Streptococcus pyogenes pSM19035 requires dynamic assembly of ATP-bound ParA and ParB on parS DNA during plasmid segregation

The accurate partitioning of Firmicute plasmid pSM19035 at cell division depends on ATP binding and hydrolysis by homodimeric ATPase δ₂ (ParA) and binding of ω₂ (ParB) to its cognate parS DNA. The 1.83 Å resolution crystal structure of δ₂ in a complex with non-hydrolyzable ATPγS reveals a unique ParA dimer assembly that permits nucleotide exchange without requiring dissociation into monomers. In vitro, δ₂ had minimal ATPase activity in the absence of ω₂ and parS DNA. However, stoichiometric amounts of ω₂ and parS DNA stimulated the δ₂ ATPase activity and mediated plasmid pairing, whereas at high (4:1) ω₂ : δ₂ ratios, stimulation of the ATPase activity was reduced and δ₂ polymerized onto DNA. Stimulation of the δ₂ ATPase activity and its polymerization on DNA required ability of ω₂ to bind parS DNA and its N-terminus. In vivo experiments showed that δ₂ alone associated with the nucleoid, and in the presence of ω₂ and parS DNA, δ₂ oscillated between the nucleoid and the cell poles and formed spiral-like structures. Our studies indicate that the molar ω₂ : δ₂ ratio regulates the polymerization properties of (δ•ATP•Mg²⁺)₂ on and depolymerization from parS DNA, thereby controlling the temporal and spatial segregation of pSM19035 before cell division.     

Self-activating G protein α subunits engage seven-transmembrane regulator of G protein signaling (RGS) proteins and a Rho guanine nucleotide exchange factor effector in the amoeba Naegleria fowleri

The free-living amoeba Naegleria fowleri is a causative agent of primary amoebic meningoencephalitis and is highly resistant to current therapies, resulting in mortality rates >97%. As many therapeutics target G protein–centered signal transduction pathways, further understanding the functional significance of G protein signaling within N. fowleri should aid future drug discovery against this pathogen. Here, we report that the N. fowleri genome encodes numerous transcribed G protein signaling components, including G protein–coupled receptors, heterotrimeric G protein subunits, regulator of G protein signaling (RGS) proteins, and candidate Gα effector proteins. We found N. fowleri Gα subunits have diverse nucleotide cycling kinetics; Nf Gα5 and Gα7 exhibit more rapid nucleotide exchange than GTP hydrolysis (i.e., “self-activating” behavior). A crystal structure of Nf Gα7 highlights the stability of its nucleotide-free state, consistent with its rapid nucleotide exchange. Variations in the phosphate binding loop also contribute to nucleotide cycling differences among Gα subunits. Similar to plant G protein signaling pathways, N. fowleri Gα subunits selectively engage members of a large seven-transmembrane RGS protein family, resulting in acceleration of GTP hydrolysis. We show Nf Gα2 and Gα3 directly interact with a candidate Gα effector protein, RGS-RhoGEF, similar to mammalian Gα_12/13 signaling pathways. We demonstrate Nf Gα2 and Gα3 each engage RGS-RhoGEF through a canonical Gα/RGS domain interface, suggesting a shared evolutionary origin with G protein signaling in the enteric pathogen Entamoeba histolytica. These findings further illuminate the evolution of G protein signaling and identify potential targets of pharmacological manipulation in N. fowleri.     

Only One ATP-binding DnaX Subunit Is Required for Initiation Complex Formation by the Escherichia coli DNA Polymerase III Holoenzyme

