The Structure of the Karrikin-Insensitive Protein (KAI2) in Arabidopsis thaliana

KARRIKIN INSENSITIVE 2 (KAI2) is an α/β hydrolase involved in seed germination and seedling development. It is essential for plant responses to karrikins, a class of butenolide compounds derived from burnt plant material that are structurally similar to strigolactone plant hormones. The mechanistic basis for the function of KAI2 in plant development remains unclear. We have determined the crystal structure of Arabidopsis thaliana KAI2 in space groups P2₁ 2₁ 2₁ (a  = 63.57 Å, b  = 66.26 Å, c  = 78.25 Å) and P2₁ (a  = 50.20 Å, b  = 56.04 Å, c  = 52.43 Å, β  = 116.12°) to 1.55 and 2.11 Å respectively. The catalytic residues are positioned within a large hydrophobic pocket similar to that of DAD2, a protein required for strigolactone response in Petunia hybrida. KAI2 possesses a second solvent-accessible pocket, adjacent to the active site cavity, which offers the possibility of allosteric regulation. The structure of KAI2 is consistent with its designation as a serine hydrolase, as well as previous data implicating the protein in karrikin and strigolactone signalling.     

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.     

Quantitative models characterizing seed germination responses to abscisic Acid and osmoticum

Mathematical models were developed to characterize the physiological bases of the responses of tomato (Lycopersicon esculentum Mill. cv T5) seed germination to water potential (ψ) and abscisic acid (ABA). Using probit analysis, three parameters were derived that can describe the germination time courses of a seed population at different ψ or ABA levels. For the response of seed germination to reduced ψ, these parameters are the mean base water potential (¯ψ_b, MPa), the standard deviation of the base water potential among seeds in the population (σ_ψb, MPa), and the “hydrotime constant” (θ_H, MPa·h). For the response to ABA, they are the log of the mean base ABA concentration ([unk]ABA_b, m), the standard deviation of the base ABA concentration among seeds in the population (σ_ABAb, log[m]), and the “ABA-time constant” (θ_ABA, log[m]·h). The values of ¯ψ_b and [unk]ABA_b provide quantitative estimates of the mean sensitivity of germination rate to ψ or ABA, whereas σ^ψ_b and σ_ABAb account for the variation in sensitivity among seeds in the population. The time constants, θ_H and θ_ABA, indicate the extent to which germination rate will be affected by a given change in ψ or ABA. Using only these parameters, germination time courses can be predicted with reasonable accuracy at any medium ψ according to the equation probit(g) = [ψ - (θ_H/t_g) - ¯ψ_b]/σ_ψb, or at any ABA concentration according to the equation probit(g) = [log[ABA] - (θ_ABA/t_g) - log[[unk]ABA_b]]/σ_ABAb, where t_g is the time to radicle emergence of percentage g, and ABA is the ABA concentration (m) in the incubation solution. In the presence of both ABA and reduced ψ, the same parameters can be used to predict seed germination time courses based upon strictly additive effects of ψ and ABA in delaying the time of radicle emergence. Further analysis indicates that ABA and ψ can act both independently and interactively to influence physiological processes preparatory for radicle growth, such as the accumulation of osmotic solutes in the embryo. The models provide quantitative values for the sensitivity of germination to ABA or ψ, allow evaluation of independent and interactive effects of the two factors, and have implications for understanding how ABA and ψ may regulate growth and development.     

Effects of Hydrophobic Macromolecular Crowders on Amyloid β (16–22) Aggregation

In Alzheimer’s disease (AD), the amyloid β (Aβ) peptide aggregates in the brain to form progressively larger oligomers, fibrils, and plaques. The aggregation process is strongly influenced by the presence of other macromolecular species, called crowders, that can exert forces on the proteins. One very common attribute of macromolecular crowders is their hydrophobicity. We examined the effect of hydrophobic crowders on protein aggregation by using discontinuous molecular dynamics (DMD) simulations in combination with an intermediate resolution protein model, PRIME20. The systems considered contained 48 Aβ (16–22) peptides and crowders with diameters of 5 Å, 20 Å, and 40 Å, represented by hard spheres or spheres with square-well/square-shoulder interactions, at a crowder volume fraction of ϕ = 0.10. Results show that low levels of crowder hydrophobicity are capable of increasing the fibrillation lag time and high levels of crowder hydrophobicity can fully prevent the formation of fibrils. The types of structures that remain during the final stages of the simulations are summarized in a global phase diagram that shows fibril, disordered oligomer, or β-sheet phases in the space spanned by crowder size and crowder hydrophobicity. In particular, at high levels of hydrophobicity, simulations with 5 Å crowders result in only disordered oligomers and simulations with 40 Å crowders result in only β-sheets. The presence of hydrophobic crowders reduces the antiparallel β-sheet content of fibrils, whereas hard sphere crowders increase it. Finally, strong hydrophobic crowders alter the secondary structure of the Aβ (16–22) monomers, bending them into a shape that is incapable of forming ordered β-sheets or fibrils. These results qualitatively agree with previous theoretical and experimental work.     

Molecular Anatomy of ParA-ParA and ParA-ParB Interactions during Plasmid Partitioning

Firmicutes multidrug resistance inc18 plasmids encode parS sites and two small homodimeric ParA-like (δ₂) and ParB-like (ω₂) proteins to ensure faithful segregation. Protein ω₂ binds to parS DNA, forming a short left-handed helix wrapped around the full parS, and interacts with δ₂. Protein δ₂ interacts with ω₂ and, in the ATP-bound form, binds to nonspecific DNA (nsDNA), forming small clusters. Here, we have mapped the ω₂·δ₂ and δ₂·δ₂ interacting domains in the δ₂ that are adjacent to but distinct from each other. The δ₂ nsDNA binding domain is essential for stimulation of ω₂·parS-mediated ATP hydrolysis. From the data presented here, we propose that δ₂ interacts with ATP, nsDNA, and with ω₂ bound to parS at near equimolar concentrations, facilitating a δ₂ structural transition. This δ₂ “activated” state overcomes its impediment in ATP hydrolysis, with the subsequent release of both of the proteins from nsDNA (plasmid unpairing).     

