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Mechanistic regulation of DNA transposon systems in mammalian cells remains poorly understood. Using modeling, biochemical, and cell-based assays, we sought to extend the recent cryoEM structural insight into the piggyBac transpososome to evaluate the previously unexplained role of the transposase N-terminus, the need for asymmetric transposon ends, and the complexity of transposase tetramer formation for transposition in mammalian cells. We found that N-terminal phosphorylation by casein kinase II inhibits transposase-DNA interaction and designed deletion of this phosphorylated domain releases inhibition thereby enhancing activity. We also found that the N-terminal domain promotes transposase dimerization in the absence of transposon DNA. N-terminal deletion enables transposition of symmetric transposon ends that was previously not achievable with piggyBac. The complex transposase tetramer needed for transposition of asymmetric transposon ends can be overcome via appending a second transposase C-terminal domain in combination with symmetric transposon ends overcoming the negative regulation by asymmetric ends. Our results demonstrate that N-terminal transposase phosphorylation and the requirement for asymmetric transposon ends both negatively regulate piggyBac transposons in mammalian cells. These novel insights into mechanism and structure of the piggyBac transposase expand its potential use for genomic applications.

Insulin-degrading enzyme (IDE) hydrolyzes monomeric polypeptides, including amyloid-β (Aβ) and HIV-1 p6. It also acts as a nonproteolytic chaperone to prevent Aβ polymerization. Here we compare interactions of Aβ and non-amyloidogenic p6 with IDE. Although both exhibited similar proteolysis rates, the binding kinetics to an inactive IDE characterized using relaxation-based NMR were remarkably different. IDE and Aβ formed a sparsely populated complex with a lifetime of milliseconds in which a short hydrophobic cleavage segment of Aβ was anchored to IDE. Strikingly, a second and more stable complex was significantly populated with a subsecond lifetime owing to multiple intermolecular contacts between Aβ and IDE. By selectively sequestering Aβ in this nonproductive complex, IDE likely increases the critical concentration required for fibrillization. In contrast, IDE and p6 formed a transient, submillisecond complex involving a single anchoring p6 motif. Modulation of intermolecular interactions, thus, allows IDE to differentiate between non-amyloidogenic and amyloidogenic substrates.

S-acylation, also known as palmitoylation, is the most abundant form of protein lipidation in humans. This reversible posttranslational modification, which targets thousands of proteins, is catalyzed by 23 members of the DHHC family of integral membrane enzymes. DHHC enzymes use fatty acyl-CoA as the ubiquitous fatty acyl donor and become autoacylated at a catalytic cysteine; this intermediate subsequently transfers the fatty acyl group to a cysteine in the target protein. Protein S-acylation intersects with almost all areas of human physiology, and several DHHC enzymes are considered as possible therapeutic targets against diseases such as cancer. These efforts would greatly benefit from a detailed understanding of the molecular basis for this crucial enzymatic reaction. Here, we combine X-ray crystallography with all-atom molecular dynamics simulations to elucidate the structure of the precatalytic complex of human DHHC20 in complex with palmitoyl CoA. The resulting structure reveals that the fatty acyl chain inserts into a hydrophobic pocket within the transmembrane spanning region of the protein, whereas the CoA headgroup is recognized by the cytosolic domain through polar and ionic interactions. Biochemical experiments corroborate the predictions from our structural model. We show, using both computational and experimental analyses, that palmitoyl CoA acts as a bivalent ligand where the interaction of the DHHC enzyme with both the fatty acyl chain and the CoA headgroup is important for catalytic chemistry to proceed. This bivalency explains how, in the presence of high concentrations of free CoA under physiological conditions, DHHC enzymes can efficiently use palmitoyl CoA as a substrate for autoacylation.

Description, Heptad repeat regions of SARS-CoV-2 spike protein transiently disrupt membrane integrity while driving cellular entry., Entry of SARS-CoV-2 into a host cell is mediated by spike, a class I viral fusion protein responsible for merging the viral and host cell membranes. Recent studies have revealed atomic-resolution models for both the postfusion 6-helix bundle (6HB) and the prefusion state of spike. However, a mechanistic understanding of the molecular basis for the intervening structural transition, important for the design of fusion inhibitors, has remained elusive. Using nuclear magnetic resonance spectroscopy and other biophysical methods, we demonstrate the presence of α-helical, membrane-bound, intermediate states of spike’s heptad repeat (HR1 and HR2) domains that are embedded at the lipid-water interface while in a slow dynamic equilibrium with the postfusion 6HB state. These results support a model where the HR domains lower the large energy barrier associated with membrane fusion by destabilizing the host and viral membranes, while 6HB formation actively drives their fusion by forcing physical proximity.

Energy-dependent Lon proteases play a key role in cellular regulation by degrading short-lived regulatory proteins and misfolded proteins in the cell. The structure of the catalytically inactive S679A mutant of Escherichia coli LonA protease (EcLon) has been determined by cryo-EM at the resolution of 3.5 Å. EcLonA without a bound substrate adopts a hexameric open-spiral quaternary structure that might represent the resting state of the enzyme. Upon interaction with substrate the open-spiral hexamer undergoes a major conformational change resulting in a compact, closed-circle hexamer as in the recent structure of a complex of Yersinia pestis LonA with a protein substrate. This major change is accomplished by the rigid-body rearrangement of the individual domains within the protomers of the complex around the hinge points in the interdomain linkers. Comparison of substrate-free and substrate-bound Lon structures allows to mark the location of putative pivotal points involved in such conformational changes., Graphical abstract In Brief The structure of the E. coli LonA protease was determined by cryo-EM. The unliganded hexameric complex adopts an open spiral quaternary structure. Upon interaction with substrate it likely undergoes a rigid-body conformational change resulting in a compact, closed-circle hexamer as reported in the Yersinia pestis Lon structure.Image 1, Highlights • E. coli LonA(S679A) protease adopts an open spiral quaternary structure. • This conformation might represent the resting state of the complex. • Upon substrate binding the open spiral will close into a circular hexamer.

