Methylation of specific nucleotides is integral for ribosomal biogenesis and serves as a common way to confer antibiotic resistance by pathogenic bacteria. Here, by determining the high-resolution structure of 30S-KsgA by cryo-EM, a state was captured, where KsgA juxtaposes between helices h44 and h45, separating them, thereby enabling remodeling of the surrounded rRNA and allowing the cognate site

Cells utilize protein-protein interaction (PPI) networks to receive, transduce, and respond to stimuli. Interaction network rewiring drives devastating diseases like cancers, making PPIs attractive targets for pharmacological intervention. Kinases are druggable nodes in PPI networks but high-throughput proteomics approaches to quantify disease-associated kinome PPI rewiring are lacking. We introduce

Phosphocholine molecules decorating bacterial cell wall teichoic acids and outer-membrane lipopolysaccharide have significant roles in adhesion to host cells, immune evasion, and persistence. Bacteria carrying the operon that performs phosphocholine decoration, synthesize phosphocholine after uptake of the choline precursor by LicB, a conserved transporter among divergent species. Streptococcus pneumoniae

The AAA+ protein KaiC is the central pacemaker for cyanobacterial circadian rhythms. Composed of two hexameric rings with tightly coupled activities, KaiC undergoes changes in autophosphorylation on its C-terminal (CII) domain that restrict binding of of clock proteins on its N-terminal (CI) domain to the evening. Here, we use cryo-electron microscopy to investigate how daytime and nighttime states

Hundreds of cytotoxic natural or synthetic lipidic compounds contain chiral alkynylcarbinol motifs, but the mechanism of action of those potential therapeutic agents remains unknown. Using a genetic screen in haploid human cells, we discovered that the enantiospecific cytotoxicity of numerous terminal alkynylcarbinols, including the highly cytotoxic dialkynylcarbinols, involves a bioactivation by HSD17B11

G protein-coupled receptors (GPCRs) translate binding of extracellular ligands into intracellular responses through conformational changes. Ligand properties are described by the maximum response (efficacy) and the agonist concentration at half-maximal response (potency). Integrating structural changes with pharmacological properties remains challenging and has not yet been performed at the resolution

During spermatogenesis, spermatogonia undergo a series of mitotic and meiotic divisions on their path to spermatozoa. To achieve this, a succession of processes requiring high proteolytic activity are in part orchestrated by the proteasome. The spermatoproteasome (s20S) is specific to the developing gametes, in which the gamete-specific α4s subunit replaces the α4 isoform found in the constitutive

Many metabolic enzymes self-assemble into micron-scale filaments to organize and regulate metabolism. The appearance of these assemblies often coincides with large metabolic changes as in development, cancer, and stress. Yeast undergo cytoplasmic acidification upon starvation, triggering the assembly of many metabolic enzymes into filaments. However, it is unclear how these filaments assemble at the

The widespread use of CRISPR/Cas9 as a programmable genome editing tool has been hindered by off-target DNA cleavage (Cong et al., 2013; Doudna, 2020; Fu et al., 2013; Jinek et al., 2013). While analysis of such off-target editing events have enabled the development of Cas9 variants with greater discrimination against mismatches (Chen et al., 2017; Kleinstiver et al., 2016; Slaymaker et al., 2016)

The type VI secretion system (T6SS) is a widespread protein export apparatus found in Gram-negative bacteria. The majority of T6SSs deliver toxic effector proteins into competitor bacteria. Yet, the structure, function, and activation of many of these effectors remains poorly understood. Here, we present the structures of the T6SS effector RhsA from Pseudomonas protegens and its cognate T6SS spike

Microcrystal electron diffraction (MicroED) is transforming the visualization of molecules from nanocrystals, rendering their three-dimensional atomic structures from previously unamenable samples. Peptidic structures determined by MicroED include naturally occurring peptides, synthetic protein fragments and peptide-based natural products. However, as a diffraction method, MicroED is beholden to the

Development of efficient therapies for COVID-19 is the focus of intense research. The cytokine release syndrome was underlined as a culprit for severe outcomes in COVID-19 patients. Interleukin-6 (IL-6) plays a crucial role in human immune responses and elevated IL-6 plasma levels have been associated with the exacerbated COVID-19 pathology. Since non-structural protein 10 (NSP10) of SARS-CoV-2 has