The DnaX complex (DnaX₃δδ′χψ) within the Escherichia coli DNA polymerase III holoenzyme serves to load the dimeric sliding clamp processivity factor, β₂, onto DNA. The complex contains three DnaX subunits, which occur in two forms: τ and the shorter γ, produced by translational frameshifting. Ten forms of E. coli DnaX complex containing all possible combinations of wild-type or a Walker A motif K51E variant τ or γ have been reconstituted and rigorously purified. DnaX complexes containing three DnaX K51E subunits do not bind ATP. Comparison of their ability to support formation of initiation complexes, as measured by processive replication by the DNA polymerase III holoenzyme, indicates a minimal requirement for one ATP-binding DnaX subunit. DnaX complexes containing two mutant DnaX subunits support DNA synthesis at about two-thirds the level of their wild-type counterparts. β₂ binding (determined functionally) is diminished 12–30-fold for DnaX complexes containing two K51E subunits, suggesting that multiple ATPs must be bound to place the DnaX complex into a conformation with maximal affinity for β₂. DNA synthesis activity can be restored by increased concentrations of β₂. In contrast, severe defects in ATP hydrolysis are observed upon introduction of a single K51E DnaX subunit. Thus, ATP binding, hydrolysis, and the ability to form initiation complexes are not tightly coupled. These results suggest that although ATP hydrolysis likely enhances β₂ loading, it is not absolutely required in a mechanistic sense for formation of functional initiation complexes.     

One rotary mechanism for F1-ATPase over ATP concentrations from millimolar down to nanomolar

F₁-ATPase is a rotary molecular motor in which the central γ-subunit rotates inside a cylinder made of α₃β₃-subunits. The rotation is driven by ATP hydrolysis in three catalytic sites on the β-subunits. How many of the three catalytic sites are filled with a nucleotide during the course of rotation is an important yet unsettled question. Here we inquire whether F₁ rotates at extremely low ATP concentrations where the site occupancy is expected to be low. We observed under an optical microscope rotation of individual F₁ molecules that carried a bead duplex on the γ-subunit. Time-averaged rotation rate was proportional to the ATP concentration down to 200 pM, giving an apparent rate constant for ATP binding of 2 × 10⁷ M⁻¹s⁻¹. A similar rate constant characterized bulk ATP hydrolysis in solution, which obeyed a simple Michaelis-Menten scheme between 6 mM and 60 nM ATP. F₁ produced the same torque of ~40 pN·nm at 2 mM, 60 nM, and 2 nM ATP. These results point to one rotary mechanism governing the entire range of nanomolar to millimolar ATP, although a switchover between two mechanisms cannot be dismissed. Below 1 nM ATP, we observed less regular rotations, indicative of the appearance of another reaction scheme.     

pi protein- and ATP-dependent transitions from 'closed' to 'open' complexes at the gamma ori of plasmid R6K

R6K-encoded π protein can bind to the seven, 22 bp tandem iterons of the γ origin. In this work, we use a variant of π, His-π·F107S, that is hyperactive in replication. In vitro, His-π·F107S-dependent local DNA melting (open complex formation) occurs in the absence of host proteins (IHF/HU or DnaA) and it is positioned in the A + T-rich region adjacent to iterons. Experiments described here examine the effects of ATP, Mg²⁺ and temperature on the opening reaction. We show that the opening of the γ origin can occur in the presence of ATP as well as AMP-PCP (a non-hydrolyzable ATP analog). This suggests that, for γ origin, ATP hydrolysis may be unnecessary for open complex formation facilitated by His-π·F107S. In the absence of ATP or Mg²⁺, His-π·F107S yielded data suggestive of distortions in the iteron attributable to DNA bending rather than DNA melting. Our findings also demonstrate that ATP and π stimulate open complex formation over a wide range of temperatures, but not at 0°C. These and other results indicate that ATP and/or Mg²⁺ are not needed for His-π·F107S binding to iterons and that ATP effects an allosteric change in the protein bound to γ origin.     

The Inhibitory Mechanism of the ζ Subunit of the F1FO-ATPase Nanomotor of Paracoccus denitrificans and Related α-Proteobacteria