Legionella pneumophila causes a potentially fatal form of pneumonia by replicating within macrophages in the Legionella-containing vacuole (LCV). Bacterial survival and proliferation within the LCV rely on hundreds of secreted effector proteins comprising high functional redundancy. The vacuolar membrane-localized MavN, hypothesized to support iron transport, is unique among effectors because loss-of-function mutations result in severe intracellular growth defects. We show here an iron starvation response by L. pneumophila after infection of macrophages that was prematurely induced in the absence of MavN, consistent with MavN granting access to limiting cellular iron stores. MavN cysteine accessibilities to a membrane-impermeant label were determined during macrophage infections, revealing a topological pattern supporting multipass membrane transporter models. Mutations to several highly conserved residues that can take part in metal recognition and transport resulted in defective intracellular growth. Purified MavN and mutant derivatives were directly tested for transporter activity after heterologous purification and liposome reconstitution. Proteoliposomes harboring MavN exhibited robust transport of Fe2+, with the severity of defect of most mutants closely mimicking the magnitude of defects during intracellular growth. Surprisingly, MavN was equivalently proficient at transporting Fe2+, Mn2+, Co2+, or Zn2+ Consequently, flooding infected cells with either Mn2+ or Zn2+ allowed collaboration with iron to enhance intracellular growth of L. pneumophila ΔmavN strains, indicating a clear role for MavN in transporting each of these ions. These findings reveal that MavN is a transition-metal-ion transporter that plays a critical role in response to iron limitation during Legionella infection.

The cell is constructed by higher-order structures and organelles through complex interactions among distinct structural constituents. The centrosome is a membraneless organelle composed of two microtubule-derived structures called centrioles and an amorphous mass of pericentriolar material. Super-resolution microscopic analyses in various organisms revealed that diverse pericentriolar material proteins are concentrically localized around a centriole in a highly organized manner. However, the molecular nature underlying these organizations remains unknown. Here we show that two human pericentriolar material scaffolds, Cep63 and Cep152, cooperatively generate a heterotetrameric α-helical bundle that functions in conjunction with its neighboring hydrophobic motifs to self-assemble into a higher-order cylindrical architecture capable of recruiting downstream components, including Plk4, a key regulator for centriole duplication. Mutations disrupting the self-assembly abrogate Plk4-mediated centriole duplication. Because pericentriolar material organization is evolutionarily conserved, this work may offer a paradigm for investigating the assembly and function of centrosomal scaffolds in various organisms., The centrosome is a membraneless organelle composed of two centrioles and an amorphous pericentriolar material but the overall centrosome organizations remains unknown. Here authors show that two scaffold proteins, Cep63 and Cep152, self-assemble into a higher-order cylindrical architecture capable of recruiting downstream components, including Plk4.

Deregulation of KRAS4b signaling pathway has been implicated in 30% of all cancers. Membrane localization of KRAS4b is an essential step for the initiation of the downstream signaling cascades that guide various cellular mechanisms. KRAS4b plasma membrane (PM) binding is mediated by the insertion of a prenylated moiety that is attached to the terminal carboxy-methylated cysteine, in addition to electrostatic interactions of its positively charged hypervariable region with anionic lipids. Calmodulin (CaM) has been suggested to selectively bind KRAS4b to act as a negative regulator of the RAS/mitogen-activated protein kinase (MAPK) signaling pathway by displacing KRAS4b from the membrane. However, the mechanism by which CaM can recognize and displace KRAS4b from the membrane is not well understood. In this study, we employed biophysical and structural techniques to characterize this mechanism in detail. We show that KRAS4b prenylation is required for bindingtoCaM and that the hydrophobic pockets of CaM can accommodate the prenylated region of KRAS4b, which might representa novel CaM-binding motif. Remarkably, prenylated KRAS4b forms a 2:1 stoichiometric complex with CaM in a nucleotide-independentmanner. The interaction between prenylated KRAS4b and CaM is enthalpically driven, and electrostatic interactions also contribute to the formation of the complex. The prenylated KRAS4b terminal KSKTKC-farnesylation and carboxy-methylation is sufficient for binding and defines the minimal CaM-binding motif. This is the same region implicated in membrane and phosphodiesterase6-δ binding. Finally, we provide a structure-based docking model by which CaM binds to prenylated KRAS4b. Our data provide new insights into the KRAS4b-CaM interaction and suggest a possible mechanism whereby CaM can regulate KRAS4b membrane localization.