The rate-limiting enzyme of salvage pathway for NAD+ synthesis, NAMPT, is aberrantly overexpressed in a variety of tumor cells and is a poor prognosis factor for patient survival. NAMPT plays a major role in tumor cell proliferation, acting concurrently as an NAD+ synthase and unexpectedly, an extracellular ligand for several tumor-promoting signaling pathways. While previous efforts to modulate NAMPT

Mislocalised membrane proteins (MLPs) present a risk to the cell due to exposed hydrophobic amino acids which cause MLPs to aggregate. Previous studies identified SGTA as a key component of the machinery that regulates the quality control of MLPs. Overexpression of SGTA promotes deubiqutination of MLPs resulting in their accumulation in cytosolic inclusions, suggesting SGTA acts in collaboration with

Type III CRISPR systems detect invading RNA, resulting in the activation of the enzymatic Cas10 subunit. The Cas10 cyclase domain generates cyclic oligoadenylate (cOA) second messenger molecules, activating a variety of effector nucleases that degrade nucleic acids to provide immunity. The prophage-encoded Vibrio metoecus type III-B (VmeCmr) locus is uncharacterised, lacks the HD nuclease domain in

DNA/RNA-gold nanoparticle (DNA/RNA-AuNP) nanoprobes have been widely employed for nanobiotechnology applications. Here we discovered that both thiolated and non-thiolated DNA/RNA can be efficiently attached to AuNPs to achieve high-stable spherical nucleic acid (SNA) within minutes under a domestic microwave (MW)-assisted heating-dry circumstance. Further studies showed that for non-thiolated DNA/RNA

The NDUFS4 knockout (KO) mouse phenotype resembles the human Complex I deficiency Leigh Syndrome. The irreversible succination of protein thiols by fumarate is increased in select regions of the NDUFS4 KO brain affected by neurodegeneration, suggesting a mechanistic role in neurodegenerative decline. We report that dihydrolipoyllysine-residue succinyltransferase (DLST), a component of the α-ketoglutarate

RAS is the most commonly mutated oncogene in human cancers and RAS-driven tumors are amongst the most difficult to treat. Historically, discovery of therapeutics targeting this protein has proven challenging due to a lack of deep hydrophobic pockets to which a small molecule might bind. The single such pocket available is normally occupied by GDP or GTP which have millimolar cellular concentrations

Understanding how point mutations can render a ligand or a drug ineffective against a given biological target is a problem of immense fundamental and practical relevance. Often the efficacy of such resistance mutations can be explained purely on a thermo-dynamic basis wherein the mutated system displays a reduced binding affinity for the ligand. However, the more perplexing and harder to explain situation

HOIL-1, a component of the Linear Ubiquitin Assembly Complex (LUBAC), ubiquitylates serine and threonine residues in proteins, forming ester bonds (Kelsall et al, 2019, PNAS 116, 13293-13298). Here we report that mice expressing the E3 ligase-inactive HOIL-1[C458S] mutant accumulate polyglucosan in brain, cardiac muscle and other organs, indicating that HOIL-1’s E3 ligase activity is essential to prevent

Crystallographic phasing recovers the phase information that is lost during a diffraction experiment. Molecular replacement is a dominant phasing method for the crystal structures in the protein data bank. In one form it uses a protein sequence to search a structure database for finding suitable templates for phasing. However, such sequence information is not always available such as when proteins

BINding ANAlyzer (BINANA) is an algorithm for identifying and characterizing protein/ligand interactions and other factors that contribute to binding. We recently updated BINANA to make the algorithm more accessible to a broader audience. We have also ported the Python3 codebase to JavaScript, thus enabling BINANA analysis in the web browser. As proof of principle, we created a web-browser application

Unique among metazoan repressive histone methyltransferases, G9a and GLP, which chiefly target histone 3 lysine 9 (H3K9), require dimerization for productive H3K9 mono (me1)- and dimethylation (me2) in vivo. Intriguingly, even though each enzyme can independently methylate H3K9, the predominant active form in vivo is a heterodimer of G9a and GLP. How dimerization influences the central H3K9 methyl

According to its primary structure, the [PITG_06016] gene encodes for one of the 7 glucokinases present in Phytophthora infestans (PiGlcK-1), the causal agent of late blight disease. Currently, there are no structural studies of any of its enzymes, being the determination of the three-dimensional (3D) structure of PiGlcK-1 a necessary contribution in the deduction of its functions, its interaction