Structural basis for the in vitro efficacy of nirmatrelvir against SARS-CoV-2 variants

The COVID-19 pandemic continues to be a public health threat with emerging variants of SARS-CoV-2. Nirmatrelvir (PF-07321332) is a reversible, covalent inhibitor targeting the main protease (M^pro) of SARS-CoV-2 and the active protease inhibitor in PAXLOVID (nirmatrelvir tablets and ritonavir tablets). However, the efficacy of nirmatrelvir is underdetermined against evolving SARS-CoV-2 variants. Here, we evaluated the in vitro catalytic activity and potency of nirmatrelvir against the M^pro of prevalent variants of concern (VOCs) or variants of interest (VOIs): Alpha (α, B.1.1.7), Beta (β, B.1.351), Delta (δ, B1.617.2), Gamma (γ, P.1), Lambda (λ, B.1.1.1.37/C37), Omicron (ο, B.1.1.529), as well as the original Washington or wildtype strain. These VOCs/VOIs carry prevalent mutations at varying frequencies in the M^pro specifically for α, β, γ (K90R), λ (G15S), and ο (P132H). In vitro biochemical enzymatic assay characterization of the enzyme kinetics of the mutant M^pros demonstrates that they are catalytically comparable to wildtype. We found that nirmatrelvir has similar potency against each mutant M^pro including P132H that is observed in the Omicron variant with a Ki of 0.635 nM as compared to a Ki of 0.933 nM for wildtype. The molecular basis for these observations were provided by solution-phase structural dynamics and structural determination of nirmatrelvir bound to the ο, λ, and β M^pro at 1.63 to 2.09 Å resolution. These in vitro data suggest that PAXLOVID has the potential to maintain plasma concentrations of nirmatrelvir many-fold times higher than the amount required to stop the SARS-CoV-2 VOC/VOI, including Omicron, from replicating in cells.     

Microsecond Time-Resolved Absorption Spectroscopy Used to Study CO Compounds of Cytochrome bd from Escherichia coli

Cytochrome bd is a tri-heme (b ₅₅₈, b ₅₉₅, d) respiratory oxygen reductase that is found in many bacteria including pathogenic species. It couples the electron transfer from quinol to O₂ with generation of an electrochemical proton gradient. We examined photolysis and subsequent recombination of CO with isolated cytochrome bd from Escherichia coli in one-electron reduced (MV) and fully reduced (R) states by microsecond time-resolved absorption spectroscopy at 532-nm excitation. Both Soret and visible band regions were examined. CO photodissociation from MV enzyme possibly causes fast (τ<1.5 µs) electron transfer from heme d to heme b ₅₉₅ in a small fraction of the protein, not reported earlier. Then the electron migrates to heme b ₅₅₈ (τ∼16 µs). It returns from the b-hemes to heme d with τ∼180 µs. Unlike cytochrome bd in the R state, in MV enzyme the apparent contribution of absorbance changes associated with CO dissociation from heme d is small, if any. Photodissociation of CO from heme d in MV enzyme is suggested to be accompanied by the binding of an internal ligand (L) at the opposite side of the heme. CO recombines with heme d (τ∼16 µs) yielding a transient hexacoordinate state (CO-Fe²⁺-L). Then the ligand slowly (τ∼30 ms) dissociates from heme d. Recombination of CO with a reduced heme b in a fraction of the MV sample may also contribute to the 30-ms phase. In R enzyme, CO recombines to heme d (τ∼20 µs), some heme b ₅₅₈ (τ∼0.2–3 ms), and finally migrates from heme d to heme b ₅₉₅ (τ∼24 ms) in ∼5% of the enzyme population. Data are consistent with the recent nanosecond study of Rappaport et al. conducted on the membranes at 640-nm excitation but limited to the Soret band. The additional phases were revealed due to differences in excitation and other experimental conditions.     

Geometrical Membrane Curvature as an Allosteric Regulator of Membrane Protein Structure and Function

Transmembrane proteins are embedded in cellular membranes of varied lipid composition and geometrical curvature. Here, we studied for the first time the allosteric effect of geometrical membrane curvature on transmembrane protein structure and function. We used single-channel optical analysis of the prototypic transmembrane β-barrel α-hemolysin (α-HL) reconstituted on immobilized single small unilamellar liposomes of different diameter and therefore curvature. Our data demonstrate that physiologically abundant geometrical membrane curvatures can enforce a dramatic allosteric regulation (1000-fold inhibition) of α-HL permeability. High membrane curvatures (1/diameter ∼1/40 nm⁻¹) compressed the effective pore diameter of α-HL from 14.2 ± 0.8 Å to 11.4 ± 0.6 Å. This reduction in effective pore area (∼40%) when combined with the area compressibility of α-HL revealed an effective membrane tension of ∼50 mN/m and a curvature-imposed protein deformation energy of ∼7 k_BT. Such substantial energies have been shown to conformationally activate, or unfold, β-barrel and α-helical transmembrane proteins, suggesting that membrane curvature could likely regulate allosterically the structure and function of transmembrane proteins in general.     

Elastic-Mathematical Theory of Cells and Mitochondria in Swelling Process: II. Effect of Temperature upon Modulus of Elasticity of Membranous Material of Egg Cells of Sea Urchin, Strongylocentrotus purpuratus, and of Oyster, Crassostrea virginica

The elastic behavior of the cell wall as a function of the temperature has been studied with particular attention being given to the swelling of egg cells of Strongylocentrotus purpuratus and Crassostrea virginica in different sea water concentrations at different temperatures. It was found that the modulus of elasticity is a nonlinear function of temperature. At about 12-13°C the modulus of elasticity (E) is constant, independent of the stress (σ) and strain (ε_ν) which exist at the cell wall; the membranous material follows Hooke's law, and E ≈ 3 × 10⁷ dyn/cm² for S. purpuratus and C. virginica. When the temperature is higher or lower than 12-13°C, the modulus of elasticity increases, and the membranous material does not follow Hooke's law, but is almost directly proportional to the stresses existing at the cell wall. On increasing the stress, the function E_σ = E(σ) approaches saturation. The corresponding stress-strain diagrams, σ = σ(ε_ν), and the graphs, E_σ = E(σ) and E_σ = E(t) are given. The cyto-elastic phenomena at the membrane are discussed.     

Crystal Structure of Allophycocyanin from Marine Cyanobacterium Phormidium sp. A09DM

Isolated phycobilisome (PBS) sub-assemblies have been widely subjected to X-ray crystallography analysis to obtain greater insights into the structure-function relationship of this light harvesting complex. Allophycocyanin (APC) is the phycobiliprotein always found in the PBS core complex. Phycocyanobilin (PCB) chromophores, covalently bound to conserved Cys residues of α- and β- subunits of APC, are responsible for solar energy absorption from phycocyanin and for transfer to photosynthetic apparatus. In the known APC structures, heterodimers of α- and β- subunits (known as αβ monomers) assemble as trimer or hexamer. We here for the first time report the crystal structure of APC isolated from a marine cyanobacterium (Phormidium sp. A09DM). The crystal structure has been refined against all the observed data to the resolution of 2.51 Å to R_work (R_free) of 0.158 (0.229) with good stereochemistry of the atomic model. The Phormidium protein exists as a trimer of αβ monomers in solution and in crystal lattice. The overall tertiary structures of α- and β- subunits, and trimeric quaternary fold of the Phormidium protein resemble the other known APC structures. Also, configuration and conformation of the two covalently bound PCB chromophores in the marine APC are same as those observed in fresh water cyanobacteria and marine red algae. More hydrophobic residues, however, constitute the environment of the chromophore bound to α-subunit of the Phormidium protein, owing mainly to amino acid substitutions in the marine protein.     

