Protocol for phospho-SrcKD: rPTPεD1 complex preparation and BLI binding assays to demonstrate their exosite interface.

Here, we present a protocol for the in vitro phosphorylation of Src kinase domain (SrcKD), preparation of phospho-SrcKD in complex with the D1 domain of rPTP epsilon (rPTPεD1), and binding assays using biolayer interferometry (BLI). We describe steps for the in vitro phosphorylation of SrcKD and preparation of the phospho-SrcKD: rPTPεD1 complex for small-angle X-ray scattering (SAXS) experiments. We then detail instructions for the BLI binding assay to determine the binding affinity between phospho-SrcKD and rPTPεD1. For complete details on the use and execution of this protocol, please refer to EswarKumar et al.1.

A structure of the relict phycobilisome from a thylakoid-free cyanobacterium.

Phycobilisomes (PBS) are antenna megacomplexes that transfer energy to photosystems II and I in thylakoids. PBS likely evolved from a basic, inefficient form into the predominant hemidiscoidal shape with radiating peripheral rods. However, it has been challenging to test this hypothesis because ancestral species are generally inaccessible. Here we use spectroscopy and cryo-electron microscopy to reveal a structure of a "paddle-shaped" PBS from a thylakoid-free cyanobacterium that likely retains ancestral traits. This PBS lacks rods and specialized ApcD and ApcF subunits, indicating relict characteristics. Other features include linkers connecting two chains of five phycocyanin hexamers (CpcN) and two core subdomains (ApcH), resulting in a paddle-shaped configuration. Energy transfer calculations demonstrate that chains are less efficient than rods. These features may nevertheless have increased light absorption by elongating PBS before multilayered thylakoids with hemidiscoidal PBS evolved. Our results provide insights into the evolution and diversification of light-harvesting strategies before the origin of thylakoids.

An integrative approach unveils a distal encounter site for rPTPε and phospho-Src complex formation.

The structure determination of protein tyrosine phosphatase (PTP): phospho-protein complexes, which is essential to understand how specificity is achieved at the amino acid level, remains a significant challenge for protein crystallography and cryoEM due to the transient nature of binding interactions. Using rPTPεD1 and phospho-SrcKD as a model system, we have established an integrative workflow to address this problem, by means of which we generate a protein:phospho-protein complex model using predetermined protein structures, SAXS and pTyr-tailored MD simulations. Our model reveals transient protein-protein interactions between rPTPεD1 and phospho-SrcKD and is supported by three independent experimental validations. Measurements of the association rate between rPTPεD1 and phospho-SrcKD showed that mutations on the rPTPεD1: SrcKD complex interface disrupts these transient interactions, resulting in a reduction in protein-protein association rate and, eventually, phosphatase activity. This integrative approach is applicable to other PTP: phospho-protein complexes and the characterization of transient protein-protein interface interactions.

PAICS ubiquitination recruits UBAP2 to trigger phase separation for purinosome assembly.

Purinosomes serve as metabolons to enhance de novo purine synthesis (DNPS) efficiency through compartmentalizing DNPS enzymes during stressed conditions. However, the mechanism underpinning purinosome assembly and its pathophysiological functions remains elusive. Here, we show that K6-polyubiquitination of the DNPS enzyme phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthetase (PAICS) by cullin-5/ankyrin repeat and SOCS box containing 11 (Cul5/ASB11)-based ubiquitin ligase plays a driving role in purinosome assembly. Upon several purinosome-inducing cues, ASB11 is upregulated by relieving the H3K9me3/HP1α-mediated transcriptional silencing, thus stimulating PAICS polyubiquitination. The polyubiquitinated PAICS recruits ubiquitin-associated protein 2 (UBAP2), a ubiquitin-binding protein with multiple stretches of intrinsically disordered regions, thereby inducing phase separation to trigger purinosome assembly for enhancing DNPS pathway flux. In human melanoma, ASB11 is highly expressed to facilitate a constitutive purinosome formation to which melanoma cells are addicted for supporting their proliferation, viability, and tumorigenesis in a xenograft model. Our study identifies a driving mechanism for purinosome assembly in response to cellular stresses and uncovers the impact of purinosome formation on human malignancies.

Structural basis for calcium-stimulating pore formation of Vibrio α-hemolysin.

