Stopped-flow Kinetic Analysis Research Articles

Structural and kinetic analysis of the monofunctional Staphylococcus aureus PBP1

Staphylococcus aureus, an ESKAPE pathogen, is a major clinical concern due to its pathogenicity and manifold antimicrobial resistance mechanisms. The commonly used β-lactam antibiotics target bacterial penicillin-binding proteins (PBPs) and inhibit crosslinking of peptidoglycan strands that comprise the bacterial cell wall mesh, initiating a cascade of effects leading to bacterial cell death. S. aureus PBP1 is involved in synthesis of the bacterial cell wall during division and its presence is essential for survival of both antibiotic susceptible and resistant S. aureus strains. Here, we present X-ray crystallographic data for S. aureus PBP1 in its apo form as well as acyl-enzyme structures with distinct classes of β-lactam antibiotics representing the penicillins, carbapenems, and cephalosporins, respectively: oxacillin, ertapenem and cephalexin. Our structural data suggest that the PBP1 active site is readily accessible for substrate, with little conformational change in key structural elements required for its covalent acylation of β-lactam inhibitors. Stopped-flow kinetic analysis and gel-based competition assays support the structural observations, with even the weakest performing β-lactams still having comparatively high acylation rates and affinities for PBP1. Our structural and kinetic analysis sheds insight into the ligand–PBP interactions that drive antibiotic efficacy against these historically useful antimicrobial targets and expands on current knowledge for future drug design and treatment of S. aureus infections.

Reductive Coupling of Nitric Oxide by Cu(I): Stepwise Formation of Mono- and Dinitrosyl Species En Route to a Cupric Hyponitrite Intermediate.

Transition-metal-mediated reductive coupling of nitric oxide (NO(g)) to nitrous oxide (N2O(g)) has significance across the fields of industrial chemistry, biochemistry, medicine, and environmental health. Herein, we elucidate a density functional theory (DFT)-supplemented mechanism of NO(g) reductive coupling at a copper-ion center, [(tmpa)CuI(MeCN)]+ (1) {tmpa = tris(2-pyridylmethyl)amine}. At -110 °C in EtOH (<-90 °C in MeOH), exposing 1 to NO(g) leads to a new binuclear hyponitrite intermediate [{(tmpa)CuII}2(μ-N2O22-)]2+ (2), exhibiting temperature-dependent irreversible isomerization to the previously characterized κ2-O,O'-trans-[(tmpa)2Cu2II(μ-N2O22-)]2+ (OOXray) complex. Complementary stopped-flow kinetic analysis of the reaction in MeOH reveals an initial mononitrosyl species [(tmpa)Cu(NO)]+ (1-(NO)) that binds a second NO molecule, forming a dinitrosyl species [(tmpa)CuII(NO)2] (1-(NO)2). The decay of 1-(NO)2 requires an available starting complex 1 to form a dicopper-dinitrosyl species hypothesized to be [{(tmpa)Cu}2(μ-NO)2]2+ (D) bearing a diamond-core motif, en route to the formation of hyponitrite intermediate 2. In contrast, exposing 1 to NO(g) in 2-MeTHF/THF (v/v 4:1) at <-80 °C leads to the newly observed transient metastable dinitrosyl species [(tmpa)CuII(NO)2] (1-(NO)2) prior to its disproportionation-mediated transformation to the nitrite product [(tmpa)CuII(NO2)]+. Our study furnishes a near-complete profile of NO(g) activation at a reduced Cu site with tripodal tetradentate ligation in two distinctly different solvents, aided by detailed spectroscopic characterization of metastable intermediates, including resonance Raman characterization of the new dinitrosyl and hyponitrite species detected.

Conformational Dynamics of Human ALKBH2 Dioxygenase in the Course of DNA Repair as Revealed by Stopped-Flow Fluorescence Spectroscopy

Elucidation of physicochemical mechanisms of enzymatic processes is one of the main tasks of modern biology. High efficiency and selectivity of enzymatic catalysis are mostly ensured by conformational dynamics of enzymes and substrates. Here, we applied a stopped-flow kinetic analysis based on fluorescent spectroscopy to investigate mechanisms of conformational transformations during the removal of alkylated bases from DNA by ALKBH2, a human homolog of Escherichia coli AlkB dioxygenase. This enzyme protects genomic DNA against various alkyl lesions through a sophisticated catalytic mechanism supported by a cofactor (Fe(II)), a cosubstrate (2-oxoglutarate), and O2. We present here a comparative study of conformational dynamics in complexes of the ALKBH2 protein with double-stranded DNA substrates containing N1-methyladenine, N3-methylcytosine, or 1,N6-ethenoadenine. By means of fluorescent labels of different types, simultaneous detection of conformational transitions in the protein globule and DNA substrate molecule was performed. Fitting of the kinetic curves by a nonlinear-regression method yielded a molecular mechanism and rate constants of its individual steps. The results shed light on overall conformational dynamics of ALKBH2 and damaged DNA during the catalytic cycle.