Enzyme I (EI) is a phosphotransferase enzyme responsible for converting phosphoenolpyruvate (PEP) into pyruvate. This reaction initiates a five-step phosphorylation cascade in the bacterial phosphotransferase (PTS) transduction pathway. Under physiological conditions, EI exists in an equilibrium between a functional dimer and an inactive monomer. The monomer–dimer equilibrium is a crucial factor regulating EI activity and the phosphorylation state of the overall PTS. Experimental studies of EI’s monomeric state have yet been hampered by the dimer’s high thermodynamic stability, which prevents its characterization by standard structural techniques. In this study, we modified the dimerization domain of EI (EIC) by mutating three amino acids involved in the formation of intersubunit salt bridges. The engineered variant forms an active dimer in solution that can bind and hydrolyze PEP. Using hydrostatic pressure as an additional perturbation, we were then able to study the complete dissociation of the variant from 1 bar to 2.5 kbar in the absence and the presence of EI natural ligands. Backbone residual dipolar couplings collected under high-pressure conditions allowed us to determine the conformational ensemble of the isolated EIC monomeric state in solution. Our calculations reveal that three catalytic loops near the dimerization interface become unstructured upon monomerization, preventing the monomeric enzyme from binding its natural substrate. This study provides an atomic-level characterization of EI’s monomeric state and highlights the role of the catalytic loops as allosteric connectors controlling both the activity and oligomerization of the enzyme.

Proline-rich domains (PRDs) are among the most prevalent signaling modules of eukaryotes but often unexplored by biophysical techniques as their heterologous recombinant expression poses significant difficulties. Using a "divide-and-conquer" approach, we present a detailed investigation of a PRD (166 residues; ∼30% prolines) belonging to a human protein ALIX, a versatile adaptor protein involved in essential cellular processes including ESCRT-mediated membrane remodeling, cell adhesion, and apoptosis. In solution, the N-terminal fragment of ALIX-PRD is dynamically disordered. It contains three tandem sequentially similar proline-rich motifs that compete for a single binding site on its signaling partner, TSG101-UEV, as evidenced by heteronuclear NMR spectroscopy. Global fitting of relaxation dispersion data, measured as a function of TSG101-UEV concentration, allowed precise quantitation of these interactions. In contrast to the soluble N-terminal portion, the C-terminal tyrosine-rich fragment of ALIX-PRD forms amyloid fibrils and viscous gels validated using dye-binding assays with amyloid-specific probes, congo red and thioflavin T (ThT), and visualized by transmission electron microscopy. Remarkably, fibrils dissolve at low temperatures (2 to 6 °C) or upon hyperphosphorylation with Src kinase. Aggregation kinetics monitored by ThT fluorescence shows that charge repulsion dictates phosphorylation-mediated fibril dissolution and that the hydrophobic effect drives fibril formation. These data illuminate the mechanistic interplay between interactions of ALIX-PRD with TSG101-UEV and polymerization of ALIX-PRD and its central role in regulating ALIX function. This study also demonstrates the broad functional repertoires of PRDs and uncovers the impact of posttranslational modifications in the modulation of reversible amyloids.

Significance The root cause of pathology in sickle cell disease is the polymerization of the mutant hemoglobin S upon deoxygenation in the tissues to form fibers. Both the amount of fiber at equilibrium and the kinetics of fiber formation depend on the partial pressure of oxygen. We show that control of polymerization by oxygen at equilibrium can be better explained by a recent extension of the famous two-state allosteric model of Monod, Wyman, and Changeux. Because of the unusual kinetics, polymerization is far out of equilibrium, which explains why patients with the disease manage to survive, while those with high levels of fetal hemoglobin and those with sickle trait (the heterozygous condition) have relatively benign conditions., The pathology of sickle cell disease is caused by polymerization of the abnormal hemoglobin S upon deoxygenation in the tissues to form fibers in red cells, causing them to deform and occlude the circulation. Drugs that allosterically shift the quaternary equilibrium from the polymerizing T quaternary structure to the nonpolymerizing R quaternary structure are now being developed. Here we update our understanding on the allosteric control of fiber formation at equilibrium by showing how the simplest extension of the classic quaternary two-state allosteric model of Monod, Wyman, and Changeux to include tertiary conformational changes provides a better quantitative description. We also show that if fiber formation is at equilibrium in vivo, the vast majority of cells in most tissues would contain fibers, indicating that it is unlikely that the disease would be survivable once the nonpolymerizing fetal hemoglobin has been replaced by adult hemoglobin S at about 1 y after birth. Calculations of sickling times, based on a recently discovered universal relation between the delay time prior to fiber formation and supersaturation, show that in vivo fiber formation is very far from equilibrium. Our analysis indicates that patients survive because the delay period allows the majority of cells to escape the small vessels of the tissues before fibers form. The enormous sensitivity of the duration of the delay period to intracellular hemoglobin composition also explains why sickle trait, the heterozygous condition, and the compound heterozygous condition of hemoglobin S with pancellular hereditary persistence of fetal hemoglobin are both relatively benign conditions.

Conformational disorder is emerging as an important feature of biopolymers, regulating a vast array of cellular functions, including signaling, phase separation, and enzyme catalysis. Here we combine NMR, crystallography, computer simulations, protein engineering, and functional assays to investigate the role played by conformational heterogeneity in determining the activity of the C-terminal domain of bacterial Enzyme I (EIC). In particular, we design chimeric proteins by hybridizing EIC from thermophilic and mesophilic organisms, and we characterize the resulting constructs for structure, dynamics, and biological function. We show that EIC exists as a mixture of active and inactive conformations and that functional regulation is achieved by tuning the thermodynamic balance between active and inactive states. Interestingly, we also present a hybrid thermophilic/mesophilic enzyme that is thermostable and more active than the wild-type thermophilic enzyme, suggesting that hybridizing thermophilic and mesophilic proteins is a valid strategy to engineer thermostable enzymes with significant low-temperature activity.