Vibrio α-hemolysins (αHLs) are ß-pore-forming toxins secreted by Vibrio pathogens, crucial for the facilitation of bacterial infections through host cell lysis. These toxins are produced as inactive precursors, requiring proteolytic maturation and membrane association for activation within host tissues. Here, we investigate Vibrio campbellii αHL (VcαHL), and establish that its hemolytic activity is significantly stimulated by calcium ions, with an EC50 that aligns with physiological calcium concentrations. Furthermore, we illustrate the vital contribution of calcium ions to the oligomerization of VcαHL on membranes. Using X-ray crystallography and cryo-electron microscopy, we decipher both the immature and assembled structures of VcαHL and elucidate the conformational changes corresponding to toxin assembly. We also identify a calcium-binding module that is integral for VcαHL's calcium-dependent activation. These findings provide insights into the regulatory mechanisms of VcαHL and have the potential to inform the development of targeted therapeutic strategies against Vibrio infections.

Visualizing the membrane disruption action of antimicrobial peptides by cryo-electron tomography.

The abuse of antibiotics has led to the emergence of multidrug-resistant microbial pathogens, presenting a pressing challenge in global healthcare. Membrane-disrupting antimicrobial peptides (AMPs) combat so-called superbugs via mechanisms different than conventional antibiotics and have good application prospects in medicine, agriculture, and the food industry. However, the mechanism-of-action of AMPs has not been fully characterized at the cellular level due to a lack of high-resolution imaging technologies that can capture cellular-membrane disruption events in the hydrated state. Previously, we reported PepD2M, a de novo-designed AMP with potent and wide-spectrum bactericidal and fungicidal activity. In this study, we use cryo-electron tomography (cryo-ET) and high-speed atomic force microscopy (HS-AFM) to directly visualize the pepD2M-induced disruption of the outer and inner membranes of the Gram-negative bacterium Escherichia coli, and compared with a well-known pore-forming peptide, melittin. Our high-resolution cryo-ET images reveal how pepD2M disrupts the E. coli membrane using a carpet/detergent-like mechanism. Our studies reveal the direct membrane-disrupting consequence of AMPs on the bacterial membrane by cryo-ET, and this information provides critical insights into the mechanisms of this class of antimicrobial agents.

A RAD51-ADP double filament structure unveils the mechanism of filament dynamics in homologous recombination.

ATP-dependent RAD51 recombinases play an essential role in eukaryotic homologous recombination by catalyzing a four-step process: 1) formation of a RAD51 single-filament assembly on ssDNA in the presence of ATP, 2) complementary DNA strand-exchange, 3) ATP hydrolysis transforming the RAD51 filament into an ADP-bound disassembly-competent state, and 4) RAD51 disassembly to provide access for DNA repairing enzymes. Of these steps, filament dynamics between the ATP- and ADP-bound states, and the RAD51 disassembly mechanism, are poorly understood due to the lack of near-atomic-resolution information of the ADP-bound RAD51-DNA filament structure. We report the cryo-EM structure of ADP-bound RAD51-DNA filaments at 3.1 Å resolution, revealing a unique RAD51 double-filament that wraps around ssDNA. Structural analysis, supported by ATP-chase and time-resolved cryo-EM experiments, reveals a collapsing mechanism involving two four-protomer movements along ssDNA for mechanical transition between RAD51 single- and double-filament without RAD51 dissociation. This mechanism enables elastic change of RAD51 filament length during structural transitions between ATP- and ADP-states.

SUB1A-1 anchors a regulatory cascade for epigenetic and transcriptional controls of submergence tolerance in rice.