THE RHEOLOGY OF THE BLOOD. III

The authors have confirmed the fact that blood serum and plasma behave rheologically like a true viscous liquid. It is true for whole blood only to a first approximation, but with this reservation they have studied the available data and extended the equation of Bingham and Durham to cover protein solutions of various concentrations and at various temperatures as well as mixtures of proteins and corpuscles present in whole blood. If Φ is the fluidity of whole blood, Φ₁ is the fluidity of water and ΔΦ = Φ – Φ₁, then ΔΦ = β₁ b ₁ + β₂ b ₂ + β₃ b ₃ + ··· where β₁, β₂, β₃, etc., are constants for the fluidity lowering of the salts, albumin, globulin, fibrinogen, and the corpuscles, etc., present in the whole blood. The conclusions from the data referred to are intended to buttress this simple equation (6).     

A GTPase Chimera Illustrates an Uncoupled Nucleotide Affinity and Release Rate,Providing Insight into the Activation Mechanism

The release of GDP from GTPases signals the initiation of a GTPase cycle, where the association of GTP triggers conformational changes promoting binding of downstream effector molecules. Studies have implicated the nucleotide-binding G5 loop to be involved in the GDP release mechanism. For example, biophysical studies on both the eukaryotic Gα proteins and the GTPase domain (NFeoB) of prokaryotic FeoB proteins have revealed conformational changes in the G5 loop that accompany nucleotide binding and release. However, it is unclear whether this conformational change in the G5 loop is a prerequisite for GDP release, or, alternatively, the movement is a consequence of release. To gain additional insight into the sequence of events leading to GDP release, we have created a chimeric protein comprised of Escherichia coli NFeoB and the G5 loop from the human Giα1 protein. The protein chimera retains GTPase activity at a similar level to wild-type NFeoB, and structural analyses of the nucleotide-free and GDP-bound proteins show that the G5 loop adopts conformations analogous to that of the human nucleotide-bound Giα1 protein in both states. Interestingly, isothermal titration calorimetry and stopped-flow kinetic analyses reveal uncoupled nucleotide affinity and release rates, supporting a model where G5 loop movement promotes nucleotide release.The hydrolysis of guanosine triphosphate (GTP) by GTPases, such as the oncoprotein p21 Ras and heterotrimeric Gα proteins, is a critical regulatory activity for cell growth and proliferation (1). Aberrant GTPases are consequently often implicated in tumorigenesis, developmental disorders, and metabolic diseases (2). Critical for the initiation of a GTPase cycle is the release of guanosine diphosphate (GDP), which allows GTP to bind and switch the protein from an inactive to an active conformation. The GTP is subsequently hydrolyzed to GDP and inorganic phosphate, returning the GTPase to an inactive conformation (3).Given that the release of GDP is the fundamental step in the initiation of a GTPase cycle, the detailed mechanism by which it is released has been under intense scrutiny. Studies using double electron-electron resonance, deuterium-exchange, Rosetta energy analysis, and electron paramagnetic resonance, have shown that the mechanism involves conformational changes in the nucleotide-coordinating G5 loop, one of five nucleotide recognition motifs (4, 5, 6, 7, 8, 9, 10, 11). Structural studies of eukaryotic Gα proteins and the intracellular TEES-type GTPase domain of the prokaryotic iron transporter FeoB (NFeoB) have also illustrated distinct conformations of the G5 loop, depending on the nucleotide-bound state (9, 12).Recently, we reported mutational studies of the G5 loop of Escherichia coli NFeoB, which illustrated a correlation between the sequence composition of the loop and the intrinsic GDP release rate (13). However, despite these observations, it is unclear whether the observed conformational changes in the G5 loop are a prerequisite for GDP release, or if the movement is a consequence of GDP release. To address this fundamental question, in this study we have used a combination of protein engineering and biophysical methods.Initially, to assess the relevance of conformational flexibility in the G5 loop, we aimed to create a protein chimera combining sequence and structural characteristics of both fast and slow GDP-releasing GTPases. We thus engineered a protein chimera using E. coli NFeoB as the scaffold (a protein with fast intrinsic GDP release) and substituted the G5 loop with that of a slow GDP-releasing protein (the human Giα1 protein; Gene ID 2770; Fig. 1 A (5)). GTP hydrolysis assays comparing wild-type (wt) NFeoB (wtNFeoB) and the protein chimera (ChiNFeoB) validated the integrity of the GTPase activities of both proteins (k_cat = 0.46 and 0.36 min⁻¹, respectively). To further assess the ChiNFeoB protein, we determined its crystal structure at 2.2 Å resolution (see Table S1 in the Supporting Material). The ChiNFeoB structure contains two molecules in the asymmetric unit, with molecule A bound to GDP. They are essentially identical to the nucleotide-bound wtNFeoB structure (root-mean-square deviation of 1.2 Å over 226 Cα atoms; Fig. 2).Open in a separate windowFigure 1Chimera model and structural comparison. (A) Illustration highlighting the chimera sequence change. (Orange) Sequence of the extended G5 loop from Giα1, which replaced the NFeoB sequence (gray). (B–F) Structural comparison of the G5 loop between (B) WT apo (PDB:3HYR) and nucleotide-bound (PDB:3HYT) NFeoB structures. (C) NFeoB nucleotide-bound and Giα1 (PDB:2ZJZ). (D) Nucleotide-bound NFeoB and chimera (Chi_GDP). (E) Nucleotide-bound chimera and Giα1. (F) Nucleotide-free (Chi_apo) and bound chimera protein. (G) Overview of the nucleotide binding site and structural overlay of chimera and Giα1 structures. To see this figure in color, go online.Open in a separate windowFigure 2Superimposition of nucleotide-bound NFeoB and chimera protein, with thermodynamic parameters. To see this figure in color, go online.However, the ChiNFeoB structure, when compared to the wtNFeoB structure, revealed an alteration in the conformation of the G5 loop, showing an extra turn on the N-terminal end of the α6 helix. This is structurally distinct from the wtFeoB protein, but with a conformation similar to that of the Giα1 protein (PDB:2ZJZ; Fig. 1, B–F). As in the crystal structures of wtNFeoB and Giα1, ChiNFeoB residues implicated in coordination of the nucleotide base maintain their positions in the G5 loop relative to GDP. In particular, residues Ala^∗150 and Thr^∗151 (NFeoB numbering, the asterisk indicates Giα1 chimera residue) are involved in electrostatic interactions with the nucleotide base moiety, analogous to the structures of both wtNFeoB and Giα1 (Fig. 1 G). Serendipitously, the second molecule in the asymmetric unit of ChiNFeoB (molecule B) was present in the nucleotide-free state. The two molecules (GDP-bound and nucleotide-free) are nearly identical (the superposition of molecules A and B yields a root-mean-square deviation of 0.36 Å over 229 Cα atoms), with the G5 loop adopting a nearly indistinguishable conformation compared to that of the GDP-bound molecule A (Fig. 1 F).Importantly, this conformation is independent of the crystallographic packing, inasmuch as the loop is not involved in any crystal contacts. In contrast, the structures of nucleotide-bound and nucleotide-free wtNFeoB illustrated a large conformational change in the G5 loop (Fig. 1 B). Hence, the substitution in the chimera extends the secondary structure of the α6 helix, and as hypothesized, the engineered ChiNFeoB protein has a G5 loop structure that is more conformationally stable than that of wtNFeoB.We subsequently measured the affinity of the ChiNFeoB protein for GDP using isothermal titration calorimetry (ITC). Nonlinear regression was used to attain the thermodynamic parameters (including the GDP binding affinity, K_a; the corresponding dissociation constant (K_d) was calculated from the equation K_d = 1/K_a). Interestingly, these measurements revealed the ChiNFeoB protein to have an almost 10-fold reduced affinity for GDP (82 vs. 9 μM measured for the WT protein; Fig. 2). In contrast, in a recent alanine scanning mutagenesis study of the G5 loop we observed a fivefold increase in affinity for GDP in a Ser¹⁵⁰Ala mutant (2 μM) (14). This mutant protein has a coordination environment for the GDP base analogous to that of the ChiNFeoB protein (Fig. 1 A), indicating that it is not the presence of an alanine at position 150 that causes the reduced GDP affinity observed for the chimera protein. Instead, the analysis by ITC and comparison with previous mutagenesis studies indicates that the GDP binding site is less accessible in the ChiNFeoB protein, likely due to the introduction of conformational rigidity that accompanies the extension of secondary structural elements within the loop (Fig. 1 D).To further evaluate the functional characteristics of the chimera protein, we used stopped-flow fluorescence assays to determine the rate of nucleotide dissociation (k_off) and association (k_on) for the ChiNFeoB protein. The association rate for the GTP analog mant-GMPPNP was determined from the slope of a linear plot of protein concentration versus the observed association constant (k_obs). The k_on for the chimera was determined to be 3.20 μM⁻¹ min⁻¹ (Supporting Material), the dissociation rate (k_off) of GDP for the chimera was determined to be 16.6 s⁻¹ (vs. 144 s⁻¹ for wtNFeoB;

