicating that the spatial and temporal regulation of methylation readers and modifiers is imperative for development and differentiation. In particular, the cross-regulation between 5-methylcytosine binders (MBD) and modifiers (Tet) has not been investigated. Here, we show that binding of Mecp2 and Mbd2 to DNA protects 5-methylcytosine from Tet1-mediated oxidation. The mechanism is not based on competition for 5-methylcytosine binding but on Mecp2 and Mbd2 directly restricting Tet1 access to DNA. We demonstrate that the efficiency of this process depends on the number of bound MBDs per DNA molecule. Accordingly, we find 5-hydroxymethylcytosine enriched at heterochromatin of Mecp2-deficient neurons of a mouse model for Rett syndrome and Tet1-induced reexpression of silenced major satellite repeats. These data unveil fundamental regulatory mechanisms of Tet enzymes and their potential pathophysiological role in Rett syndrome. Importantly, it suggests that Mecp2 and Mbd2 have an essential physiological role as guardians of the epigenome.

Visualization of the Nucleolus in Living Cells with Cell-Penetrating Fluorescent Peptides.

The nucleolus is the hallmark of nuclear compartmentalization and has been shown to exert multiple roles in cellular metabolism besides its main function as the place of ribosomal RNA synthesis and assembly of ribosomes. The nucleolus plays also a major role in nuclear organization as the largest compartment within the nucleus. The prominent structure of the nucleolus can be detected using contrast light microscopy providing an approximate localization of the nucleolus, but this approach does not allow to determine accurately the three-dimensional structure of the nucleolus in cells and tissues. Immunofluorescence staining with antibodies specific to nucleolar proteins albeit very useful is time consuming, normally antibodies recognize their epitopes only within a small range of species and is applicable only in fixed cells. Here, we present a simple method to selectively and accurately label this ubiquitous subnuclear compartment in living cells of a large range of species using a fluorescently labeled cell-penetrating peptide.

Can Specific Protein-Lipid Interactions Stabilize an Active State of the Beta 2 Adrenergic Receptor?

G-protein-coupled receptors are eukaryotic membrane proteins with broad biological and pharmacological relevance. Like all membrane-embedded proteins, their location and orientation are influenced by lipids, which can also impact protein function via specific interactions. Extensive simulations totaling 0.25 ms reveal a process in which phospholipids from the membrane's cytosolic leaflet enter the empty G-protein binding site of an activated ß2 adrenergic receptor and form salt-bridge interactions that inhibit ionic lock formation and prolong active-state residency. Simulations of the receptor embedded in an anionic membrane show increased lipid binding, providing a molecular mechanism for the experimental observation that anionic lipids can enhance receptor activity. Conservation of the arginine component of the ionic lock among Rhodopsin-like G-protein-coupled receptors suggests that intracellular lipid ingression between receptor helices H6 and H7 may be a general mechanism for active-state stabilization.

Principles of protein targeting to the nucleolus.

The nucleolus is the hallmark of nuclear compartmentalization and has been shown to exert multiple roles in cellular metabolism besides its main function as the place of rRNA synthesis and assembly of ribosomes. Nucleolar proteins dynamically localize and accumulate in this nuclear compartment relative to the surrounding nucleoplasm. In this study, we have assessed the molecular requirements that are necessary and sufficient for the localization and accumulation of peptides and proteins inside the nucleoli of living cells. The data showed that positively charged peptide entities composed of arginines alone and with an isoelectric point at and above 12.6 are necessary and sufficient for mediating significant nucleolar accumulation. A threshold of 6 arginines is necessary for peptides to accumulate in nucleoli, but already 4 arginines are sufficient when fused within 15 amino acid residues of a nuclear localization signal of a protein. Using a pH sensitive dye, we found that the nucleolar compartment is particularly acidic when compared to the surrounding nucleoplasm and, hence, provides the ideal electrochemical environment to bind poly-arginine containing proteins. In fact, we found that oligo-arginine peptides and GFP fusions bind RNA in vitro. Consistent with RNA being the main binding partner for arginines in the nucleolus, we found that the same principles apply to cells from insects to man, indicating that this mechanism is highly conserved throughout evolution.

Complete topology of the RNF complex from Vibrio cholerae.