Copy‐out/paste‐in transposition is a major bacterial DNA mobility pathway. It contributes significantly to the emergence of antibiotic resistance, often by upregulating expression of downstream genes upon integration. Unlike other transposition pathways, it requires both asymmetric and symmetric strand transfer steps. Here, we report the first structural study of a copy‐out/paste‐in transposase and demonstrate its ability to catalyze all pathway steps in vitro. X‐ray structures of ISC th4 transposase, a member of the IS 256 family of insertion sequences, bound to DNA substrates corresponding to three sequential steps in the reaction reveal an unusual asymmetric dimeric transpososome. During transposition, an array of N‐terminal domains binds a single transposon end while the catalytic domain moves to accommodate the varying substrates. These conformational changes control the path of DNA flanking the transposon end and the generation of DNA‐binding sites. Our results explain the asymmetric outcome of the initial strand transfer and show how DNA binding is modulated by the asymmetric transposase to allow the capture of a second transposon end and to integrate a circular intermediate., Structures of ISC th4 transposase/DNA complexes at different stages of the transposition pathway provide first insight into transpososome architecture and reaction mechanism of this major bacterial DNA mobility pathway.

Human profilin I reduces aggregation and concomitant toxicity of the polyglutamine-containing N-terminal region of the huntingtin protein encoded by exon 1 (htt ex1 ) and responsible for Huntington’s disease. Here, we investigate the interaction of profilin with htt ex1 using NMR techniques designed to quantitatively analyze the kinetics and equilibria of chemical exchange at atomic resolution, including relaxation dispersion, exchange-induced shifts, and lifetime line broadening. We first show that the presence of two polyproline tracts in htt ex1 , absent from a shorter huntingtin variant studied previously, modulates the kinetics of the transient branched oligomerization pathway that precedes nucleation, resulting in an increase in the populations of the on-pathway helical coiled-coil dimeric and tetrameric species (τ ex ≤ 50 to 70 μs), while leaving the population of the off-pathway (nonproductive) dimeric species largely unaffected (τ ex ∼750 μs). Next, we show that the affinity of a single molecule of profilin to the polyproline tracts is in the micromolar range ( K diss ∼ 17 and ∼ 31 μM), but binding of a second molecule of profilin is negatively cooperative, with the affinity reduced ∼11-fold. The lifetime of a 1:1 complex of htt ex1 with profilin, determined using a shorter huntingtin variant containing only a single polyproline tract, is shown to be on the submillisecond timescale ( τ ex ∼ 600 μs and K diss ∼ 50 μM). Finally, we demonstrate that, in stable profilin–htt ex1 complexes, the productive oligomerization pathway, leading to the formation of helical coiled-coil htt ex1 tetramers, is completely abolished, and only the pathway resulting in “nonproductive” dimers remains active, thereby providing a mechanistic basis for how profilin reduces aggregation and toxicity of htt ex1 .

The bacterial phosphotransferase system (PTS) is a signal transduction pathway that couples phosphoryl transfer to active sugar transport across the cell membrane. The PTS is initiated by phosphorylation of enzyme I (EI) by phosphoenolpyruvate (PEP). The EI phosphorylation state determines the phosphorylation states of all other PTS components and is thought to play a central role in the regulation of several metabolic pathways and to control the biology of bacterial cells at multiple levels, for example, affecting virulence and biofilm formation. Given the pivotal role of EI in bacterial metabolism, an improved understanding of the mechanisms controlling its activity could inform future strategies for bioengineering and antimicrobial design. Here, we report an enzymatic assay, based on Selective Optimized Flip Angle Short Transient (SOFAST) NMR experiments, to investigate the effect of the small-molecule metabolite α-ketoglutarate (αKG) on the kinetics of the EI-catalyzed phosphoryl transfer reaction. We show that at experimental conditions favoring the monomeric form of EI, αKG promotes dimerization and acts as an allosteric stimulator of the enzyme. However, when the oligomerization state of EI is shifted toward the dimeric species, αKG functions as a competitive inhibitor of EI. We developed a kinetic model that fully accounted for the experimental data and indicated that bacterial cells might use the observed interplay between allosteric stimulation and competitive inhibition of EI by αKG to respond to physiological fluctuations in the intracellular environment. We expect that the mechanism for regulating EI activity revealed here is common to several other oligomeric enzymes.

Cryo-electron microscopy and X-ray crystallography have shown that the pre- and post-fusion states of the HIV-1 gp41 viral coat protein, although very different from one another, each adopt C3 symmetric structures. A stable homotrimeric structure for the transmembrane domain (TM) also was modeled and supported by experimental data. For a C3 symmetric structure, alignment in an anisotropic medium must be axially symmetric, with the unique axis of the alignment tensor coinciding with the C3 axis. However, NMR residual dipolar couplings (RDCs) measured under three different alignment conditions were found to be incompatible with C3 symmetry. Subsequent measurements by paramagnetic relaxation enhancement, analytical ultracentrifugation, and DEER EPR, indicate that the transmembrane domain is monomeric. 15N NMR relaxation data and RDCs show that TM is highly ordered and uninterrupted for a total length of 32 residues, extending well into the membrane proximal external region.

The HIV-1 virion structural polyprotein, Gag, is directed to particle assembly sites at the plasma membrane by its N-terminal Matrix (MA) domain. MA also binds to host tRNAs. To understand the molecular basis of MA-tRNA interaction and its potential function, we present a co-crystal structure of HIV-1 MA-tRNA(Lys3) complex. The structure reveals a specialized group of MA basic and aromatic residues preconfigured to recognize the distinctive structure of the tRNA elbow. Mutational, crosslinking, fluorescence and NMR analyses show that the crystallographically defined interface drives MA-tRNA binding in solution and living cells. The structure indicates that MA is unlikely to bind tRNA and membrane simultaneously. Accordingly, single amino-acid substitutions that abolish MA-tRNA binding caused striking redistribution of Gag to the plasma membrane and reduced HIV-1 replication. Thus, HIV-1 exploits host tRNAs to occlude a membrane localization signal and control the subcellular distribution of its major structural protein.