A Gs-RhoGEF interaction: An old G protein finds a new job

The heterotrimeric G proteins are known to have a variety of downstream effectors, but G_s was long thought to be specifically coupled to adenylyl cyclases. A new study indicates that activated G_s can also directly interact with a guanine nucleotide exchange factor for Rho family small GTPases, PDZ-RhoGEF. This novel interaction mediates activation of the small G protein Cdc42 by G_s-coupled GPCRs, inducing cytoskeletal rearrangements and formation of filopodia-like structures. Furthermore, overexpression of a minimal PDZ-RhoGEF fragment can down-regulate cAMP signaling, suggesting that this effector competes with canonical signaling. This first demonstration that the Gα_s subfamily regulates activity of Rho GTPases extends our understanding of Gα_s activity and establishes RhoGEF coupling as a universal Gα function.

The canonical G protein pathway consists of a cell surface receptor, a heterotrimeric G protein, and an effector protein that controls signaling within the cells. This fundamental paradigm, familiar to every biologist, is rooted in discoveries by the laboratories of Sutherland, Rodbell, and Gilman, which in the 1970s and 1980s dissected biochemical mechanisms of adenylyl cyclase activation by hormones. Their breakthrough came after experiments showing that the G protein G_s is essential to transfer agonist stimulation from the receptor to adenylyl cyclase (1). This G protein consists of the ∼42-kDa α subunit, which binds and hydrolyzes GTP, and the permanently associated dimer of 35-kDa β and ∼10-kDa γ subunits (Gβγ). Their findings helped establish a canonical model in which the agonist-bound receptor causes the G protein to release GDP, and the heterotrimer dissociates into Gα-GTP and free Gβγ; in this state, the G protein can activate its effector (i.e. Gα_s will activate adenylyl cyclase until GTP is hydrolyzed). Although the rod photoreceptor G protein, transducin, was discovered by that time (2), the ubiquitously expressed G_s can be considered the founding member of the G protein family.The subsequent cloning and identification of the other three families (G_i, G_q, and G₁₂) completed the rough map of G protein–mediated transduction. These initial studies suggested that the α subunits were responsible for activation of one type of effector (e.g. Gα_s for adenylyl cyclase and cAMP; Gα_q for phospholipase C, phosphoinositides, and Ca²⁺; and Gα_i for ion channels and inhibition of adenylyl cyclase), whereas the free Gβγ complexes interact with a remarkably large number of binding partners, including some effector enzymes and ion channels (3). Later, Gα₁₂ and Gα₁₃ were found to regulate a distinct type of effectors, the RhoGEFs (4, 5). These multidomain proteins contain pleckstrin homology (PH) domains, which facilitate their membrane localization, and Dbl homology (DH) domains, which catalyze GDP-for-GTP exchange (guanine nucleotide exchange factor; GEF) in the Rho family of small (∼20-kDa) G proteins. At the time, the G₁₂-RhoGEF pathway seemed odd as it contained two G proteins: the receptor-activated “large” G₁₂ class protein and the “small” Rho G protein, which is activated by RhoGEF. However, it was then discovered that Gα_q could activate a RhoGEF called Trio (6), and that Gβγ complexes activate other RhoGEFs, indicating that this pathway, if unusual, is at least popular. Gα_s, however, mostly appeared to be faithful to its originally determined role—to stimulate adenylyl cyclase(s)—possibly contributing to the enduring perception that regulation of a second messenger–generating enzyme is the “real” function of a heterotrimeric G protein.In the current issue of JBC, Castillo-Kauil et al. (7) force a reexamination of the existing canon, presenting data that show Gα_s can also interact with a specific RhoGEF, in this case PDZ-RhoGEF (PRG). The authors made this discovery as part of an examination of the regulation of cell shape by the Rho family. They began by expressing a series of short constructs of three RhoGEF proteins, p115RhoGEF, PRG, and LARG, all of which activated RhoA as expected, promoting cell contraction. However, they noticed that the DH/PH domain of PRG also activated Cdc42 and induced filopodia-like cell protrusions. To investigate which G protein is responsible for activation of this Cdc42-mediated pathway, they overexpressed constitutively active mutants of different Gα subunits. These mutants are stabilized in the active GTP-bound state due to substitution of the glutamine residue crucial for GTP hydrolysis. Surprisingly, the PRG-Cdc42 pathway was stimulated by Gα_sQ227L, the one Gα subtype not known for interaction with RhoGEFs. Furthermore, they showed that binding of PRG to Cdc42 was promoted only by G_s-coupled receptors, and not by G_q- or G_i-coupled GPCRs. The authors then investigated the PRG site responsible for the interaction with Gα_s, narrowing it down to the isolated PRG DH and PH domains and their linker region. A construct encompassing these domains was able to inhibit (i) GPCR-mediated activation of Cdc42, (ii) the Gα_sQ227L-promoted interaction of PRG with Cdc42, and (iii) some protein phosphorylation events downstream of the canonical cAMP pathway. Taken together, their work identifies PRG as a novel effector for G_s; the Gα_s-PRG interaction mediates activation of Rho family protein Cdc42, leading to cytoskeletal remodeling.The unexpected results of Castillo-Kauil et al. open up new opportunities to explore this mechanism at different levels of biology. The experiments described in the paper were performed in vitro using cultured cells, imaging, and pulldown of protein complexes containing the overexpressed Gα_s Q227L mutant. Considering the multitude of G_s-coupled receptors and RhoGEFs in the body (8, 9), it will be important to understand the physiological context where the new G_s-mediated pathway plays a significant role. This will require experimentation in vivo and possibly reevaluation of the phenotypes associated with known pathogenic mutations in Gα_s (GNAS) and other relevant genes. At the molecular level, it would be important to delineate the biochemical mechanisms of Gα_s interaction with PRG. For example, at what stage of the GTP/GDP cycle does Gα_s bind to PRG: in the GTP-bound state, which also activates adenylate cyclase, or in the transition state (i.e. just before the terminal phosphate of GTP is removed)? Indeed, there is precedent for proteins that bind preferentially with the transition state—specifically RGS proteins, which accelerate the GTPase reaction. Another possibility is that, by analogy with p115RhoGEF, which stimulates GTPase activity of Gα₁₂ and Gα₁₃, PRG (and other RhoGEFs with similar DH-PH sequences) can influence interaction of Gα_s with nucleotides, Gβγ, and other partners.Since defining the receptor, G protein, and effector as the three essential members of the G protein pathway, researchers have discovered many additional proteins that regulate the amplitude and duration of the stimulus and/or participate in cross-talk with other signaling circuits. These “new” proteins include arrestins, receptor kinases, nonreceptor exchange factors, GTPase-activating proteins, special chaperones, etc. Thus, in a way, discovering a novel binding partner for a signaling molecule is not as surprising as it would have been 20 years ago. However, the new partner identified by Castillo-Kauil et al. makes the result of extra significance; until now, we knew that three of four G protein subfamilies could regulate Rho GTPases by activating RhoGEFs: G₁₂ and G_q via their α subunits and G_i via the Gβγ subunits (10). The demonstration that the G_s subfamily is no exception shows that activation of RhoGEFs by heterotrimeric G proteins may be a truly universal mechanism (Fig. 1). The significance of this insight is that the multitude of biological processes regulated by Rho-GTPase networks can potentially respond to the entire repertoire of GPCR-mediated stimuli.Open in a separate windowFigure 1.Activation of the Rho family by heterotrimeric G proteins. The Rho family of small GTPases is activated by RhoGEF proteins, some of which can be stimulated by heterotrimeric G proteins. Of four families of heterotrimeric G proteins, three (G₁₂, G_q, and G_i, shown in shades of gray) were known to activate certain RhoGEFs. The new results (highlighted in orange) (7) show that G_s, the G protein known to stimulate production of cAMP, can also stimulate a particular RhoGEF; this suggests that the Rho GTPases can potentially be stimulated by the multitude of signals from the entire class of GPCRs, including those coupled to G_s. IP₃, inositol 1,4,5-trisphosphate.

Funding and additional information—This work was supported in part by National Institutes of Health Grant R56DK119262 (to V. Z. S.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

Abbreviations—The abbreviations used are:

PH

pleckstrin homology

DH

Dbl homology

GEF

guanine nucleotide exchange factor

PRG

PDZ-RhoGEF

GPCR

G protein–coupled receptor.

     

Cellular and Physiological Effects of Dietary Supplementation with β-Hydroxy-β-Methylbutyrate (HMB) and β-Alanine in Late Middle-Aged Mice

There is growing evidence that severe decline of skeletal muscle mass and function with age may be mitigated by exercise and dietary supplementation with protein and amino acid ingredient technologies. The purposes of this study were to examine the effects of the leucine catabolite, beta-hydroxy-beta-methylbutyrate (HMB), in C₂C₁₂ myoblasts and myotubes, and to investigate the effects of dietary supplementation with HMB, the amino acid β-alanine and the combination thereof, on muscle contractility in a preclinical model of pre-sarcopenia. In C₂C₁₂ myotubes, HMB enhanced sarcoplasmic reticulum (SR) calcium release beyond vehicle control in the presence of all SR agonists tested (KCl, P<0.01; caffeine, P = 0.03; ionomycin, P = 0.03). HMB also improved C₂C₁₂ myoblast viability (25 μM HMB, P = 0.03) and increased proliferation (25 μM HMB, P = 0.04; 125 μM HMB, P<0.01). Furthermore, an ex vivo muscle contractility study was performed on EDL and soleus muscle from 19 month old, male C57BL/6nTac mice. For 8 weeks, mice were fed control AIN-93M diet, diet with HMB, diet with β-alanine, or diet with HMB and β-alanine. In β-alanine fed mice, EDL muscle showed a 7% increase in maximum absolute force compared to the control diet (202 ± 3vs. 188± 5 mN, P = 0.02). At submaximal frequency of stimulation (20 Hz), EDL from mice fed HMB plus β-alanine showed an 11% increase in absolute force (88.6 ± 2.2 vs. 79.8 ± 2.4 mN, P = 0.025) and a 13% increase in specific force (12.2 ± 0.4 vs. 10.8 ± 0.4 N/cm², P = 0.021). Also in EDL muscle, β-alanine increased the rate of force development at all frequencies tested (P<0.025), while HMB reduced the time to reach peak contractile force (TTP), with a significant effect at 80 Hz (P = 0.0156). In soleus muscle, all experimental diets were associated with a decrease in TTP, compared to control diet. Our findings highlight beneficial effects of HMB and β-alanine supplementation on skeletal muscle function in aging mice.     