Regulation of translation is a fundamental facet of the cellular response to rapidly changing external conditions. Specific RNA-binding proteins (RBPs) co-ordinate the translational regulation of distinct mRNA cohorts during stress. To identify RBPs with previously under-appreciated roles in translational control, we used polysome profiling and mass spectrometry to identify and quantify proteins associated

Cryptochrome (CRY) entrains the fly circadian clock by binding to Timeless (TIM) in light and triggering its degradation. Undocking of a helical C-terminal tail (CTT) in response to photoreduction of the CRY flavin cofactor gates TIM binding. A generally-applicable Select Western-blot-Free Tagged-protein Interaction (SWFTI) assay enables quantification of CRY binding to TIM in dark and light. The assay

The solution structure of SARS-CoV-2 nonstructural protein 7 (nsp7) at pH 7.0 has been determined by NMR spectroscopy. nsp7 is conserved in the coronavirinae subfamily and is an essential co-factor of the viral RNA-dependent RNA polymerase for active and processive replication. Similar to the previously deposited structures of SARS-CoV-1 nsp7 at acidic and basic conditions, SARS-CoV-2 nsp7 has a helical

Caloric Restriction (CR) extends lifespan and augments cellular stress-resistance from yeast to primates, making CR an attractive strategy for organ protection in the clinic. Translation of CR to patients is complex, due to problems regarding adherence, feasibility and safety concerns in frail patients. Novel tailored dietary regimens, which modulate the dietary composition of macro- and micronutrients

The membrane-embedded γ-secretase complex processively cleaves within the transmembrane domain of amyloid precursor protein (APP) to produce 37-to-43-residue amyloid β-peptides (Aβ) of Alzheimer’s disease (AD). Despite its importance in pathogenesis, the mechanism of processive proteolysis by γ-secretase remains poorly understood. Here, mass spectrometry and western blotting were used to quantify the

Analysis of mucin type O-glycans linked to serine/threonine of glycoproteins is technically challenging, in part, due to a lack of effective enzymatic tools that enable their analysis. Recently, several O-glycan-specific endoproteases that can cleave the protein adjacent to the appended glycan have been described. Despite significant progress in understanding the biochemistry of these enzymes, known

The precise maintenance of PTEN dosage is crucial for tumor suppression across a wide variety of cancers. Post-transcriptional regulation of Pten heavily relies on regulatory elements encoded by its 3’UTR. We previously reported the important diversity of 3’UTR isoforms of Pten mRNAs produced through alternative polyadenylation (APA). Here, we reveal the direct regulation of Pten APA by the mammalian

Cellular transmembrane (TM) proteins are essential sentries of the cell facilitating cell-cell communication, internal signaling, and solute transport. Reconstituting functional TM proteins into model membranes remains a challenge due to the difficulty of expressing hydrophobic TM domains and the required use of detergents. Herein, we use a intein-mediated ligation strategy to semisynthesize bitopic

The dueling roles of H2S as an endogenously synthesized respiratory substrate and as a toxin, raise questions as to how it is cleared when the electron transport chain is inhibited. Sulfide quinone oxidoreductase (SQOR) is a mitochondrial inner membrane flavoprotein that catalyzes the first step in the H2S oxidation pathway and uses coenzyme Q (CoQ) as an electron acceptor. However, complex IV poisoning

LOW GERMINATION STIMULANT 1 (LGS1) plays an important role in strigolactones (SLs) biosynthesis and Striga resistance in sorghum but the catalytic function remains unclear. Using the recently developed SL-producing microbial consortia, we examined the activities of sorghum MAX1 analogs and LGS1. Surprisingly, SbMAX1d (accession # XP_002458367) synthesized 18-hydroxy-carlactonoic acid (18-hydroxy-CLA)

Breast cancer is one the most aggressive cancer worldwide, especially Pakistan due to limited therapeutic options. This study was conducted to repurpose the use of selective serotonin reuptake inhibitors (SSRIs), in the treatment of breast cancers, and merit to pursue drug re-positioning in oncology. Anti-proliferative activity of SSRIs, such as fluoxetine, paroxetine, and sertraline hydrochloride