Analyses of pre-steady-state kinetics and isotope effects of the γ-elimination reaction catalyzed by Citrobacter freundii methionine γ-lyase

Methionine γ-lyase (MGL) is a pyridoxal 5′-phosphate–dependent enzyme catalyzing γ-elimination in l-methionine. Pyridoxal 5′-phosphate–dependent enzymes have unique spectral properties that allow to monitor sequential formation and decomposition of various intermediates via the detection of absorbance changes. The kinetic mechanism of the γ-elimination reaction catalyzed by Citrobacter freundii MGL was elucidated here by fast stopped-flow kinetic analysis. Single-wavelength detection of characteristic absorbance changes enabled us to compare transformations of intermediates in the course of the reaction with different substrates. The influence of various γ-substituents in the substrate on the formation of key intermediates was estimated. Kinetic isotope effects of α- and β-protons were determined using deuterium-substituted l-methionine. Contributions of amino acid residues Tyr113 and Tyr58 located in the active site on the formation and decomposition of reaction intermediates were identified too. α-Aminocrotonate formation is the rate-limiting step of the enzymatic γ-elimination reaction. Kinetic isotope effects strongly support concerted reaction mechanisms of transformation between an external aldimine and a ketimine intermediate as well as a ketimine intermediate and an unsaturated ketimine.

The ATPase mechanism of myosin 15, the molecular motor mutated in DFNB3 human deafness

Cochlear hair cells each possess an exquisite bundle of actin-based stereocilia that detect sound. Unconventional myosin 15 (MYO15) traffics and delivers critical molecules required for stereocilia development and thus is essential for building the mechanosensory hair bundle. Mutations in the human MYO15A gene interfere with stereocilia trafficking and cause hereditary hearing loss, DFNB3, but the impact of these mutations is not known, as MYO15 itself is poorly characterized. To learn more, we performed a kinetic study of the ATPase motor domain to characterize its mechanochemical cycle. Using the baculovirus–Sf9 system, we purified a recombinant minimal motor domain (S1) by coexpressing the mouse MYO15 ATPase, essential and regulatory light chains that bind its IQ domains, and UNC45 and HSP90A chaperones required for correct folding of the ATPase. MYO15 purified with either UNC45A or UNC45B coexpression had similar ATPase activities (kcat = ∼ 6 s−1 at 20 °C). Using stopped-flow and quenched-flow transient kinetic analyses, we measured the major rate constants describing the ATPase cycle, including ATP, ADP, and actin binding; hydrolysis; and phosphate release. Actin-attached ADP release was the slowest measured transition (∼12 s−1 at 20 °C), although this did not rate-limit the ATPase cycle. The kinetic analysis shows the MYO15 motor domain has a moderate duty ratio (∼0.5) and weak thermodynamic coupling between ADP and actin binding. These findings are consistent with MYO15 being kinetically adapted for processive motility when oligomerized. Our kinetic characterization enables future studies into how deafness-causing mutations affect MYO15 and disrupt stereocilia trafficking necessary for hearing.

Identification of the viral RNA promoter stem loop A (SLA)-binding site on Zika virus polymerase NS5

Zika virus has recently emerged as an important human pathogen that has spread to more than 60 countries. Infection of a pregnant woman with Zika virus can cause severe brain malformations in the child such as microcephaly and other birth defects. Despite the medical importance of Zika virus infection, the mechanism of viral replication, a process commonly targeted by antiviral therapeutics, is not well understood. Stem-loop A (SLA), located in the 5′ untranslated region of the viral genome, acts as a promotor for viral replication and thus is critical for recognition of the viral genome by the viral polymerase NS5. However, how NS5 engages SLA is not clear. We have quantitatively examined the intrinsic affinities between Zika virus SLA and NS5, and identified the SLA-binding site on NS5. Amino acid substitutions in the thumb subdomain of the RNA-dependent RNA polymerase (RdRp) and the methyltransferase (MTase) domain reduced SLA-binding affinity, indicating that they each are part of the SLA-binding site. Furthermore, stopped-flow kinetic analysis of Zika NS5-, RdRp- and MTase–SLA interactions identified distinct intermediates during NS5 and SLA complex formation. These data suggest a model for SLA recognition and the initiation of flaviviral replication by NS5.