Comparative studies of bile salts. 5α-Chimaerol, a new bile alcohol from the white sucker Catostomus commersoni Lacépède

1. G.l.c. examination of bile alcohols prepared from the sucker Catostomus commersoni Lacépède (family Catostomidae) showed that although 5α-cyprinol (5α-cholestane-3α,7α,12α,26,27-pentol) was a minor constituent, the principal bile alcohol was an undescribed substance, probably present in the bile as the C-26 sulphate ester, whose i.r., n.m.r. and mass spectra agreed with the structure 5α-cholestane-3α,7α,12α,24,26-pentol. 2. M_D studies suggest that this 5α-chimaerol is the 24(+), 25S enantiomer and that 5β-chimaerol (chimaerol) from Chimaera monstrosa bile also has the 24(+), 25S configuration. These findings imply that bile alcohol biosynthesis in suckers and chimaeras includes stereospecific oxidation of cholesterol at C-26. 3. C. commersoni bile acids (present in minor amounts) probably consist largely of 3α,7α,12α-trihydroxy-5α-cholan-24-oic acid (allocholic acid). 4. 5α-Chimaerol sulphate and 5α-cyprinol sulphate are probably biochemically equivalent as bile salts, and can be considered as arising by parallel evolution.     

Angiotensin II Stimulates Thick Ascending Limb Superoxide Production via Protein Kinase Cα-dependent NADPH Oxidase Activation

Angiotensin II (Ang II) stimulates thick ascending limb (TAL) O production, but the receptor(s) and signaling mechanism(s) involved are unknown. The effect of Ang II on O is generally attributed to the AT₁ receptor. In some cells, Ang II stimulates protein kinase C (PKC), whose α isoform (PKCα) can activate NADPH oxidase. We hypothesized that in TALs, Ang II stimulates O via AT₁ and PKCα-dependent NADPH oxidase activation. In rat TALs, 1 nm Ang II stimulated O from 0.76 ± 0.17 to 1.97 ± 0.21 nmol/min/mg (p < 0.001). An AT₁ antagonist blocked the stimulatory effect of Ang II on O (0.87 ± 0.25 nmol/min/mg; p < 0.006), whereas an AT₂ antagonist had no effect (2.16 ± 0.133 nmol/min/mg; p < 0.05 versus vehicle). Apocynin, an NADPH oxidase inhibitor, blocked Ang II-stimulated O by 90% (p < 0.01). Ang II failed to stimulate O in TALs from p47^phox−/− mice (p < 0.02). Monitored by fluorescence resonance energy transfer, Ang II increased PKC activity from 0.02 ± 0.03 to 0.13 ± 0.02 arbitrary units (p < 0.03). A general PKC inhibitor, GF109203X, blocked the effect of Ang II on O (1.47 ± 0.21 versus 2.72 ± 0.47 nmol/min/mg with Ang II alone; p < 0.03). A PKCα- and β-selective inhibitor, Gö6976, also blocked the stimulatory effect of Ang II on O (0.59 ± 0.15 versus 2.05 ± 0.28 nmol/min/mg with Ang II alone; p < 0.001). To distinguish between PKCα and PKCβ, we used tubules expressing dominant-negative PKCα or -β. In control TALs, Ang II stimulated O by 2.17 ± 0.44 nmol/min/mg (p < 0.011). In tubules expressing dominant-negative PKCα, Ang II failed to stimulate O (change: −0.30 ± 0.27 nmol/min/mg). In tubules expressing dominant-negative PKCβ1, Ang II stimulated O by 2.08 ± 0.69 nmol/min/mg (p < 0.002). We conclude that Ang II stimulates TAL O production via activation of AT₁ receptors and PKCα-dependent NADPH oxidase.     

Hepcidin Bound to α2-Macroglobulin Reduces Ferroportin-1 Expression and Enhances Its Activity at Reducing Serum Iron Levels

Hepcidin regulates iron metabolism by down-regulating ferroportin-1 (Fpn1). We demonstrated that hepcidin is complexed to the blood transport protein, α₂-macroglobulin (α₂M) (Peslova, G., Petrak, J., Kuzelova, K., Hrdy, I., Halada, P., Kuchel, P. W., Soe-Lin, S., Ponka, P., Sutak, R., Becker, E., Huang, M. L., Suryo Rahmanto, Y., Richardson, D. R., and Vyoral, D. (2009) Blood 113, 6225–6236). However, nothing is known about the mechanism of hepcidin binding to α₂M or the effects of the α₂M·hepcidin complex in vivo. We show that decreased Fpn1 expression can be mediated by hepcidin bound to native α₂M and also, for the first time, hepcidin bound to methylamine-activated α₂M (α₂M-MA). Passage of high molecular weight α₂M·hepcidin or α₂M-MA·hepcidin complexes (≈725 kDa) through a Sephadex G-25 size exclusion column retained their ability to decrease Fpn1 expression. Further studies using ultrafiltration indicated that hepcidin binding to α₂M and α₂M-MA was labile, resulting in some release from the protein, and this may explain its urinary excretion. To determine whether α₂M-MA·hepcidin is delivered to cells via the α₂M receptor (Lrp1), we assessed α₂M uptake and Fpn1 expression in Lrp1^−/− and Lrp1^+/+ cells. Interestingly, α₂M·hepcidin or α₂M-MA·hepcidin demonstrated similar activities at decreasing Fpn1 expression in Lrp1^−/− and Lrp1^+/+ cells, indicating that Lrp1 is not essential for Fpn1 regulation. In vivo, hepcidin bound to α₂M or α₂M-MA did not affect plasma clearance of α₂M/α₂M-MA. However, serum iron levels were reduced to a significantly greater extent in mice treated with α₂M·hepcidin or α₂M-MA·hepcidin relative to unbound hepcidin. This effect could be mediated by the ability of α₂M or α₂M-MA to retard kidney filtration of bound hepcidin, increasing its half-life. A model is proposed that suggests that unlike proteases, which are irreversibly bound to activated α₂M, hepcidin remains labile and available to down-regulate Fpn1.     