Due to their unique properties, tunable size, and ability to provide a near native lipid environment, nanodiscs have found widespread use for the structural and functional studies of reconstituted membrane proteins. They have also been developed, albeit in a few applications, for therapeutic and biomedical use. For these studies and applications, it is essential to characterize the nanodisc preparations in terms of their monodispersity, size, and composition, as these can influence the properties of the membrane protein of interest. Of the many biophysical methods utilized for the study and characterization of nanodiscs, we show that analytical ultracentrifugation is able to report on sample homogeneity, shape, size, composition, and membrane protein stoichiometry or oligomerization state in a direct and simple fashion. The method is truly versatile and does not require nanodisc modification or disassembly.

Bacterial MinD and MinE form a standing oscillatory wave which positions the cell division inhibitor MinC, that binds MinD, everywhere on the membrane except at the midpoint of the cell, ensuring midcell positioning of the cytokinetic septum. During this process MinE undergoes fold switching as it interacts with different partners. We explore the exchange dynamics between major and excited states of the MinE dimer in 3 forms using 15 N relaxation dispersion NMR: the full-length protein (6-stranded β-sheet sandwiched between 4 helices) representing the resting state; a 10-residue N-terminal deletion (Δ10) mimicking the membrane-binding competent state where the N-terminal helix is detached to interact with membrane; and N-terminal deletions of either 30 (Δ30) or 10 residues with an I24N mutation (Δ10/I24N), in which the β1-strands at the dimer interface are extruded and available to bind MinD, leaving behind a 4-stranded β-sheet. Full-length MinE samples 2 “excited” states: The first is similar to a full-length/Δ10 heterodimer; the second, also sampled by Δ10, is either similar to or well along the pathway toward the 4-stranded β-sheet form. Both Δ30 and Δ10/I24N sample 2 excited species: The first may involve destabilization of the β3- and β3′-strands at the dimer interface; changes in the second are more extensive, involving further disruption of secondary structure, possibly representing an ensemble of states on the pathway toward restoration of the resting state. The quantitative information on MinE conformational dynamics involving these excited states is crucial for understanding the oscillation pattern self-organization by MinD–MinE interaction dynamics on the membrane.

Neurofibromin is the protein product of the NF1 gene which is mutated in the Rasopathy disease Neurofibromatosis Type I. Defects in NF1 lead to aberrant signaling through the RAS-MAPK pathway due to disruption of the Neurofibromin GTPase-activating function on RAS family small GTPases. Very little is known about the function of the majority of Neurofibromin—to date, biochemical and structural data exist only for the GAP domain and the region containing a Sec-PH motif. To better understand the role of this large protein, we carried out a series of biochemical and biophysical studies which demonstrate that full length Neurofibromin forms a high-affinity dimer. Neurofibromin dimerization also occurs in cells, and likely has biological and clinical implications. Analysis of purified full-length and truncated variants of Neurofibromin by negative stain electron microscopy reveals the overall architecture of the dimer and predicts the potential interactions which contribute to the dimer interface. Structures resembling high-affinity full-length dimers could be reconstituted by mixing N- and C-terminal protein domains in vitro. Taken together these data suggest how Neurofibromin dimers might form and be stabilized within the cell.

Anthrax toxin activator (AtxA) is the master virulence gene regulator of Bacillus anthracis. It regulates genes on the chromosome as well as the pXO1 and pXO2 plasmids. It is not clear how AtxA regulates these genes, and direct binding of AtxA to its targets has not been shown. It has been previously suggested that AtxA and other proteins in the Mga/AtxA global transcriptional regulators family bind to the curvature of their DNA targets, although this has never been experimentally proven. Using electrophoretic mobility shift assays, we demonstrate that AtxA binds directly to the promoter region of pagA upstream of the RNA polymerase binding site. We also demonstrate that in vitro, CO(2) appears to have no role in AtxA binding. However, phosphomimetic and phosphoablative substitutions in the phosphotransferase system (PTS) regulation domains (PRDs) do appear to influence AtxA binding and pagA regulation. In silico, in vitro, and in vivo analyses demonstrate that one of two hypothesized stem-loops located upstream of the RNA polymerase binding site in the pagA promoter region is important for AtxA binding in vitro and pagA regulation in vivo. Our study clarifies the mechanism by which AtxA interacts with one of its targets. IMPORTANCE Anthrax toxin activator (AtxA) regulates the major virulence genes in Bacillus anthracis. The bacterium produces the anthrax toxins, and understanding the mechanism of toxin production may facilitate the development of therapeutics for B. anthracis infection. Since the discovery of AtxA 25 years ago, the mechanism by which it regulates its targets has largely remained a mystery. Here, we provide evidence that AtxA binds to the promoter region of the pagA gene encoding the main central protective antigen (PA) component of the anthrax toxin. These data suggest that AtxA binding plays a direct role in gene regulation. Our work also assists in clarifying the role of CO(2) in AtxA’s gene regulation and provides more evidence for the role of AtxA phosphorylation in virulence gene regulation.