Most rice (Oryza sativa) cultivars cannot survive under prolonged submergence. However, some O. sativa ssp. indica cultivars, such as FR13A, are highly tolerant owing to the SUBMERGENCE 1A-1 (SUB1A-1) allele, which encodes a Group VII ethylene-responsive factor (ERFVII) protein; other submergence-intolerant cultivars contain a SUB1A-2 allele. The two alleles differ only by a single substitution at the 186th amino acid position from serine in SUB1A-1 to proline in SUB1A-2 resulting in only SUB1A-1 being able to be phosphorylated. Two other ERFVIIs, ERF66 and ERF67, function downstream of SUB1A-1 to form a regulatory cascade in response to submergence stress. Here, we show that SUB1A-1, but not SUB1A-2, interacts with ADA2b of the ADA2b-GCN5 acetyltransferase complex, in which GCN5 functions as a histone acetyltransferase. Phosphorylation of SUB1A-1 at serine 186 enhances the interaction of SUB1A-1 with ADA2b. ADA2b and GCN5 expression was induced under submergence, suggesting that these two genes might play roles in response to submergence stress. In transient assays, binding of SUB1A-1 to the ERF67 promoter and ERF67 transcription were highly induced when SUB1A-1 was expressed together with the ADA2b-GCN5 acetyltransferase complex. Taken together, these results suggest that phospho-SUB1A-1 recruits the ADA2-GCN5 acetyltransferase complex to modify the chromatin structure of the ERF66/ERF67 promoter regions and activate gene expression, which in turn enhances rice submergence tolerance.

An ATP-sensitive phosphoketolase regulates carbon fixation in cyanobacteria.

Regulation of CO2 fixation in cyanobacteria is important both for the organism and global carbon balance. Here we show that phosphoketolase in Synechococcus elongatus PCC7942 (SeXPK) possesses a distinct ATP-sensing mechanism, where a drop in ATP level allows SeXPK to divert precursors of the RuBisCO substrate away from the Calvin-Benson-Bassham cycle. Deleting the SeXPK gene increased CO2 fixation particularly during light-dark transitions. In high-density cultures, the Δxpk strain showed a 60% increase in carbon fixation and unexpectedly resulted in sucrose secretion without any pathway engineering. Using cryo-EM analysis, we discovered that these functions were enabled by a unique allosteric regulatory site involving two subunits jointly binding two ATP, which constantly suppresses the activity of SeXPK until the ATP level drops. This magnesium-independent ATP allosteric site is present in many species across all three domains of life, where it may also play important regulatory functions.

Suppression of the human malic enzyme 2 modifies energy metabolism and inhibits cellular respiration.

Human mitochondrial NAD(P)+-dependent malic enzyme (ME2) is well-known for its role in cell metabolism, which may be involved in cancer or epilepsy. We present potent ME2 inhibitors based on cyro-EM structures that target ME2 enzyme activity. Two structures of ME2-inhibitor complexes demonstrate that 5,5'-Methylenedisalicylic acid (MDSA) and embonic acid (EA) bind allosterically to ME2's fumarate-binding site. Mutagenesis studies demonstrate that Asn35 and the Gln64-Tyr562 network are required for both inhibitors' binding. ME2 overexpression increases pyruvate and NADH production while decreasing the cell's NAD+/NADH ratio; however, ME2 knockdown has the opposite effect. MDSA and EA inhibit pyruvate synthesis and thus increase the NAD+/NADH ratio, implying that these two inhibitors interfere with metabolic changes by inhibiting cellular ME2 activity. ME2 silence or inhibiting ME2 activity with MDSA or EA decreases cellular respiration and ATP synthesis. Our findings suggest that ME2 is crucial for mitochondrial pyruvate and energy metabolism, as well as cellular respiration, and that ME2 inhibitors could be useful in the treatment of cancer or other diseases that involve these processes.

Structures of honeybee-infecting Lake Sinai virus reveal domain functions and capsid assembly with dynamic motions.

Understanding the structural diversity of honeybee-infecting viruses is critical to maintain pollinator health and manage the spread of diseases in ecology and agriculture. We determine cryo-EM structures of T = 4 and T = 3 capsids of virus-like particles (VLPs) of Lake Sinai virus (LSV) 2 and delta-N48 LSV1, belonging to tetraviruses, at resolutions of 2.3-2.6 Å in various pH environments. Structural analysis shows that the LSV2 capsid protein (CP) structural features, particularly the protruding domain and C-arm, differ from those of other tetraviruses. The anchor loop on the central ß-barrel domain interacts with the neighboring subunit to stabilize homo-trimeric capsomeres during assembly. Delta-N48 LSV1 CP interacts with ssRNA via the rigid helix α1', α1'-α1 loop, ß-barrel domain, and C-arm. Cryo-EM reconstructions, combined with X-ray crystallographic and small-angle scattering analyses, indicate that pH affects capsid conformations by regulating reversible dynamic particle motions and sizes of LSV2 VLPs. C-arms exist in all LSV2 and delta-N48 LSV1 VLPs across varied pH conditions, indicating that autoproteolysis cleavage is not required for LSV maturation. The observed linear domino-scaffold structures of various lengths, made up of trapezoid-shape capsomeres, provide a basis for icosahedral T = 4 and T = 3 architecture assemblies. These findings advance understanding of honeybee-infecting viruses that can cause Colony Collapse Disorder.