The ζ subunit is a novel inhibitor of the F₁F_O-ATPase of Paracoccus denitrificans and related α-proteobacteria. It is different from the bacterial (ϵ) and mitochondrial (IF₁) inhibitors. The N terminus of ζ blocks rotation of the γ subunit of the F₁-ATPase of P. denitrificans (Zarco-Zavala, M., Morales-Ríos, E., Mendoza-Hernández, G., Ramírez-Silva, L., Pérez-Hernández, G., and García-Trejo, J. J. (2014) FASEB J. 24, 599–608) by a hitherto unknown quaternary structure that was first modeled here by structural homology and protein docking. The F₁-ATPase and F₁-ζ models of P. denitrificans were supported by cross-linking, limited proteolysis, mass spectrometry, and functional data. The final models show that ζ enters into F₁-ATPase at the open catalytic α_E/β_E interface, and two partial γ rotations lock the N terminus of ζ in an “inhibition-general core region,” blocking further γ rotation, while the ζ globular domain anchors it to the closed α_DP/β_DP interface. Heterologous inhibition of the F₁-ATPase of P. denitrificans by the mitochondrial IF₁ supported both the modeled ζ binding site at the α_DP/β_DP/γ interface and the endosymbiotic α-proteobacterial origin of mitochondria. In summary, the ζ subunit blocks the intrinsic rotation of the nanomotor by inserting its N-terminal inhibitory domain at the same rotor/stator interface where the mitochondrial IF₁ or the bacterial ϵ binds. The proposed pawl mechanism is coupled to the rotation of the central γ subunit working as a ratchet but with structural differences that make it a unique control mechanism of the nanomotor to favor the ATP synthase activity over the ATPase turnover in the α-proteobacteria.     

Designer proteins that competitively inhibit Gαq by targeting its effector site

During signal transduction, the G protein, Gα_q, binds and activates phospholipase C-β isozymes. Several diseases have been shown to manifest upon constitutively activating mutation of Gα_q, such as uveal melanoma. Therefore, methods are needed to directly inhibit Gα_q. Previously, we demonstrated that a peptide derived from a helix-turn-helix (HTH) region of PLC-β3 (residues 852–878) binds Gα_q with low micromolar affinity and inhibits Gα_q by competing with full-length PLC-β isozymes for binding. Since the HTH peptide is unstructured in the absence of Gα_q, we hypothesized that embedding the HTH in a folded protein might stabilize the binding-competent conformation and further improve the potency of inhibition. Using the molecular modeling software Rosetta, we searched the Protein Data Bank for proteins with similar HTH structures near their surface. The candidate proteins were computationally docked against Gα_q, and their surfaces were redesigned to stabilize this interaction. We then used yeast surface display to affinity mature the designs. The most potent design bound Gα_q/i with high affinity in vitro (K_D = 18 nM) and inhibited activation of PLC-β isozymes in HEK293 cells. We anticipate that our genetically encoded inhibitor will help interrogate the role of Gα_q in healthy and disease model systems. Our work demonstrates that grafting interaction motifs into folded proteins is a powerful approach for generating inhibitors of protein–protein interactions.     

Substrate Specificities and Conformational Flexibility of 3-Ketosteroid 9α-Hydroxylases

KshA is the oxygenase component of 3-ketosteroid 9α-hydroxylase, a Rieske oxygenase involved in the bacterial degradation of steroids. Consistent with its role in bile acid catabolism, KshA1 from Rhodococcus rhodochrous DSM43269 had the highest apparent specificity (k_cat/K_m) for steroids with an isopropyl side chain at C17, such as 3-oxo-23,24-bisnorcholesta-1,4-diene-22-oate (1,4-BNC). By contrast, the KshA5 homolog had the highest apparent specificity for substrates with no C17 side chain (k_cat/K_m >10⁵ s⁻¹ m⁻¹ for 4-estrendione, 5α-androstandione, and testosterone). Unexpectedly, substrates such as 4-androstene-3,17-dione (ADD) and 4-BNC displayed strong substrate inhibition (K_iS ∼100 μm). By comparison, the cholesterol-degrading KshA_Mtb from Mycobacterium tuberculosis had the highest specificity for CoA-thioesterified substrates. These specificities are consistent with differences in the catabolism of cholesterol and bile acids, respectively, in actinobacteria. X-ray crystallographic structures of the KshA_Mtb·ADD, KshA1·1,4-BNC-CoA, KshA5·ADD, and KshA5·1,4-BNC-CoA complexes revealed that the enzymes have very similar steroid-binding pockets with the substrate''s C17 oriented toward the active site opening. Comparisons suggest Tyr-245 and Phe-297 are determinants of KshA1 specificity. All enzymes have a flexible 16-residue “mouth loop,” which in some structures completely occluded the substrate-binding pocket from the bulk solvent. Remarkably, the catalytic iron and α-helices harboring its ligands were displaced up to 4.4 Å in the KshA5·substrate complexes as compared with substrate-free KshA, suggesting that Rieske oxygenases may have a dynamic nature similar to cytochrome P450.     