Neurofibromin is a tumor suppressor encoded by the NF1 gene, which is mutated in Rasopathy disease neurofibromatosis type I. Defects in NF1 lead to aberrant signaling through the RAS–mitogen-activated protein kinase pathway due to disruption of the neurofibromin GTPase-activating function on RAS family small GTPases. Very little is known about the function of most of the neurofibromin protein; to date, biochemical and structural data exist only for its GAP domain and a region containing a Sec-PH motif. To better understand the role of this large protein, here we carried out a series of biochemical and biophysical experiments, including size-exclusion chromatography–multiangle light scattering (SEC-MALS), small-angle X-ray and neutron scattering, and analytical ultracentrifugation, indicating that full-length neurofibromin forms a high-affinity dimer. We observed that neurofibromin dimerization also occurs in human cells and likely has biological and clinical implications. Analysis of purified full-length and truncated neurofibromin variants by negative-stain EM revealed the overall architecture of the dimer and predicted the potential interactions that contribute to the dimer interface. We could reconstitute structures resembling high-affinity full-length dimers by mixing N- and C-terminal protein domains in vitro. The reconstituted neurofibromin was capable of GTPase activation in vitro, and co-expression of the two domains in human cells effectively recapitulated the activity of full-length neurofibromin. Taken together, these results suggest how neurofibromin dimers might form and be stabilized within the cell.

Legionella pneumophila causes a potentially fatal form of pneumonia by replicating within macrophages in the Legionella-containing vacuole (LCV). Bacterial survival and proliferation within the LCV rely on hundreds of secreted effector proteins comprising high functional redundancy. The vacuolar membrane-localized MavN, hypothesized to support iron transport, is unique among effectors because loss-of-function mutations result in severe intracellular growth defects. We show here an iron starvation response by L. pneumophila after infection of macrophages that was prematurely induced in the absence of MavN, consistent with MavN granting access to limiting cellular iron stores. MavN cysteine accessibilities to a membrane-impermeant label were determined during macrophage infections, revealing a topological pattern supporting multipass membrane transporter models. Mutations to several highly conserved residues that can take part in metal recognition and transport resulted in defective intracellular growth. Purified MavN and mutant derivatives were directly tested for transporter activity after heterologous purification and liposome reconstitution. Proteoliposomes harboring MavN exhibited robust transport of Fe(2+), with the severity of defect of most mutants closely mimicking the magnitude of defects during intracellular growth. Surprisingly, MavN was equivalently proficient at transporting Fe(2+), Mn(2+), Co(2+), or Zn(2+). Consequently, flooding infected cells with either Mn(2+) or Zn(2+) allowed collaboration with iron to enhance intracellular growth of L. pneumophila ΔmavN strains, indicating a clear role for MavN in transporting each of these ions. These findings reveal that MavN is a transition-metal-ion transporter that plays a critical role in response to iron limitation during Legionella infection.

Common experimental approaches for characterizing structural conversion processes such as protein folding and self-assembly do not report on all aspects of the evolution from an initial state to the final state. Here, we demonstrate an approach that is based on rapid mixing, freeze-trapping, and low-temperature solid-state NMR (ssNMR) with signal enhancements from dynamic nuclear polarization (DNP). Experiments on the folding and tetramerization of the 26-residue peptide melittin following a rapid pH jump show that multiple aspects of molecular structure can be followed with millisecond time resolution, including secondary structure at specific isotopically labeled sites, intramolecular and intermolecular contacts between specific pairs of labeled residues, and overall structural order. DNP-enhanced ssNMR data reveal that conversion of conformationally disordered melittin monomers at low pH to α-helical conformations at neutral pH occurs on nearly the same timescale as formation of antiparallel melittin dimers, about 6 to 9 ms for 0.3 mM melittin at 24 °C in aqueous solution containing 20% (vol/vol) glycerol and 75 mM sodium phosphate. Although stopped-flow fluorescence data suggest that melittin tetramers form quickly after dimerization, ssNMR spectra show that full structural order within melittin tetramers develops more slowly, in ∼60 ms. Time-resolved ssNMR is likely to find many applications to biomolecular structural conversion processes, including early stages of amyloid formation, viral capsid formation, and protein-protein recognition.

The N-terminal region of the huntingtin protein, encoded by exon-1, comprises an amphiphilic domain (htt NT ), a polyglutamine (Q n ) tract, and a proline-rich sequence. Polyglutamine expansion results in an aggregation-prone protein responsible for Huntington’s disease. Here, we study the earliest events involved in oligomerization of a minimalistic construct, htt NT Q 7 , which remains largely monomeric over a sufficiently long period of time to permit detailed quantitative NMR analysis of the kinetics and structure of sparsely populated ( ≲ 2% ) oligomeric states, yet still eventually forms fibrils. Global fitting of concentration-dependent relaxation dispersion, transverse relaxation in the rotating frame, and exchange-induced chemical shift data reveals a bifurcated assembly mechanism in which the NMR observable monomeric species either self-associates to form a productive dimer (τ ex ∼ 30 μs, K diss ∼ 0.1 M) that goes on to form a tetramer ( τ ex ≲ 25 μs; K diss ∼ 22 μM), or exchanges with a “nonproductive” dimer that does not oligomerize further (τ ex ∼ 400 μs; K diss ∼ 0.3 M). The excited state backbone chemical shifts are indicative of a contiguous helix (residues 3–17) in the productive dimer/tetramer, with only partial helical character in the nonproductive dimer. A structural model of the productive dimer/tetramer was obtained by simulated annealing driven by intermolecular paramagnetic relaxation enhancement data. The tetramer comprises a D 2 symmetric dimer of dimers with largely hydrophobic packing between the helical subunits. The structural model, validated by EPR distance measurements, illuminates the role of the htt NT domain in the earliest stages of prenucleation and oligomerization, before fibril formation.