Alginate Lyases from Alginate-Degrading Vibrio splendidus 12B01 Are Endolytic

Alginate lyases are enzymes that degrade alginate through β-elimination of the glycosidic bond into smaller oligomers. We investigated the alginate lyases from Vibrio splendidus 12B01, a marine bacterioplankton species that can grow on alginate as its sole carbon source. We identified, purified, and characterized four polysaccharide lyase family 7 alginates lyases, AlyA, AlyB, AlyD, and AlyE, from V. splendidus 12B01. The four lyases were found to have optimal activity between pH 7.5 and 8.5 and at 20 to 25°C, consistent with their use in a marine environment. AlyA, AlyB, AlyD, and AlyE were found to exhibit a turnover number (k_cat) for alginate of 0.60 ± 0.02 s⁻¹, 3.7 ± 0.3 s⁻¹, 4.5 ± 0.5 s⁻¹, and 7.1 ± 0.2 s⁻¹, respectively. The K_m values of AlyA, AlyB, AlyD, and AlyE toward alginate were 36 ± 7 μM, 22 ± 5 μM, 60 ± 2 μM, and 123 ± 6 μM, respectively. AlyA and AlyB were found principally to cleave the β-1,4 bonds between β-d-mannuronate and α-l-guluronate and subunits; AlyD and AlyE were found to principally cleave the α-1,4 bonds involving α-l-guluronate subunits. The four alginate lyases degrade alginate into longer chains of oligomers.     

Quantifying Interactions of a Membrane Protein Embedded in a Lipid Nanodisc using Fluorescence Correlation Spectroscopy

Using fluorescence correlation spectroscopy, we measured a dissociation constant of 20 nM between EGFP-labeled LcrV from Yersinia pestis and its cognate membrane-bound protein YopB inserted into a lipid nanodisc. The combination of fluorescence correlation spectroscopy and nanodisc technologies provides a powerful approach to accurately measure binding constants of interactions between membrane bound and soluble proteins in solution. Straightforward sample preparation, acquisition, and analysis procedures make this combined technology attractive for accurately measuring binding kinetics for this important class of protein-protein interactions.Interactions involving membrane proteins are integral to a multitude of cellular processes, including signal transduction, energy production and conversion, cell adhesion, and foreign molecule identification. More than half of all pharmaceutical drugs target membrane proteins, further illustrating their importance in human health (1). Due to this, there is a continuing high demand for methods that can screen, validate, and quantify interactions involving membrane proteins. Unfortunately, the quantitative techniques available to characterize protein-protein interactions are most often directed toward soluble proteins, and are often difficult to apply to membrane proteins (2). Recently, advances in lipid nanodisc technologies, often referred to as reconstituted high-density lipoproteins, have enabled biophysical and biochemical studies of solubilized membrane proteins. The nativelike lipid environment of nanodiscs maintains protein functionality, opening a realm of possibilities in analyzing membrane protein function and dynamics in solution (3).Nanodiscs are discoidal cell membrane mimetics that are 8–20 nm in diameter, consisting of a lipid bilayer stabilized by two peripheral apolipoprotein A-I proteins (4). These particles provide an excellent alternative to traditional lipid-based platforms (e.g., liposomes) for membrane protein solubilization and interrogation. The diameter of nanodisc can be engineered to accommodate different-sized membrane proteins by varying lipid composition. As such, nanodiscs represent an important platform for expression, isolation, and study of functional membrane proteins and the multiprotein complexes they form. Several groups have successfully reconstituted a variety of membrane proteins, including bacteriorhodopsin (5), G-protein coupled receptors (6), and cytochrome P450 (7) to name a few. To produce solubilized, discrete membrane proteins, we utilized a cell-free expression approach to embed membrane proteins directly into nanodiscs formed in situ. This approach allows for enhanced purification and rapid labeling of proteins of interest where traditional approaches are unsuccessful (8). Fluorescence correlation spectroscopy (FCS) analysis coupled with nanodisc technology has been successfully used to monitor small ligand binding interactions with membrane proteins (6,8) and to measure lipid-protein interactions at the single molecule level (9). Here, we show that FCS can be used to measure interactions between soluble proteins and cognate membrane proteins inserted into nanodiscs produced using cell-free expression methodologies.FCS uses correlation analysis of fluorescence arising from randomly diffusing molecules to measure diffusion constants, and hence hydrodynamic radii, of species in solution. Diffusion times measured by FCS are inversely proportional to diffusion constants. A shift to slower diffusion time indicates an increase in the hydrodynamic radius due to binding. Due to this size sensitivity, FCS has been proposed repeatedly as a method to quantify molecular interactions in solution (10). However, an important difficulty in FCS arises when characterizing interactions between two proteins of similar size. The diffusion time is inversely proportional to the cube root of the molecular mass τ_D ∼ M^−1/3. Doubling the mass results in only a factor-of-1.3 increase in diffusion time, but a factor of 1.6 is required to resolve two species (11). As we demonstrate, this difficulty is overcome by using FCS to measure the interactions between labeled soluble proteins and membrane proteins supported within a nanodisc. The much larger size of the membrane protein-nanodisc complex (Fig. 1 A) relative to most soluble proteins provides the necessary differential in diffusion times to easily resolve bound and free species by FCS.Open in a separate windowFigure 1(A) Model of YopB (blue) inserted into a 10-nm nanodisc with cognate protein LcrV (red) labeled with EGFP (green). The molecular masses used in this model are: LcrV (35 kDa), EGFP (27 kDa), YopB (42 kDa, monomer), and nanodisc-YopB complex (258 kDa). The small LcrV binds to the much larger YopB-nanodisc complex, resulting in a significant shift in the autocorrelation curves to longer diffusion times. (B) Hypothetical autocorrelation curves expected for a series of titration experiments measured using FCS. Increased binding is measured as an increase in diffusion time. For intermediate binding, diffusion components are detectable as illustrated (red, green, and blue curves). To see this figure in color, go online.Binding kinetics are readily measured using FCS by titrating increasing amounts of unlabeled cognate protein, resulting in a series of autocorrelation curves similar to those modeled in a hypothetical example in Fig. 1 B. For 0% binding (black curve), only the labeled soluble protein is present, producing