RNF is a redox-driven ion (Na(+) and in one case possibly H(+)) transporter present in many prokaryotes. It has been proposed that RNF performs a variety of reactions in different organisms, delivering low-potential reducing equivalents for specific cellular processes. RNF shares strong homology with the Na(+)-pumping respiratory enzyme Na(+)-NQR, although there are significant differences in subunit and redox cofactor composition. Here we report a topological analysis of the six subunits of RNF from Vibrio cholerae. Although individual subunits from other organisms have previously been studied, this is the first complete, experimentally derived, analysis of RNF from any one source. This has allowed us to identify and confirm key properties of RNF. The putative NADH binding site in RnfC is located on the cytoplasmic side of the membrane. FeS centers in RnfB and RnfC are also located on the cytoplasmic side. However, covalently attached FMNs in RnfD and RnfG are both located in the periplasm. RNF also contains a number of acidic residues that correspond to functionally important groups in Na(+)-NQR. The acidic residues involved in Na(+) uptake and many of those implicated in Na(+) translocation are topologically conserved. The topology of RNF closely matches the topology represented in the newly published structure of Na(+)-NQR, consistent with the close relation between the two enzymes. The topology of RNF is discussed in the context of the current structural model of Na(+)-NQR, and the proposed functionality of the RNF complex itself.

Covalent attachment of cyclic TAT peptides to GFP results in protein delivery into live cells with immediate bioavailability.

The delivery of free molecules into the cytoplasm and nucleus by using arginine-rich cell-penetrating peptides (CPPs) has been limited to small cargoes, while large cargoes such as proteins are taken up and trapped in endocytic vesicles. Based on recent work, in which we showed that the transduction efficiency of arginine-rich CPPs can be greatly enhanced by cyclization, the aim was to use cyclic CPPs to transport full-length proteins, in this study green fluorescent protein (GFP), into the cytosol of living cells. Cyclic and linear CPP-GFP conjugates were obtained by using azido-functionalized CPPs and an alkyne-functionalized GFP. Our findings reveal that the cyclic-CPP-GFP conjugates are internalized into live cells with immediate bioavailability in the cytosol and the nucleus, whereas linear CPP analogues do not confer GFP transduction. This technology expands the application of cyclic CPPs to the efficient transport of functional full-length proteins into live cells.

A novel cell permeable DNA replication and repair marker.

Proliferating Cell Nuclear Antigen (PCNA) is a key protein in DNA replication and repair. The dynamics of replication and repair in live cells is usually studied introducing translational fusions of PCNA. To obviate the need for transfection and bypass the problem of difficult to transfect and/or short lived cells, we have now developed a cell permeable replication and/or repair marker. The design of this marker has three essential molecular components: (1) an optimized artificial PCNA binding peptide; (2) a cell-penetrating peptide, derived from the HIV-1 Trans Activator of Transcription (TAT); (3) an in vivo cleavable linker, linking the two peptides. The resulting construct was taken up by human, hamster and mouse cells within minutes of addition to the media. Inside the cells, the cargo separated from the vector peptide and bound PCNA effectively. Both replication and repair sites could be directly labeled in live cells making it the first in vivo cell permeable peptide marker for these two fundamental cellular processes. Concurrently, we also introduced a quick peptide based PCNA staining method as an alternative to PCNA antibodies for immunofluorescence applications. In summary, we present here a versatile tool to instantaneously label repair and replication processes in fixed and live cells.

Fundamental molecular mechanism for the cellular uptake of guanidinium-rich molecules.

Guanidinium-rich molecules, such as cell-penetrating peptides, efficiently enter living cells in a non-endocytic energy-independent manner and transport a wide range of cargos, including drugs and biomarkers. The mechanism by which these highly cationic molecules efficiently cross the hydrophobic barrier imposed by the plasma membrane remains a fundamental open question. Here, a combination of computational results and in vitro and live-cell experimental evidence reveals an efficient energy-independent translocation mechanism for arginine-rich molecules. This mechanism unveils the essential role of guanidinium groups and two universal cell components: fatty acids and the cell membrane pH gradient. Deprotonated fatty acids in contact with the cell exterior interact with guanidinium groups, leading to a transient membrane channel that facilitates the transport of arginine-rich peptides toward the cell interior. On the cytosolic side, the fatty acids become protonated, releasing the peptides and resealing the channel. This fundamental mechanism appears to be universal across cells from different species and kingdoms.

Altered spatio-temporal dynamics of RNase H2 complex assembly at replication and repair sites in Aicardi-Goutières syndrome.

Ribonuclease H2 plays an essential role for genome stability as it removes ribonucleotides misincorporated into genomic DNA by replicative polymerases and resolves RNA/DNA hybrids. Biallelic mutations in the genes encoding the three RNase H2 subunits cause Aicardi-Goutières syndrome (AGS), an early-onset inflammatory encephalopathy that phenotypically overlaps with the autoimmune disorder systemic lupus erythematosus. Here we studied the intracellular dynamics of RNase H2 in living cells during DNA replication and in response to DNA damage using confocal time-lapse imaging and fluorescence cross-correlation spectroscopy. We demonstrate that the RNase H2 complex is assembled in the cytosol and imported into the nucleus in an RNase H2B-dependent manner. RNase H2 is not only recruited to DNA replication foci, but also to sites of PCNA-dependent DNA repair. By fluorescence recovery after photobleaching, we demonstrate a high mobility and fast exchange of RNase H2 at sites of DNA repair and replication. We provide evidence that recruitment of RNase H2 is not only PCNA-dependent, mediated by an interaction of the B subunit with PCNA, but also PCNA-independent mediated via the catalytic domain of the A subunit. We found that AGS-associated mutations alter complex formation, recruitment efficiency and exchange kinetics at sites of DNA replication and repair suggesting that impaired ribonucleotide removal contributes to AGS pathogenesis.