In Situ Structure Determination of Bacterial Surface Nanomachines Using Cryo-Electron Tomography.

Bacterial surface nanomachines are often refractory to structural determination in their intact form due to their extensive association with the cell envelope preventing them from being properly purified for traditional structural biology methods. Cryo-electron tomography (cryo-ET) is an emerging branch of cryo-electron microscopy that can visualize supramolecular complexes directly inside frozen-hydrated cells in 3D at nanometer resolution, therefore posing a unique capability to study the intact structures of bacterial surface nanomachines in situ and reveal their molecular association with other cellular components. Furthermore, the resolution of cryo-ET is continually improving alongside methodological advancement. Here, using the type IV pilus machine in Myxococcus xanthus as an example, we describe a step-by-step workflow for in situ structure determination including sample preparation and screening, microscope and camera tuning, tilt series acquisition, data processing and tomogram reconstruction, subtomogram averaging, and structural analysis.

Enzymology and Dynamics by Cryogenic Electron Microscopy.

Cryogenic electron microscopy (cryo-EM) has revolutionized the field of structural biology, particularly in solving the structures of large protein complexes or cellular machineries that play important biological functions. This review focuses on the contribution and future potential of cryo-EM in related emerging applications-enzymatic mechanisms and dynamic processes. Work on these subjects can benefit greatly from the capability of cryo-EM to solve the structures of specific protein complexes in multiple conditions, including variations in the buffer condition, ligands, and temperature, and to capture multiple conformational states, conformational change intermediates, and reaction intermediates. These studies can expand the structural landscape of specific proteins or protein complexes in multiple dimensions and drive new advances in the fields of enzymology and dynamic processes. The advantages and complementarity of cryo-EM relative to X-ray crystallography and nuclear magnetic resonance with regard to these applications are also addressed.

The macromolecular complexes of histones affect protein arginine methyltransferase activities.

Histone arginine methylation is a key post-translational modification that mediates epigenetic events that activate or repress gene transcription. Protein arginine methyltransferases (PRMTs) are the driving force for the process of arginine methylation, and the core histone proteins have been shown to be substrates for most PRMT family members. However, previous reports of the enzymatic activities of PRMTs on histones in the context of nucleosomes seem contradictory. Moreover, what governs nucleosomal substrate recognition of different PRMT members is not understood. We sought to address this key biological question by examining how different macromolecular contexts where the core histones reside may regulate arginine methylation catalyzed by individual PRMT members (i.e., PRMT1, PRMT3, PRMT4, PRMT5, PRMT6, PRMT7, and PRMT8). Our results demonstrated that the substrate context exhibits a huge impact on the histone arginine methylation activity of PRMTs. Although all the tested PRMTs methylate multiple free histones individually, they show a preference for one particular histone substrate in the context of the histone octamer. We found that PRMT1, PRMT3, PRMT5, PRMT6, PRMT7, and PRMT8 preferentially methylate histone H4, whereas PRMT4/coactivator-associated arginine methyltransferase 1 prefers histone H3. Importantly, neither reconstituted nor cell-extracted mononucleosomes could be methylated by any PRMTs tested. Structural analysis suggested that the electrostatic interaction may play a mechanistic role in priming the substrates for methylation by PRMT enzymes. Taken together, this work expands our knowledge on the molecular mechanisms of PRMT substrate recognition and has important implications for understanding cellular dynamics and kinetics of histone arginine methylation in regulating gene transcription and other chromatin-templated processes.

Pharmacophore-based screening of diamidine small molecule inhibitors for protein arginine methyltransferases.