a single component correlation curve with a diffusion time of 0.1 ms. On the autocorrelation curve with a single component, the diffusion time is the time at which the autocorrelation amplitude decreases by half. For 100% binding (cyan curve), the amount of nanodisc with inserted membrane protein is well above the dissociation constant so that all available soluble protein is bound.Again, a single component correlation curve is found, but now with a longer diffusion time (10 ms in this hypothetical example). Intermediate cases have two components, corresponding to free and bound protein. The relative amplitudes of the components obtained by fitting to two component models in these cases can be used to obtain a binding curve. For binding interactions, the amount of ligand in the free and bound state can be separated by their diffusion time and quantified as a function of concentration. The fraction of bound protein is calculated by fitting the correlation to two components. The first component f₀(τ) is the correlation of the free protein. The second component g₀(τ) is the correlation when the protein is bound. The total correlation function ish(τ) = αf₀(τ) + βg₀(τ), (1)where f₀(τ) = 1/(1 + τ/τ_f) and g₀(τ) = 1/(1 + τ/τ_b). The values α and β are, respectively, the amplitudes of the correlation function for free and bound protein, with the corresponding diffusion times τ_f and τ_b. The ratio F = β/(α + β) gives the fraction bound.We investigated by FCS the interaction of two Yersinia pestis proteins: soluble EGFP-labeled LcrV and membrane-bound YopB (Fig. 1 A). YopB is an effector protein involved in host cell invasion and disarming the cell’s defense. Although the structure of YopB is largely unknown, it is thought to exist as a dimer (12). Delivery of YopB to the host cell is regulated by the Type III secretion system (T3SS) (i.e., injectisome). The needle tip of the injectisome contains the LcrV protein. When Y. pestis comes in contact with a cell membrane, the injectisome forms a pore through the membrane to facilitate the diffusion of effector proteins into the host cell. Multiple lines of evidence suggest that pores are formed only through the direct interaction of YopB with LcrV (13).The FCS diffusion times of free LcrV (τ_f; Fig. 2 A, black curve) and fully bound LcrV (τ_b; [YopB] = 10 μM) were found to be 140 and 630 μs, respectively. Because no measurable change in diffusion time was detected above [YopB] = 1 μM (Fig. 2, green curve), LcrV was assumed to be completely bound at [YopB] = 10 μM. The diffusion times of free and bound LcrV were determined by fitting the autocorrelation curves to a one-component model (χ² ∼ 1 for each diffusion time). These diffusion times correspond to average hydrodynamic radii of 2.5 nm for free LcrV and 11.1 nm for the LcrV + YopB-nanodisc complex, as calculated by the Einstein-Stokes equation. No significant change in diffusion time was detected when LcrV was incubated with 2 μM of nanodiscs lacking the YopB protein (Fig. 2 A, gray curve), suggesting that LcrV alone is unable to interact directly with a lipid bilayer.Open in a separate windowFigure 2(A) FCS autocorrelation curves of 1 nM EGFP-labeled LcrV protein in the absence and presence of YopB at increasing concentrations. The diffusion time of freely diffusing LcrV is τ_D,free = 140 s (black curve). At [YopB] = 1 μM where LcrV is completely bound, the FCS curve is dominated by a single component (green curve). The autocorrelation curves at intermediate concentrations (in particular [YopB] = 25 and 50 nM, blue and cyan curves) contain contributions from two diffusing components. A control experiment was performed with 2 μM of nanodisc lacking the YopB protein (gray curve). No significant change in diffusion time was detected. (B) The fraction of bound LcrV as a function of YopB concentration (log scale). This fraction is calculated by fitting the FCS curves shown in Fig. 2A with Eq. 1. The dissociation constant extracted is K_d = 20.45 ± 2.0 nM. Error bars represent an average over six FCS measurements. To see this figure in color, go online.To extract the dissociation constant (K_D), a titration was performed over a range of YopB concentrations (10 pM to 1 μM), obtained by varying the concentration of YopB-nanodisc complexed. Having determined the values of the diffusion times for free (τ_f) and bound (τ_b) protein, we fitted the autocorrelation curves to a two-species correlation function with Eq. 1. The brightness per LcrV molecule was between 2170 and 2280 photons/(s × molecule) for the entire titration, validating the use of Eq. 1. The only varying parameters were the correlation amplitudes α (free protein) and β (bound protein). Intermediate binding was observed between 10 and 100 nM, because two correlation components are resolved. At [YopB] = 50 nM (cyan curve), the ratio β/α was 1.5, indicating that more than half of LcrV was bound. For the two-component model in these intermediate cases, the χ² value was between 1 and 1.3, indicating a good fit. In contrast, fitting the data to a one-component model resulted in a poor fit (χ² > 7), justifying the use of the two-species model. Binding curves were fitted to the equation y = x/(K_D + x), where y is the fraction of bound LcrV, x is the YopB concentration, and K_d the dissociation constant.Fitting this model with a least-squares algorithm (Fig. 2 B), we extracted K_d = 20.5 ± 2.0 nM. To quantify the statistical error, three measurements were recorded for 2 min, and the entire titration was repeated. Importantly, these data provide not only a quantitative binding affinity for the YopB-LcrV interaction, but support the hypothesis that LcrV requires direct interaction with YopB, not just a lipid bilayer, to promote pore formation.In conclusion, the coupling of FCS with nanodisc technology provides a facile yet powerful tool to quantitatively measure interactions involving membrane proteins in solution. FCS can simultaneously detect the presence of both free and bound species without the need for surface immobilization of the cognate proteins. Cell-free coexpression of both membrane protein and apolipoprotein components in the presence of lipids is a facile methodology for producing functional, soluble, nanodisc-supported membrane proteins. Labeling the soluble cognate proteins with EGFP provides a simple path to obtaining fluorescent, single-labeled proteins compatible with FCS. The combination of FCS with nanodisc technology presented here provides not only new key data for modeling the invasion process of Y. pestis, but can also be generalized to study interactions between most other soluble and membrane proteins. Such methods have been lacking, yet are critical for understanding interaction networks, e.g., signal transduction cascades.See the Supporting Material for additional detail on the methodology used.     