Structural insights into the catalytic mechanism of cysteine (hydroxyl) lyase from the hydrogen sulfide-producing oral pathogen, Fusobacterium nucleatum.

Hydrogen sulfide (H2S) plays important roles in the pathogenesis of periodontitis. Oral pathogens typically produce H2S from l-cysteine in addition to pyruvate and [Formula: see text] However, fn1055 from Fusobacterium nucleatum subsp. nucleatum ATCC 25586 encodes a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the production of H2S and l-serine from l-cysteine and H2O, an unusual cysteine (hydroxyl) lyase reaction (β-replacement reaction). To reveal the reaction mechanism, the crystal structure of substrate-free Fn1055 was determined. Based on this structure, a model of the l-cysteine-PLP Schiff base suggested that the thiol group forms hydrogen bonds with Asp232 and Ser74, and the substrate α-carboxylate interacts with Thr73 and Gln147 Asp232 is a unique residue to Fn1055 and its substitution to asparagine (D232N) resulted in almost complete loss of β-replacement activity. The D232N structure obtained in the presence of l-cysteine contained the α-aminoacrylate-PLP Schiff base in the active site, indicating that Asp232 is essential for the addition of water to the α-aminoacrylate to produce the l-serine-PLP Schiff base. Rapid-scan stopped-flow kinetic analyses showed an accumulation of the α-aminoacrylate intermediate during the reaction cycle, suggesting that water addition mediated by Asp232 is the rate-limiting step. In contrast, mutants containing substitutions of other active-site residues (Ser74, Thr73, and Gln147) exhibited reduced β-replacement activity by more than 100-fold. Finally, based on the structural and biochemical analyses, we propose a mechanism of the cysteine (hydroxyl) lyase reaction by Fn1055. The present study leads to elucidation of the H2S-producing mechanism in F. nucleatum.

Acid-Triggered O-O Bond Heterolysis of a Nonheme FeIII (OOH) Species for the Stereospecific Hydroxylation of Strong C-H Bonds.

A novel hydroperoxoiron(III) species [FeIII (OOH)(MeCN)(PyNMe3 )]2+ (3) has been generated by reaction of its ferrous precursor [FeII (CF3 SO3 )2 (PyNMe3 )] (1) with hydrogen peroxide at low temperatures. This species has been characterized by several spectroscopic techniques and cryospray mass spectrometry. Similar to most of the previously described low-spin hydroperoxoiron(III) compounds, 3 behaves as a sluggish oxidant and it is not kinetically competent for breaking weak C-H bonds. However, triflic acid addition to 3 causes its transformation into a much more reactive compound towards organic substrates that is capable of oxidizing unactivated C-H bonds with high stereospecificity. Stopped-flow kinetic analyses and theoretical studies provide a rationale for the observed chemistry, a triflic-acid-assisted heterolytic cleavage of the O-O bond to form a putative strongly oxidizing oxoiron(V) species. This mechanism is reminiscent to that observed in heme systems, where protonation of the hydroperoxo intermediate leads to the formation of the high-valent [(Porph. )FeIV (O)] (Compound I).

Structural Identification and Kinetic Analysis of the in Vitro Products Formed by Reaction of Bisphenol A-3,4-quinone with N-Acetylcysteine and Glutathione.