Protein arginine methyltransferases (PRMTs) are essential epigenetic and post-translational regulators in eukaryotic organisms. Dysregulation of PRMTs is intimately related to multiple types of human diseases, particularly cancer. Based on the previously reported PRMT1 inhibitors bearing the diamidine pharmacophore, we performed virtual screening to identify additional amidine-associated structural analogs. Subsequent enzymatic tests and characterization led to the discovery of a top lead K313 (2-(4-((4-carbamimidoylphenyl)amino)phenyl)-1H-indole-6-carboximidamide), which possessed low-micromolar potency with biochemical IC50 of 2.6 µM for human PRMT1. Limited selectivity was observed over some other PRMT isoforms such as CARM1 and PRMT7. Molecular modeling and inhibition pattern studies suggest that K313 is a nonclassic noncompetitive inhibitor to PRMT1. K313 significantly inhibited cell proliferation and reduced the arginine asymmetric dimethylation level in the leukaemia cancer cells.

Single nucleotide variants lead to dysregulation of the human mitochondrial NAD(P)+-dependent malic enzyme.

Human mitochondrial NAD(P)+-dependent malic enzyme (ME2) is well recognized to associate with cancer cell metabolism, and the single nucleotide variants (SNVs) of ME2 may play a role in enzyme regulation. Here we reported that the SNVs of ME2 occurring in the allosteric sites lead to inactivation or overactivation of ME2. Two ME2-SNVs, ME2_R67Q and ME2-R484W, that demonstrated inactivating or overactivating enzyme activities of ME2, respectively, have different impact toward the cells. The cells with overactivating SNV enzyme, ME2_R484W, grow more rapidly and are more resistant to cellular senescence than the cells with wild-type or inactivating SNV enzyme, ME2_R67Q. Crystal structures of these two ME2-SNVs reveal that ME2_R67Q was an inactivating "dead form," and ME2_R484W was an overactivating "closed form" of the enzyme. The resolved ME2-SNV structures provide a molecular basis to explain the abnormal kinetic properties of these SNV enzymes.

Identification of fidelity-governing factors in human recombinases DMC1 and RAD51 from cryo-EM structures.

Both high-fidelity and mismatch-tolerant recombination, catalyzed by RAD51 and DMC1 recombinases, respectively, are indispensable for genomic integrity. Here, we use cryo-EM, MD simulation and functional analysis to elucidate the structural basis for the mismatch tolerance of DMC1. Structural analysis of DMC1 presynaptic and postsynaptic complexes suggested that the lineage-specific Loop 1 Gln244 (Met243 in RAD51) may help stabilize DNA backbone, whereas Loop 2 Pro274 and Gly275 (Val273/Asp274 in RAD51) may provide an open "triplet gate" for mismatch tolerance. In support, DMC1-Q244M displayed marked increase in DNA dynamics, leading to unobservable DNA map. MD simulation showed highly dispersive mismatched DNA ensemble in RAD51 but well-converged DNA in DMC1 and RAD51-V273P/D274G. Replacing Loop 1 or Loop 2 residues in DMC1 with RAD51 counterparts enhanced DMC1 fidelity, while reciprocal mutations in RAD51 attenuated its fidelity. Our results show that three Loop 1/Loop 2 residues jointly enact contrasting fidelities of DNA recombinases.

A Carbohydrate-Binding Protein from the Edible Lablab Beans Effectively Blocks the Infections of Influenza Viruses and SARS-CoV-2.

The influenza virus hemagglutinin (HA) and coronavirus spike (S) protein mediate virus entry. HA and S proteins are heavily glycosylated, making them potential targets for carbohydrate binding agents such as lectins. Here, we show that the lectin FRIL, isolated from hyacinth beans (Lablab purpureus), has anti-influenza and anti-SARS-CoV-2 activity. FRIL can neutralize 11 representative human and avian influenza strains at low nanomolar concentrations, and intranasal administration of FRIL is protective against lethal H1N1 infection in mice. FRIL binds preferentially to complex-type N-glycans and neutralizes viruses that possess complex-type N-glycans on their envelopes. As a homotetramer, FRIL is capable of aggregating influenza particles through multivalent binding and trapping influenza virions in cytoplasmic late endosomes, preventing their nuclear entry. Remarkably, FRIL also effectively neutralizes SARS-CoV-2, preventing viral protein production and cytopathic effect in host cells. These findings suggest a potential application of FRIL for the prevention and/or treatment of influenza and COVID-19.