Certhrax Toxin,an Anthrax-related ADP-ribosyltransferase from Bacillus cereus

We identified Certhrax, the first anthrax-like mART toxin from the pathogenic G9241 strain of Bacillus cereus. Certhrax shares 31% sequence identity with anthrax lethal factor from Bacillus anthracis; however, we have shown that the toxicity of Certhrax resides in the mART domain, whereas anthrax uses a metalloprotease mechanism. Like anthrax lethal factor, Certhrax was found to require protective antigen for host cell entry. This two-domain enzyme was shown to be 60-fold more toxic to mammalian cells than anthrax lethal factor. Certhrax localizes to distinct regions within mouse RAW264.7 cells by 10 min postinfection and is extranuclear in its cellular location. Substitution of catalytic residues shows that the mART function is responsible for the toxicity, and it binds NAD⁺ with high affinity (K_D = 52.3 ± 12.2 μm). We report the 2.2 Å Certhrax structure, highlighting its structural similarities and differences with anthrax lethal factor. We also determined the crystal structures of two good inhibitors (P6 (K_D = 1.7 ± 0.2 μm, K_i = 1.8 ± 0.4 μm) and PJ34 (K_D = 5.8 ± 2.6 μm, K_i = 9.6 ± 0.3 μm)) in complex with Certhrax. As with other toxins in this family, the phosphate-nicotinamide loop moves toward the NAD⁺ binding site with bound inhibitor. These results indicate that Certhrax may be important in the pathogenesis of B. cereus.