Kinetic diversity of single-channel burst openings underlying persistent Na(+) current in entorhinal cortex neurons

The kinetic diversity of burst openings responsible for the persistent Na⁺ current (I_NaP) in entorhinal cortex neurons was examined by separately analyzing single bursts. Although remarkable kinetic variability was observed among bursts in terms of intraburst opening probability and mean open and closed times, the values of time constants describing intraburst open times (τ_o(b)s) and closed times (τ_c(b)s) were distributed around well-defined peaks. At −40 mV, τ_o(b) peaks were found at ~0.34 (τ_o(b)1) and 0.77 (τ_o(b)2) ms, and major τ_c(b) peaks were found at ~0.24 (τ_c(b)1) and 0.54 (τ_c(b)2) ms. In ~80% of the bursts two preferential gating modes were found that consisted of a combination of either τ_o(b)1 and τ_c(b)2 (“intraburst mode 1”), or τ_o(b)2 and τ_c(b)1 (“intraburst mode 2”). Individual channels could switch between different gating modalities, but normally tended to maintain a specific gating mode for long periods. Mean burst duration also displayed considerable variability. At least three time constants were found to describe burst duration, and the frequencies at which each of the corresponding “bursting states” occurred varied in different channels. Short-lasting bursting states were preferentially associated with intraburst mode 1, whereas very-long-lasting bursts tended to gate according to mode 2 only or other modes that included considerably longer mean open times. These results show that I_NaP channels can generate multiple intraburst open and closed states and bursting states, but these different kinetic states tend to combine in definite ways to produce a limited number of prevalent, well-defined gating modalities. Modulation of distinct gating modalities in individual Na⁺ channels may be a powerful form of plasticity to influence neuronal excitability and function.     

Structure of Oxalacetate Acetylhydrolase,a Virulence Factor of the Chestnut Blight Fungus

Oxalacetate acetylhydrolase (OAH), a member of the phosphoenolpyruvate mutase/isocitrate lyase superfamily, catalyzes the hydrolysis of oxalacetate to oxalic acid and acetate. This study shows that knock-out of the oah gene in Cryphonectria parasitica, the chestnut blight fungus, reduces the ability of the fungus to form cankers on chestnut trees, suggesting that OAH plays a key role in virulence. OAH was produced in Escherichia coli and purified, and its catalytic rates were determined. Oxalacetate is the main OAH substrate, but the enzyme also acts as a lyase of (2R,3S)-dimethyl malate with ∼1000-fold lower efficacy. The crystal structure of OAH was determined alone, in complex with a mechanism-based inhibitor, 3,3-difluorooxalacetate (DFOA), and in complex with the reaction product, oxalate, to a resolution limit of 1.30, 1.55, and 1.65 Å, respectively. OAH assembles into a dimer of dimers with each subunit exhibiting an (α/β)₈ barrel fold and each pair swapping the 8th α-helix. An active site “gating loop” exhibits conformational disorder in the ligand-free structure. To obtain the structures of the OAH·ligand complexes, the ligand-free OAH crystals were soaked briefly with DFOA or oxalacetate. DFOA binding leads to ordering of the gating loop in a conformation that sequesters the ligand from the solvent. DFOA binds in a gem-diol form analogous to the oxalacetate intermediate/transition state. Oxalate binds in a planar conformation, but the gating loop is largely disordered. Comparison between the OAH structure and that of the closely related enzyme, 2,3-dimethylmalate lyase, suggests potential determinants of substrate preference.     