The human chaperonin Hsp60 is thought to play a role in the progression of Alzheimer’s disease by mitigating against intracellular β-amyloid stress. Here, we show that the bacterial homolog GroEL (51% sequence identity) reduces the neurotoxic effects of amyloid-β(1–42) (Aβ42) on human neural stem cell-derived neuronal cultures. To understand the mechanism of GroEL-mediated abrogation of neurotoxicity, we studied the interaction of Aβ42 with GroEL using a variety of biophysical techniques. Aβ42 binds to GroEL as a monomer with a lifetime of ∼1 ms, as determined from global analysis of multiple relaxation-based NMR experiments. Dynamic light scattering demonstrates that GroEL dissolves small amounts of high–molecular-weight polydisperse aggregates present in fresh soluble Aβ42 preparations. The residue-specific transverse relaxation rate profile for GroEL-bound Aβ42 reveals the presence of three anchor-binding regions (residues 16–21, 31–34, and 40–41) located within the hydrophobic GroEL-consensus binding sequences. Single-molecule FRET analysis of Aβ42 binding to GroEL results in no significant change in the FRET efficiency of a doubly labeled Aβ42 construct, indicating that Aβ42 samples a random coil ensemble when bound to GroEL. Finally, GroEL substantially slows down the disappearance of NMR visible Aβ42 species and the appearance of Aβ42 protofibrils and fibrils as monitored by electron and atomic force microscopies. The latter observations correlate with the effect of GroEL on the time course of Aβ42-induced neurotoxicity. These data provide a physical basis for understanding how Hsp60 may serve to slow down the progression of Alzheimer’s disease.

Summary The repeating structural unit of metazoan chromatin is the chromatosome, a nucleosome bound to a linker histone, H1. There are 11 human H1 isoforms with diverse cellular functions, but how they interact with the nucleosome remains elusive. Here, we determined the cryoelectron microscopy (cryo-EM) structures of chromatosomes containing 197 bp DNA and three different human H1 isoforms, respectively. The globular domains of all three H1 isoforms bound to the nucleosome dyad. However, the flanking/linker DNAs displayed substantial distinct dynamic conformations. Nuclear magnetic resonance (NMR) and H1 tail-swapping cryo-EM experiments revealed that the C-terminal tails of the H1 isoforms mainly controlled the flanking DNA orientations. We also observed partial ordering of the core histone H2A C-terminal and H3 N-terminal tails in the chromatosomes. Our results provide insights into the structures and dynamics of the chromatosomes and have implications for the structure and function of chromatin.

Linker histones bind to the nucleosome and regulate the structure and function of chromatin. We have previously shown that the globular domains of chicken H5 and Drosophila H1 linker histones bind to the nucleosome with on- or off-dyad modes, respectively. To explore the determinant for the distinct binding modes, we investigated the binding of a mutant globular domain of H5 to the nucleosome. This mutant, termed GH5_pMut, includes substitutions of five globular domain residues of H5 with the corresponding residues in the globular domain of Drosophila H1. The residues at these five positions play important roles in nucleosome binding by either H5 or Drosophila H1. NMR and spin-labeling experiments showed that GH5_pMut bound to the nucleosome off the dyad. We further found that the nucleosome array condensed by either the GH5_pMut or the globular domain of Drosophila H1 displayed a similar sedimentation coefficient, whereas the same nucleosome array condensed by the wild-type globular domain of H5 showed a much larger sedimentation coefficient. Moreover, NMR and spin-labeling results from the study of the nucleosome in complex with the full-length human linker histone H1.0, whose globular domain shares high sequence conservation with the corresponding globular domain of H5, are consistent with an on-dyad binding mode. Taken together, our results suggest that a small number of residues in the globular domain of a linker histone can control its binding location on the nucleosome and higher-order chromatin structure.

The role of the zinc finger protein CTCF in organizing the genome within the nucleus is now well established. Widely separated sites on DNA, occupied by both CTCF and the cohesin complex, make physical contacts that create large loop domains. Additional contacts between loci within those domains, often also mediated by CTCF, tend to be favored over contacts between loci in different domains. A large number of studies during the past 2 years have addressed the questions: How are these loops generated? What are the effects of disrupting them? Are there rules governing large-scale genome organization? It now appears that the strongest and evolutionarily most conserved of these CTCF interactions have specific rules for the orientation of the paired CTCF sites, implying the existence of a nonequilibrium mechanism of generation. Recent experiments that invert, delete, or inactivate one of a mating CTCF pair result in major changes in patterns of organization and gene expression in the surrounding regions. What remain to be determined are the detailed molecular mechanisms for re-establishing loop domains and maintaining them after replication and mitosis. As recently published data show, some mechanisms may involve interactions with noncoding RNAs as well as protein cofactors. Many CTCF sites are also involved in other functions such as modulation of RNA splicing and specific regulation of gene expression, and the relationship between these activities and loop formation is another unanswered question that should keep investigators occupied for some time.

It has long been suggested that chromatin may form a fiber with a diameter of ~30 nm that suppresses transcription. Despite nearly four decades of study, the structural nature of the 30-nm chromatin fiber and conclusive evidence of its existence in vivo remain elusive. The key support for the existence of specific 30 nm chromatin fiber structures is based on the determination of the structures of reconstituted nucleosome arrays using X-ray crystallography and single particle cryo-electron microscopy coupled with glutaraldehyde chemical cross-linking. Here we report the characterization of these nucleosome arrays in solution using analytical ultracentrifugation, nuclear magnetic resonance, and small angle X-ray scattering. We found that the physical properties of these nucleosome arrays in solution are not consistent with formation of just a few discrete structures of nucleosome arrays. In addition, we obtained a crystal of the nucleosome in complex with the globular domain of linker histone H5 that shows a new form of nucleosome packing and suggests a plausible alternative compact conformation for nucleosome arrays. Taken together, our results challenge the key evidence for the existence of a limited number of structures of reconstituted nucleosome arrays in solution by revealing that the reconstituted nucleosome arrays are actually best described as an ensemble of various conformations with a zigzagged arrangement of nucleosomes. Our finding has implications for understanding the structure and function of chromatin in vivo.