Visualization and targeted disruption of protein interactions in living cells.

Protein-protein interactions are the basis of all processes in living cells, but most studies of these interactions rely on biochemical in vitro assays. Here we present a simple and versatile fluorescent-three-hybrid (F3H) strategy to visualize and target protein-protein interactions. A high-affinity nanobody anchors a GFP-fusion protein of interest at a defined cellular structure and the enrichment of red-labelled interacting proteins is measured at these sites. With this approach, we visualize the p53-HDM2 interaction in living cells and directly monitor the disruption of this interaction by Nutlin 3, a drug developed to boost p53 activity in cancer therapy. We further use this approach to develop a cell-permeable vector that releases a highly specific peptide disrupting the p53 and HDM2 interaction. The availability of multiple anchor sites and the simple optical readout of this nanobody-based capture assay enable systematic and versatile analyses of protein-protein interactions in practically any cell type and species.

Direct homo- and hetero-interactions of MeCP2 and MBD2.

Epigenetic marks like methylation of cytosines at CpG dinucleotides are essential for mammalian development and play a major role in the regulation of gene expression and chromatin architecture. The methyl-cytosine binding domain (MBD) protein family recognizes and translates this methylation mark. We have recently shown that the level of MeCP2 and MBD2, two members of the MBD family, increased during differentiation and their ectopic expression induced heterochromatin clustering in vivo. As oligomerization of these MBD proteins could constitute a factor contributing to the chromatin clustering effect, we addressed potential associations among the MBD family performing a series of different interaction assays in vitro as well as in vivo. Using recombinant purified MBDs we found that MeCP2 and MBD2 showed the stronger self and cross association as compared to the other family members. Besides demonstrating that these homo- and hetero-interactions occur in the absence of DNA, we could confirm them in mammalian cells using co-immunoprecipitation analysis. Employing a modified form of the fluorescent two-hybrid protein-protein interaction assay, we could clearly visualize these associations in single cells in vivo. Deletion analysis indicated that the region of MeCP2 comprising amino acids 163-309 as well the first 152 amino acids of MBD2 are the domains responsible for MeCP2 and MBD2 associations. Our results strengthen the possibility that MeCP2 and MBD2 direct interactions could crosslink chromatin fibers and therefore give novel insight into the molecular mechanism of MBD mediated global heterochromatin architecture.

Cube-octameric silsesquioxane-mediated cargo peptide delivery into living cancer cells.

Cube octameric silsesquioxanes (COSS) are among the smallest nanoparticles known to date with a diameter of only 0.7 nm. We describe a COSS-based delivery system which allows for the drug targeting in human cells. It comprises a siloxane core with seven pendant aminopropyl groups and a fluorescently labeled peptidic ligand attached to one cage corner via a reversible disulfide bond to ensure its intracellular release. Bimodal amplitude-modulated atomic force microscopy (AFM) experiments revealed the formation of dendritic COSS structures by a self-assembly of single particles on negatively charged surfaces. Nuclear targeting was demonstrated in HeLa cells by selective binding of released p21(Cip1/Waf1)-derived cargo peptide to PCNA, a protein involved in DNA replication and repair.

Histone hypoacetylation is required to maintain late replication timing of constitutive heterochromatin.

The replication of the genome is a spatio-temporally highly organized process. Yet, its flexibility throughout development suggests that this process is not genetically regulated. However, the mechanisms and chromatin modifications controlling replication timing are still unclear. We made use of the prominent structure and defined heterochromatic landscape of pericentric regions as an example of late replicating constitutive heterochromatin. We manipulated the major chromatin markers of these regions, namely histone acetylation, DNA and histone methylation, as well as chromatin condensation and determined the effects of these altered chromatin states on replication timing. Here, we show that manipulation of DNA and histone methylation as well as acetylation levels caused large-scale heterochromatin decondensation. Histone demethylation and the concomitant decondensation, however, did not affect replication timing. In contrast, immuno-FISH and time-lapse analyses showed that lowering DNA methylation, as well as increasing histone acetylation, advanced the onset of heterochromatin replication. While dnmt1(-)(/)(-) cells showed increased histone acetylation at chromocenters, histone hyperacetylation did not induce DNA demethylation. Hence, we propose that histone hypoacetylation is required to maintain normal heterochromatin duplication dynamics. We speculate that a high histone acetylation level might increase the firing efficiency of origins and, concomitantly, advances the replication timing of distinct genomic regions.