The extraction and hydrolysis of steroid monoglucuronides

1. A method in use for the extraction of urinary steroid conjugates has been applied to study the recovery of synthetic steroid monoglucuronides from aqueous solution. 2. In the presence of dissolved ammonium sulphate (50g./100ml.), ether–ethanol (3:1, v/v, 3×0·5vol.) extracted the monoglucuronides of steroids of the C₁₈, C₁₉ and C₂₁ series, quantitatively at values pH2–9. 3. The hydrolysis of the synthetic steroid monoglucuronides by β-glucuronidase (Patella vulgata) has been examined with reference to the pH value of the medium, enzyme concentration and substrate concentration. 4. The rate of hydrolysis of steroid monoglucuronides was dependent upon steroid structure and upon site of conjugation. 5. The rate of hydrolysis of the monoglucuronides decreased in the order C-3 (phenolic) >C-3β>C-17β>C-3α.     

Molecular insights into the primary nucleation of polymorphic amyloid β dimers in DOPC lipid bilayer membrane

Alzheimer''s disease (AD) pathology is characterized by loss of memory cognitive and behavioral deterioration. One of the hallmarks of AD is amyloid β (Aβ) plaques in the brain that consists of Aβ oligomers and fibrils. It is accepted that oligomers, particularly dimers, are toxic species that are produced extracellularly and intracellularly in membranes. It is believed that the disruption of membranes by polymorphic Aβ oligomers is the key for the pathology of AD. This is a first study that investigate the effect of polymorphic “α‐helix/random coil” and “fibril‐like” Aβ dimers on 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC) membrane. It has been found that the DOPC membrane promotes Aβ_1–42 “fibril‐like” dimers and impedes Aβ_1–42 “α‐helix/random coil” dimers. The N‐termini domains within Aβ_1–42 dimers play a role in Aβ aggregation in membrane milieus. In addition, the aromatic π–π interactions (involving residues F19 and F20 in Aβ_1–42) are the driving forces for the hydrophobic interactions that initiate the primary nucleation of polymorphic Aβ_1–42 dimers within DOPC membrane. Finally, the DOPC bilayer membrane thickness is locally decreased, and it is disrupted by an embedded distinct Aβ_1–42 dimer, due to relatively large contacts between Aβ_1–42 monomers and the DOPC membrane. This study reveals insights into the molecular mechanisms by which polymorphic early‐stage Aβ_1–42 dimers have distinct impacts on DOPC membrane.     

Structure and Kinetic Investigation of Streptococcus pyogenes Family GH38 α-Mannosidase

Background

The enzymatic hydrolysis of α−mannosides is catalyzed by glycoside hydrolases (GH), termed α−mannosidases. These enzymes are found in different GH sequence–based families. Considerable research has probed the role of higher eukaryotic “GH38” α−mannosides that play a key role in the modification and diversification of hybrid N-glycans; processes with strong cellular links to cancer and autoimmune disease. The most extensively studied of these enzymes is the Drosophila GH38 α−mannosidase II, which has been shown to be a retaining α−mannosidase that targets both α−1,3 and α−1,6 mannosyl linkages, an activity that enables the enzyme to process GlcNAc(Man)₅(GlcNAc)₂ hybrid N-glycans to GlcNAc(Man)₃(GlcNAc)₂. Far less well understood is the observation that many bacterial species, predominantly but not exclusively pathogens and symbionts, also possess putative GH38 α−mannosidases whose activity and specificity is unknown.

Methodology/Principal Findings

Here we show that the Streptococcus pyogenes (M1 GAS SF370) GH38 enzyme (Spy1604; hereafter SpGH38) is an α−mannosidase with specificity for α−1,3 mannosidic linkages. The 3D X-ray structure of SpGH38, obtained in native form at 1.9 Å resolution and in complex with the inhibitor swainsonine (K _i 18 µM) at 2.6 Å, reveals a canonical GH38 five-domain structure in which the catalytic “–1” subsite shows high similarity with the Drosophila enzyme, including the catalytic Zn²⁺ ion. In contrast, the “leaving group” subsites of SpGH38 display considerable differences to the higher eukaryotic GH38s; features that contribute to their apparent specificity.