Bisphenol A (BPA) has received considerable attention as an endocrine disrupting chemical and a possible substrate for genotoxic metabolites. BPA metabolism leads to formation of electrophilic o-quinones cable of binding to DNA and other endogenous nucleophiles. We have structurally identified the products resulting from the reaction of bisphenol A-3,4-quinone (BPAQ) with N-acetylcysteine (NAC) and glutathione (GSH). The major and minor isomers are both the result of 1,6-conjugate addition and are produced almost instantly in high yield. Reactions using 1.3 equiv of GSH showed the presence of a bis-glutathionyl adduct which was not observed using higher GSH concentration relative to BPAQ. NAC reactions with BPAQ showed no bis-N-acetylcysteinyl adducts. Stopped-flow kinetic analysis reveals the 1,6-conjugate additions to be reversible with a forward free energy of activation of 9.2 and 7.8 kcal/mol for the NAC and GSH reactions, respectively. The bimolecular forward rate constant at 19.4 °C was approximately three time faster for GSH compared to NAC, 1547 vs 496 M-1 s-1. The free energy of activation for the reverse reactions were similar, 11.7 and 11.2 kcal/mol for NAC and GSH, respectively. We plan to use this model system to further explore the mechanism of adduct formation between sulfur nucleophiles and o-quinones and the resulting chemical properties of both NAC and GSH adducts.

New insights into the mechanism of nickel superoxide degradation from studies of model peptides

A series of small, catalytically active metallopeptides, which were derived from the nickel superoxide dismutase (NiSOD) active site were employed to study the mechanism of superoxide degradation especially focusing on the role of the axial imidazole ligand. In the literature, there are contradicting propositions about the catalytic importance of the N-terminal histidine. Therefore, we studied the stability and activity of a set of eight NiSOD model peptides, which represent the major model systems discussed in the literature to date, yet differing in their length and their Ni-coordination. UV-Vis-coupled stopped-flow kinetic measurements and mass spectrometry analysis unveiled their high oxidation sensitivity in the presence of oxygen and superoxide resulting into a much faster Ni(II)-peptide degradation for the amine/amide Ni(II) coordination than for the catalytically inactive bis-amidate Ni(II) coordination. With respect to these results we determined the catalytic activities for all NiSOD mimics studied herein, which turned out to be in almost the same range of about 2 × 106 M−1 s−1. From these experiments, we concluded that the amine/amide Ni(II) coordination is clearly the key factor for catalytic activity. Finally, we were able to clarify the role of the N-terminal histidine and to resolve the contradictory literature propositions, reported in previous studies.

Switching between Inner- and Outer-Sphere PCET Mechanisms of Small-Molecule Activation: Superoxide Dismutation and Oxygen/Superoxide Reduction Reactivity Deriving from the Same Manganese Complex.

Readily exchangeable water molecules are commonly found in the active sites of oxidoreductases, yet the overwhelming majority of studies on small-molecule mimics of these enzymes entirely ignores the contribution of water to the reactivity. Studies of how these enzymes can continue to function in spite of the presence of highly oxidizing species are likewise limited. The mononuclear MnII complex with the potentially hexadentate ligand N-(2-hydroxy-5-methylbenzyl)-N,N',N'-tris(2-pyridinylmethyl)-1,2-ethanediamine (LOH) was previously found to act as both a H2O2-responsive MRI contrast agent and a mimic of superoxide dismutase (SOD). Here, we studied this complex in aqueous solutions at different pH values in order to determine its (i) acid-base equilibria, (ii) coordination equilibria, (iii) substitution lability and operative mechanisms for water exchange, (iv) redox behavior and ability to participate in proton-coupled electron transfer (PCET) reactions, (v) SOD activity and reductive activity toward both oxygen and superoxide, and (vi) mechanism for its transformation into the binuclear MnII complex with (H)OL-LOH and its hydroxylated derivatives. The conclusions drawn from potentiometric titrations, low-temperature mass spectrometry, temperature- and pressure-dependent 17O NMR spectroscopy, electrochemistry, stopped-flow kinetic analyses, and EPR measurements were supported by the structural characterization and quantum chemical analysis of proposed intermediate species. These comprehensive studies enabled us to determine how transiently bound water molecules impact the rate and mechanism of SOD catalysis. Metal-bound water molecules facilitate the PCET necessary for outer-sphere SOD activity. The absence of the water ligand, conversely, enables the inner-sphere reduction of both superoxide and dioxygen. The LOH complex maintains its SOD activity in the presence of •OH and MnIV-oxo species by channeling these oxidants toward the synthesis of a functionally equivalent binuclear MnII species.