Untargeted metabolomics via mass spectrometry (MS) can reveal several 100,000 molecular features in a single sample, most of which may represent unidentified metabolites, posing significant challenges to data analysis. We here introduce Metaboseek, an open-source analysis platform designed for untargeted comparative metabolomics, and demonstrate its utility to elucidate functions of a conserved fat

Mycobacterium tuberculosis peptidoglycan (PG) is atypical as its synthesis involves a new enzyme class, L,D-transpeptidases. Prior studies of L,D-transpeptidases have identified only the catalytic site that binds to peptide moiety of the PG substrate or ß-lactam antibiotics. This insight was leveraged to develop mechanism of its activity and inhibition by ß-lactams. Here we report identification of

Nonenzymatic template-directed RNA copying using chemically activated nucleotides is thought to have played a key role in the emergence of genetic information on the early Earth. A longstanding question concerns the number and nature of different environments that might have been necessary to enable all of the steps from nucleotide synthesis to RNA replication. Here we explore three sequential steps

The molecular motor myosin undergoes a series of major structural transitions during its force-producing motor cycle. The underlying mechanism and its coupling to ATP hydrolysis and actin binding is only partially understood, mostly due to sparse structural data on actin-bound states of myosin. Here, we report 26 high-resolution cryo-EM structures of the actomyosin-V complex in the strong-ADP, rigor

The functional properties of alginates are dictated by the monomer composition and molecular weight distribution. Mannuronan C-5 epimerases determine the former by epimerizing β-D-mannuronic acid residues (M) into α-L-guluronic acid residues (G). The molecular weight is affected by alginate lyases, which cleave alginate chains through β-elimination. The reaction mechanisms for the epimerization and

In bacterial defense and genome editing applications, the CRISPR-associated protein Cas9 searches millions of DNA base pairs to locate a 20-nucleotide, guide-RNA-complementary target sequence that abuts a protospacer-adjacent motif (PAM)1. Target capture requires Cas9 to unwind DNA at candidate sequences using an unknown ATP-independent mechanism2,3. Here we show that Cas9 sharply bends and undertwists

Assembly of ribosomal subunits occurs via parallel pathways, which accelerate the process and render it more robust. Nonetheless, in vitro analyses have also demonstrated that some assembly pathways are dead-ends, presumably due to rRNA misfolding. If and how these non-productive pathways are avoided during assembly in vivo remains unknown. Here we use a combination of biochemical, genetic, proteomic

Human autosomal recessive mutations in the PINK1 gene are causal for Parkinson’s disease (PD). PINK1 encodes a mitochondrial localised protein kinase that is a master-regulator of mitochondrial quality control pathways. Structural studies to date have elaborated the mechanism of how mutations located within the kinase domain disrupt PINK1 function, however, the molecular mechanism of PINK1 mutations

Metabolism forms a complex, interdependent network, and perturbations can have indirect effects that are pervasive. Identifying these patterns and their consequences is difficult, particularly when the effects occur across canonical pathways, and these difficulties have long acted as a bottleneck in metabolic data analysis. This challenge is compounded by technical limitations in metabolomics approaches

Hemoglobin oxidation due to oxidative stress and disease conditions leads to generation of ROS (reactive oxygen species) and membrane attachment of hemoglobin in-vivo, where its redox activity leads to peroxidative damage of membrane lipids and proteins. Spectrin, the major component of the RBC membrane skeleton, is known to interact with hemoglobin and, here this interaction is shown to increase hemoglobin

Septins are part of the cytoskeleton and polymerize into non-polar filaments of heteromeric hexamers or octamers. They belong to the class of P-loop GTPases but the roles of GTP binding and hydrolysis on filament formation and dynamics are not well understood.

Living cells move and change their shape because signaling chemical reactions modify the state of their cytoskeleton; an active gel that converts chemical energy into mechanical forces. To create life-like materials, it is thus key to engineer chemical pathways that drive active gels. Here, we describe the preparation of DNA-responsive surfaces that control the activity of a cytoskeletal active gel

Spermidine and other polyamines alleviate oxidative stress, yet excess spermidine seems toxic to Escherichia coli unless it is neutralized by SpeG, an enzyme for the spermidine N-acetyl transferase function. Besides, a specific mechanism of SpeG function conferring pathogenic fitness to Staphylococcus aureus USA300 strain is unknown. Here, we provide evidence that although spermidine mitigates oxidative