Conclusions/Significance

Although the in vivo function of this streptococcal GH38 α−mannosidase remains unknown, it is shown to be an α−mannosidase active on N-glycans. SpGH38 lies on an operon that also contains the GH84 hexosaminidase (Spy1600) and an additional putative glycosidase. The activity of SpGH38, together with its genomic context, strongly hints at a function in the degradation of host N- or possibly O-glycans. The absence of any classical signal peptide further suggests that SpGH38 may be intracellular, perhaps functioning in the subsequent degradation of extracellular host glycans following their initial digestion by secreted glycosidases.     

Studies on the congenitally goitrous sheep. The iodinated compounds of serum, and circulating thyroid-stimulating hormone

1. A group of normal and congenitally goitrous Merino sheep were investigated to identify the metabolic defect present in the abnormal animals. 2. Protein-bound iodine concentrations of serum from goitrous animals (average 5·7μg./100ml.) were higher than normal (average 4·2μg./100ml.; P 0·001), but the hormonal iodine measured as butanol-extractable ¹³¹I was low in the serum of goitrous (average 40·3% of protein-bound ¹³¹I) compared with that of normal (84·2%; P 0·02) sheep. The non-hormonal iodine of the serum of goitrous sheep appeared to include iodotyrosines and iodinated protein. 3. Starch-gel-electrophoretic separations of sera from normal and goitrous sheep after ¹³¹I injection (100–500μc) showed no qualitative differences in the radioactivity of protein components. No significant differences in thyroxine-binding in vitro by serum proteins of normal and goitrous sheep were observed. 4. The clearance rates of ¹³¹I-labelled iodotyrosines (t_½ 1·2–2·9hr.) and iodothyronines (t_½ 33·5–47·4hr.) were similar in normal and goitrous sheep. 5. The concentration of circulating thyroid-stimulating hormone was significantly higher (P<0·01 in three sheep, P<0·05 in one sheep) in goitrous sheep. 6. The congenital goitre appears to be due to compensatory hypertrophy of the gland resulting from an inability to synthesize an adequate supply of thyroid hormone.     

Calcium Regulates Molecular Interactions of Otoferlin with Soluble NSF Attachment Protein Receptor (SNARE) Proteins Required for Hair Cell Exocytosis

Mutations in otoferlin, a C2 domain-containing ferlin family protein, cause non-syndromic hearing loss in humans (DFNB9 deafness). Furthermore, transmitter secretion of cochlear inner hair cells is compromised in mice lacking otoferlin. In the present study, we show that the C2F domain of otoferlin directly binds calcium (K_D = 267 μm) with diminished binding in a pachanga (D1767G) C2F mouse mutation. Calcium was found to differentially regulate binding of otoferlin C2 domains to target SNARE (t-SNARE) proteins and phospholipids. C2D–F domains interact with the syntaxin-1 t-SNARE motif with maximum binding within the range of 20–50 μm Ca²⁺. At 20 μm Ca²⁺, the dissociation rate was substantially lower, indicating increased binding (K_D = ∼10⁻⁹) compared with 0 μm Ca²⁺ (K_D = ∼10⁻⁸), suggesting a calcium-mediated stabilization of the C2 domain·t-SNARE complex. C2A and C2B interactions with t-SNAREs were insensitive to calcium. The C2F domain directly binds the t-SNARE SNAP-25 maximally at 100 μm and with reduction at 0 μm Ca²⁺, a pattern repeated for C2F domain interactions with phosphatidylinositol 4,5-bisphosphate. In contrast, C2F did not bind the vesicle SNARE protein synaptobrevin-1 (VAMP-1). Moreover, an antibody targeting otoferlin immunoprecipitated syntaxin-1 and SNAP-25 but not synaptobrevin-1. As opposed to an increase in binding with increased calcium, interactions between otoferlin C2F domain and intramolecular C2 domains occurred in the absence of calcium, consistent with intra-C2 domain interactions forming a “closed” tertiary structure at low calcium that “opens” as calcium increases. These results suggest a direct role for otoferlin in exocytosis and modulation of calcium-dependent membrane fusion.