Stopped-flow kinetic analysis of the oxidation of semicarbazide by hexachloroiridate(IV)

A kinetic analysis of the oxidation of semicarbazide (SEM) by the single-electron oxidant [IrCl6]2− has been carried out by stopped-flow spectrometric techniques. The reaction proved to be first order each in [IrCl6 2−] and [SEM]tot, leading to overall second-order kinetics. The variation in the observed second-order rate constant k′ with pH was explored over the pH range of 0–7.11. Spectrophotometric titration revealed a stoichiometry of Δ[IrCl6 2−]/Δ[SEM]tot = 4:1 for the redox reaction. On the basis of the rate law, the redox stoichiometry, and the rapid scan spectra, a reaction mechanism is proposed which involves parallel attacks of [IrCl6]2− on both H2NCONHNH3 + and H2NCONHNH2 as rate-determining steps, followed by several rapid reactions. The rate expression, derived from the reaction mechanism, was utilized to simulate the k′–pH profile yielding a virtually perfect fit and indicating that the reaction path involving H2NCONHNH3 + does not make a significant contribution to the overall rate. The reaction between [IrCl6]2− and H2NCONHNH2 was further studied as a function of both temperature and ionic strength. From the temperature dependence, activation parameters were obtained as: ∆H 2 ‡  = 34.9 ± 1.5 kJ mol−1 and ∆S 2 ‡  = −78 ± 5 J K−1 mol−1. The observed ionic strength dependence suggests that the rate-determining step is between [IrCl6]2− and a neutral species of SEM. Hence, both the temperature and ionic strength dependency studies are in good agreement with the proposed reaction mechanism, in which the rate-determining step involves an outer sphere electron transfer.

Effect of Allosteric Mutations on Conformational Equilibrium of DHFR

Allosteric mutations, distal from the binding site of DHFR from B. stearothermophilus, were previously found to alter the ligand binding specificity. In this work, we investigate how the structural conformational equilibrium of DHFR is affected when one of these mutations is introduced to the enzyme. H38N and Y127A mutations are introduced to previously developed, fluorescently labeled, conformationally-sensitive Bs DHFR (C73A/S131CMDCC DHFR). Stopped-flow kinetic analysis is currently used to study the kinetics of protein conformational motions associated with methotrexate (MTX) binding. This approach allows us to examine the conformational changes associated with MTX binding for the mutant and compares them to the data for the wild type enzyme. The data will also reveal information about the conformational equilibrium preceding ligand binding. Understanding of the effect of these distal mutations on the conformational equilibrium will provide insights on the role of allosteric regions in ligand binding specificity and inform design and repurposing of selective DHFR drug compounds

Translational control 1995-2015: unveiling molecular underpinnings and roles in human biology.

The last 20 years have seen tremendous progress in deciphering the fundamental mechanisms of protein synthesis and molecular pathways of translational control, and in implicating these processes in development, neurobiology, and etiologyof human diseases. Muchof this success can be attributed to the introduction of new technologies, such as next-generation RNA sequencing and advances in cryo-EM; and also to development of powerful in vitro systems recapitulating complex in vivo phenomena with purified components. Also critical have been strategic interactions between experts in different disciplines of biology, such as the wedding of biochemistry with genetics and structural biology, and of translation biochemistry with neurobiology and medicine. RNA has contributed to this progress by publishing key findings on molecular mechanisms of protein synthesis and the role of translational control in regulating gene expression in diverse organisms. Thebasic reactions of the elongationand termination stages of protein synthesis are highly conserved throughout all three kingdoms of life. Because they occur in the catalytic center of the ribosome, their molecular mechanisms were illuminated by the stunning successes of X-ray crystallography and cryo-electron microscopy in producing high-resolution structures of both empty ribosomes and a myriad of ribosome complexes with mRNA, tRNAs, or elongation, termination, or ribosome recycling factors trapped in different stages of the process. Recent innovations in cryo-EM technology are revealing ribosome structures at nearly 3 A resolution; and new algorithms for sorting structurally distinct molecules means that sample heterogeneity (an anathema to crystallography) is now exploited to identify different conformational states in the same preparation. The solved ribosome structures revealed the path of mRNA and the A, P, and E binding sites for the tRNA, as it progress through the elongation cycle from aminoacyltRNA, to peptidyl-tRNA to deacylated tRNA, located at the interface between the large and small subunits; as well as the peptidyl transferase center wherepeptide bond formation occurs. As the decoding center of the ribosome is comprised almost entirely of rRNA, it became clear that the ribosome is a ribozyme. The protein factors involved in translocation of mRNA and the tRNAs through the decoding sites (EF-G/ EF2), in recognizing stop codons and hydrolyzing peptidyltRNA (RF1/eRF1), and splitting apart ribosomal subunits following termination (bacterial RRF) all exploit striking structural mimicry of tRNA to fit into the A site. Interestingly, RF1 was shown to contain a protein element that “reads” the stop codon and another that reaches into the peptidyl transferase center to hydrolyze the peptidyl-tRNA linkage. The peptidyl-tRNA and aminoacyl-tRNA are anchored in the precise locations required for peptide bond formation, but the ribosome is not simply a rigid catalytic platform, but undergoes conformational changes in selecting the correct EF1A·GTP·aminoacyl-tRNA ternary complex for each codon, and displays inter-subunit ratcheting during translocation and termination. GTP binding and hydrolysis by the elongation factors evoke cyclic conformational changes that drive the process forward. The use of fluorescently labeled reactants and stopped-flow kinetic analysis allowed a detailed dissection of different steps in the processes of aminoacyltRNA selection in the A site, with proofreading of the codon:anticodon interaction; and of translocation of tRNAs through the decoding center catalyzed by EF2. Single-molecule approaches also contributed importantly to this progress, by identifying intermediate states too transient to be detected by ensemble kinetic experiments or structural biology. In other developments, eIF5A and its bacterial counterpart EF-P were shown to stimulate elongation specifically through stretches of proline codons, and both factors occupy the E-site and communicate directly with the peptidyl transferase center. Interestingly, RRF is replaced in eukaryotes and archaea by an ATP-binding cassette protein, ABCE1/ Rli1, that becomes wedged between the large and small subunits and uses ATP hydrolysis to split apart the subunits. ABCE1/Rli1also functions with RF1 paralog Dom34 to