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system is composed of a Cas12a effector that acts as a deoxyribonucleic acid (DNA)-cleaving endonuclease and a crispr ribonucleic acid (crRNA) that guides the effector to the target DNA. It is considered a key molecule for inducing target-specific gene editing in various living systems. Here, we improved the efficiency and

The conserved Tweety homolog (TTYH) family consists of three paralogs in vertebrates, displaying a ubiquitous expression pattern. Although considered as ion channels for almost two decades, recent structural and functional analyses refuted this role. Intriguingly, while all paralogs, studied following detergent solubilization, shared a dimeric stoichiometry, their spatial organization differed. Here

Transmembrane signaling proteins couple extracytosolic sensors to cytosolic effectors. Here, we examine how binding of Mg2+ to the sensor domain of an E. coli two component histidine kinase (HK), PhoQ, modulates its cytoplasmic kinase domain. We use cysteine-crosslinking and reporter-gene assays to simultaneously and independently probe the signaling state of PhoQ’s sensor and autokinase domains in

Transition metals, such as zinc, are essential micronutrients in all organisms, but also highly toxic in excessive amounts. Heavy-metal transporting P-type (PIB) ATPases are crucial for homeostasis, conferring cellular detoxification and redistribution through transport of these ions across cellular membranes. No structural information is available for the PIB-4-ATPases, the subclass with the broadest

The peroxisomal very long chain fatty acid (VLCFA) transporter ABCD1 is central to cellular fatty acid catabolism and lipid biosynthesis. Its dysfunction underlies toxic cytosolic accumulation of VLCFAs, progressive nervous system demyelination, and neurological impairments including the potentially fatal disease X-linked adrenoleukodystrophy (X-ALD). Molecular details underlying substrate recognition

β-Coronaviruses such as SARS-CoV-2 hijack coatomer protein-I (COPI) for spike protein retrograde trafficking to the progeny assembly site in endoplasmic reticulum-Golgi intermediate compartment (ERGIC). However, limited residue-level details are available into how the spike interacts with COPI. Here we identify a novel extended COPI binding motif in the spike that encompasses the canonical K-x-H dibasic

Single-cell proteomics workflows have considerably improved in sensitivity and reproducibility to characterize yet unknown biological phenomena. With the emergence of multiplexed single-cell proteomics, studies increasingly present single-cell measurements in conjunction with an abundant congruent carrier to improve precursor selection and enhance identifications. While these extreme carrier spikes

Protein hydroxylation is a post translational modification happens on various amino acids, which is catalyzed by the oxoglutarate and oxygen dependent dioxygenases. The best characterized hydroxylated protein is the hypoxia inducible factor (HIF), which is degraded by VHL/elongin C/elongin B/cullin 2/RBX1 (VCB/CR) E3 complex under normal oxygen conditions. Hypoxia or inhibitors (including FG4592 and

N-degron E3 ubiquitin ligases recognize specific residues at the N-termini of substrates. Although molecular details of N-degron recognition are known for several E3 ligases, the range of N-terminal motifs that can bind a given E3 substrate binding domain remains unclear. Here, studying the Gid4 and Gid10 substrate receptor subunits of yeast “GID”/human “CTLH” multiprotein E3 ligases, whose known substrates

The membrane-bound transcription factor Nrf1 (i.e., encoded by Nfe2l1) is activated by sensing glucose deprivation, cholesterol excess, proteasomal inhibition and oxidative stress, and then mediates distinct signaling responses in order to maintain cellular homeostasis. Herein, we found that Nrf1 stability and transactivity are both enhanced by USP19, a ubiquitin-specific protease tail-anchored in

The effects of UV light on the skin have been extensively investigated. However, systematic information about how exposure to UVA light, the least energetic but the most abundant UV radiation reaching the Earth, shapes the subcellular organization of proteins is lacking. Using subcellular fractionation, mass-spectrometry-based proteomics, machine learning algorithms, immunofluorescence, and functional

Hedgehog (Hh) signaling ligands undergo carboxy terminal sterylation through specialized autoprocessing, called cholesterolysis. Sterylation is brought about intramolecularly in a single turn-over by an enzymatic domain, called HhC. HhC is found in precursor Hh proteins only. Through cholesterolysis, HhC is cleaved from the precursor. Attempts to identify molecules that inhibit intramolecular cleavage/sterylation