Lectins are carbohydrate-binding proteins unrelated to antibodies or enzymes. While carbohydrates are present on all cells and pathogens, lectins are also ubiquitous in nature and their interactions with glycans mediate countless biological and physical interactions. Due to the multivalency found in both lectins and their glycan-binding partners, complete characterization of these interactions can be complex and typically requires the use of multiple complimentary techniques. In this chapter, we provide a general strategy and protocols for chemical and biophysical approaches that can be used to characterize carbohydrate-mediated interactions in the context of individual oligosaccharides, as part of a glycoprotein, and ending with visualization of interactions with whole virions.

Recognizing centromere by kinetochore The kinetochore proteins CENP-N and CENP-C recognize the histone H3 variant CENP-A in the centromeric nucleosome. This ensures proper kinetochore assembly and accurate segregation of chromosomes. Chittori et al. describe the cryo-electron microscopy structure of the human CENP-A nucleosome-CENP-N complex. The interaction of CENP-N with CENP-A and the nucleosomal DNA together ensure specific and stable centromeric nucleosome recognition. Mutational analyses using both human and Xenopus CENP-A and CENP-N proteins suggest that the proteins have coevolved to preserve the interacting surfaces. Science , this issue p. 339

Hfq proteins in Gram-negative bacteria play important roles in bacterial physiology and virulence, mediated by binding of the Hfq hexamer to small RNAs and/or mRNAs to post-transcriptionally regulate gene expression. However, the physiological role of Hfqs in Gram-positive bacteria is less clear. Bacillus anthracis, the causative agent of anthrax, uniquely expresses three distinct Hfq proteins, two from the chromosome (Hfq1, Hfq2) and one from its pXO1 virulence plasmid (Hfq3). The protein sequences of Hfq1 and 3 are evolutionarily distinct from those of Hfq2 and of Hfqs found in other Bacilli. Here, the quaternary structure of each B. anthracis Hfq protein, as produced heterologously in Escherichia coli, was characterized. While Hfq2 adopts the expected hexamer structure, Hfq1 does not form similarly stable hexamers in vitro. The impact on the monomer-hexamer equilibrium of varying Hfq C-terminal tail length and other sequence differences among the Hfqs was examined, and a sequence region of the Hfq proteins that was involved in hexamer formation was identified. It was found that, in addition to the distinct higher-order structures of the Hfq homologs, they give rise to different phenotypes. Hfq1 has a disruptive effect on the function of E. coli Hfq in vivo, while Hfq3 expression at high levels is toxic to E. coli but also partially complements Hfq function in E. coli. These results set the stage for future studies of the roles of these proteins in B. anthracis physiology and for the identification of sequence determinants of phenotypic complementation.

Linker histones bind to the nucleosome and regulate the structure of chromatin and gene expression. Despite more than three decades of effort, the structural basis of nucleosome recognition by linker histones remains elusive. Here, we report the crystal structure of the globular domain of chicken linker histone H5 in complex with the nucleosome at 3.5 A resolution, which is validated using nuclear magnetic resonance spectroscopy. The globular domain sits on the dyad of the nucleosome and interacts with both DNA linkers. Our structure integrates results from mutation analyses and previous cross-linking and fluorescence recovery after photobleach experiments, and it helps resolve the long debate on structural mechanisms of nucleosome recognition by linker histones. The on-dyad binding mode of the H5 globular domain is different from the recently reported off-dyad binding mode of Drosophila linker histone H1. We demonstrate that linker histones with different binding modes could fold chromatin to form distinct higher-order structures.

Sedimentation equilibrium (SE) analytical ultracentrifugation (AUC) is a gold standard for the rigorous determination of macromolecular buoyant molar masses and the thermodynamic study of reversible interactions in solution. A significant experimental drawback is the long time required to attain SE, which is usually on the order of days. We have developed a method for time-optimized SE (toSE) with defined time-varying centrifugal fields that allow SE to be attained in a significantly (up to 10-fold) shorter time than is usually required. To achieve this, numerical Lamm equation solutions for sedimentation in time-varying fields are computed based on initial estimates of macromolecular transport properties. A parameterized rotor-speed schedule is optimized with the goal of achieving a minimal time to equilibrium while limiting transient sample preconcentration at the base of the solution column. The resulting rotor-speed schedule may include multiple over- and underspeeding phases, balancing the formation of gradients from strong sedimentation fluxes with periods of high diffusional transport. The computation is carried out in a new software program called TOSE, which also facilitates convenient experimental implementation. Further, we extend AUC data analysis to sedimentation processes in such time-varying centrifugal fields. Due to the initially high centrifugal fields in toSE and the resulting strong migration, it is possible to extract sedimentation coefficient distributions from the early data. This can provide better estimates of the size of macromolecular complexes and report on sample homogeneity early on, which may be used to further refine the prediction of the rotor-speed schedule. In this manner, the toSE experiment can be adapted in real time to the system under study, maximizing both the information content and the time efficiency of SE experiments.