Reduction of ormaplatin by a dithiol model compound for the active site of thioredoxin: stopped-flow kinetic analysis

Ormaplatin ([Pt(dach)Cl4]) is a prototypical of Pt(IV) anticancer prodrug; its reduction by 3,6-dioxa-1,8-octanedithiol (DODT, a model compound for the active site of thioredoxin), has been characterized at 25.0 °C and in a wide pH range by rapid scan spectral and kinetic analysis. The reduction follows overall second-order kinetics, being first-order each in [Pt(IV)] and in [DODT]; the observed second-order constant k′ versus pH profile has been established. A 1:1 reaction stoichiometry is obtained, indicating that DODT is oxidized to its intramolecular disulfide; these characteristics are consistent with the redox properties of thioredoxin. A reaction mechanism has been proposed which involves the three protolytic species of DODT attacking [Pt(dach)Cl4] in parallel as rate-determining steps. The rate equation is derived from the reaction mechanism and rate constants of the rate-determining steps have been evaluated. The thiol–thiolate and dithiolate forms of DODT are 105–106 times more reactive than the dithiol form. Comparisons of the rate constants highlight that the electron density of the sulfur atom plays a key role in determining the reactivity of DODT toward ormaplatin and similar Pt(IV) compounds. It is concluded that the reduced thioredoxin is a strong competitor to other thiol-containing compounds in the reduction processes of Pt(IV) anticancer prodrugs.

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.

Human immunoglobulin E flexes between acutely bent and extended conformations.

Crystallographic and solution studies have shown that IgE molecules are acutely bent in their Fc region. Crystal structures reveal the Cε2 domain pair folded back onto the Cε3-Cε4 domains, but is the molecule exclusively bent or can the Cε2 domains adopt extended conformations and even “flip” from one side of the molecule to the other? We report the crystal structure of IgE-Fc captured in a fully extended, symmetrical conformation and show by molecular dynamics, calorimetry, stopped-flow kinetic, SPR and FRET analyses, that the antibody can indeed adopt such extended conformations in solution. This diversity of conformational states available to IgE-Fc offers a new perspective on IgE function in allergen recognition, as part of the B cell receptor and as a therapeutic target in allergic disease.

Lipopolysaccharide-induced Lung Injury Involves the Nitration-mediated Activation of RhoA

Acute lung injury (ALI) is characterized by increased endothelial hyperpermeability. Protein nitration is involved in the endothelial barrier dysfunction in LPS-exposed mice. However, the nitrated proteins involved in this process have not been identified. The activation of the small GTPase RhoA is a critical event in the barrier disruption associated with LPS. Thus, in this study we evaluated the possible role of RhoA nitration in this process. Mass spectroscopy identified a single nitration site, located at Tyr(34) in RhoA. Tyr(34) is located within the switch I region adjacent to the nucleotide-binding site. Utilizing this structure, we developed a peptide designated NipR1 (nitration inhibitory peptide for RhoA 1) to shield Tyr(34) against nitration. TAT-fused NipR1 attenuated RhoA nitration and barrier disruption in LPS-challenged human lung microvascular endothelial cells. Further, treatment of mice with NipR1 attenuated vessel leakage and inflammatory cell infiltration and preserved lung function in a mouse model of ALI. Molecular dynamics simulations suggested that the mechanism by which Tyr(34) nitration stimulates RhoA activity was through a decrease in GDP binding to the protein caused by a conformational change within a region of Switch I, mimicking the conformational shift observed when RhoA is bound to a guanine nucleotide exchange factor. Stopped flow kinetic analysis was used to confirm this prediction. Thus, we have identified a new mechanism of nitration-mediated RhoA activation involved in LPS-mediated endothelial barrier dysfunction and show the potential utility of "shielding" peptides to prevent RhoA nitration in the management of ALI.

Human IgE flips between two acutely bent structuresviaan ensemble of extended conformations

Immunoglobulin E (IgE) antibodies mediate allergic reactions, and their binding to FcaRI is responsible for longterm sensitisation of mast cells and basophils [1]. Disruption of this interaction is a validated strategy for therapeutic intervention in allergic diseases including asthma [2]. IgE is known to display an acutely and asymmetrically bent conformation in the Fc region through which it binds to FcaRI; this bend becomes even more acute upon receptor engagement, as shown both crystallographically and in solution [3-7]. We report the crystal structure of a complex formed between IgE-Fc and two bound Fab fragments of a inhibitory anti-IgE antibody, and show that IgE-Fc can also adopt a totally extended conformation. The IgE-Fc has adopted a completely symmetrical conformation that requires an “unbending” of approximately 120° from the previously characterised structure. Molecular dynamics simulation reveals a series of stable conformations for free IgE-Fc that suggest a pathway from the acutely bent crystal structure through stable, extended conformations close to that seen in the Fab complex. We show by ITC, stoppedflow kinetic and FRET analyses that IgE-Fc adopts extended conformations in solution, and that these are an intrinsic property of IgEFc, not induced by Fab binding. We propose that IgE-Fc passes through these extended conformations as it flips between two bent conformations in which the CΣ2 domains fold back on opposite faces of the CΣ3-CΣ4 domains. The ability of IgE to exist in both bent and extended conformations may be essential for allergen recognition by IgE-Fc when bound to FcΣRI on the surface of mast cells, and as the B cell receptor respectively. Understanding the full range of conformations accessible to the free IgE molecule is also key to developing IgE-targeted therapies for allergic disease.

Mechanism of Inducible Nitric-oxide Synthase Dimerization Inhibition by Novel Pyrimidine Imidazoles

Overproduction of nitric oxide (NO) by inducible nitric-oxide synthase (iNOS) has been etiologically linked to several inflammatory, immunological, and neurodegenerative diseases. As dimerization of NOS is required for its activity, several dimerization inhibitors, including pyrimidine imidazoles, are being evaluated for therapeutic inhibition of iNOS. However, the precise mechanism of their action is still unclear. Here, we examined the mechanism of iNOS inhibition by a pyrimidine imidazole core compound and its derivative (PID), having low cellular toxicity and high affinity for iNOS, using rapid stopped-flow kinetic, gel filtration, and spectrophotometric analysis. PID bound to iNOS heme to generate an irreversible PID-iNOS monomer complex that could not be converted to active dimers by tetrahydrobiopterin (H4B) and l-arginine (Arg). We utilized the iNOS oxygenase domain (iNOSoxy) and two monomeric mutants whose dimerization could be induced (K82AiNOSoxy) or not induced (D92AiNOSoxy) with H4B to elucidate the kinetics of PID binding to the iNOS monomer and dimer. We observed that the apparent PID affinity for the monomer was 11 times higher than the dimer. PID binding rate was also sensitive to H4B and Arg site occupancy. PID could also interact with nascent iNOS monomers in iNOS-synthesizing RAW cells, to prevent their post-translational dimerization, and it also caused irreversible monomerization of active iNOS dimers thereby accomplishing complete physiological inhibition of iNOS. Thus, our study establishes PID as a versatile iNOS inhibitor and therefore a potential in vivo tool for examining the causal role of iNOS in diseases associated with its overexpression as well as therapeutic control of such diseases.