Structural investigation of the GlmS ribozyme bound to Its catalytic cofactor

The GlmS riboswitch is located in the 5'-untranslated region of the gene encoding glucosamine-6-phosphate (GlcN6P) synthetase. The GlmS riboswitch is a ribozyme with activity triggered by binding of the metabolite GlcN6P. Presented here is the structure of the GlmS ribozyme (2.5 A resolution) with GlcN6P bound in the active site. The GlmS ribozyme adopts a compact double pseudoknot tertiary structure, with two closely packed helical stacks. Recognition of GlcN6P is achieved through coordination of the phosphate moiety by two hydrated magnesium ions as well as specific nucleobase contacts to the GlcN6P sugar ring. Comparison of this activator bound and the previously published apoenzyme complex supports a model in which GlcN6P does not induce a conformational change in the RNA, as is typical of other riboswitches, but instead functions as a catalytic cofactor for the reaction. This demonstrates that RNA, like protein enzymes, can employ the chemical diversity of small molecules to promote catalytic activity.     

Kinetic measurements of phosphoglucose isomerase and phosphomannose isomerase by direct analysis of phosphorylated aldose–ketose isomers using tandem mass spectrometry

A mass spectrometry based method for the direct determination of kinetic constants for phosphoglucose isomerase (PGI) and phosphomannose isomerase (PMI) is described. PGI catalyzes the interconversion between glucose-6-phosphate (Glc6P) and fructose-6-phosphate (Fru6P) and PMI performs the same function between mannose-6-phosphate (Man6P) and Fru6P. These two enzymes are essential in the pathways of glycolytic or oxidative metabolism of carbohydrates and have been considered as potential therapeutic targets. Traditionally, they are assayed either by spectrophotometric detection of Glc6P with one or more coupling enzymes or by a colorimetric detection of Fru6P. However, no suitable assay for Man6P has been developed yet to study the reaction of PMI in the direction from Fru6P to Man6P. In the work presented herein, a general assay for the isomeric substrate-product pair between Glc6P and Fru6P or between Man6P and Fru6P was developed, with the aim of directly studying the kinetics of PGI and PMI in both directions. The 6-phosphorylated aldose and ketose isomers were distinguished based on their corresponding tandem mass spectra (MS²) obtained on a quadrupole ion trap mass spectrometer, and a multicomponent quantification method was utilized to determine the composition of binary mixtures. Using this method, the conversion between Fru6P and Glc6P and that between Fru6P and Man6P are directly monitored. The equilibrium constants for the reversible reactions catalyzed by PGI and PMI are measured to be 0.3 and 1.1, respectively, and the kinetic parameters for both substrates of PGI and PMI are also determined. The values of K_M and V_max for Fru6P as substrate of PMI are reported to be 0.15 mM and 7.78 μmol/(min mg), respectively. All other kinetic parameters measured correlate well with those obtained using traditional methods, demonstrating the accuracy and reliability of this assay.     

Synthesis and evaluation of glucosamine-6-phosphate analogues as activators of glmS riboswitch

The glmS riboswitch is a ribozyme found in numerous Gram-positive bacteria and responds to the cellular concentrations of glucosamine 6-phosphate (GlcN6P). Given the importance of GlcN6P for cell wall biosynthesis, the glmS riboswitch has become a new drug target for the development of antibiotics. Herein, we describe the efficient synthesis of three GlcN6P analogues and their evaluation on inducing self-cleavage of the glmS riboswitch from Bacillus subtilis. Our results provide valuable information for further elucidation of the structure-activity relationships and drug design for glmS riboswitch antibiotics.     

Ligand requirements for glmS ribozyme self-cleavage

Natural RNA catalysts (ribozymes) perform essential reactions in biological RNA processing and protein synthesis, whereby catalysis is intrinsic to RNA structure alone or in combination with metal ion cofactors. The recently discovered glmS ribozyme is unique in that it functions as a glucosamine-6-phosphate (GlcN6P)-dependent catalyst believed to enable "riboswitch" regulation of amino-sugar biosynthesis in certain prokaryotes. However, it is unclear whether GlcN6P functions as an effector or coenzyme to promote ribozyme self-cleavage. Herein, we demonstrate that ligand is absolutely requisite for glmS ribozyme self-cleavage activity. Furthermore, catalysis both requires and is dependent upon the acid dissociation constant (pKa) of the amine functionality of GlcN6P and related compounds. The data demonstrate that ligand is integral to catalysis, consistent with a coenzyme role for GlcN6P and illustrating an expanded capacity for biological RNA catalysis.     

Automated Assay for Fructose 2,6-Bisphosphate

Abstract

An automated procedure for the determination of Fructose 2,6-bisphosphate (Fru 2,6-P₂) is described. The method based on the degree of activation of the enzyme Pyrophosphate: D-fructose 6-phosphate 1-phosphotransferase from potato tuber by Fru 2,6-P₂, uses a discrete-sample automatic analyzer to perform the procedure and to monitor the spectrophotometric changes. The detection limit of Fru 2,6-P₂ levels is 0.016 pmol (6.25 × 10^?14 mol ml^?1). The coefficients of variation observed when assaying 0.5 nM of Fru 2,6-P₂ in the reaction mixture, 1.34% and 7.39% (n=10) for the automated and manual method are significantly different. Thus a 5-fold higher precision is shown by the automatic method. The same precision is observed when assaying biological samples from starved or refed animals.

In conclusion the assay is as sensitive as the manual spectrophotometric method previously described, but it shows higher precision and decreases the processing time allowing routine determinations. It is specially useful when purifying 6-phosphofructo 2-kinase or assaying a large number of tissue samples for Fru 2,6-P₂.     

The glmS ribozyme tunes the catalytically critical pK(a) of its coenzyme glucosamine-6-phosphate

The glmS ribozyme riboswitch is the first known natural catalytic RNA that employs a small-molecule cofactor. Binding of glucosamine-6-phosphate (GlcN6P) uncovers the latent self-cleavage activity of the RNA, which adopts a catalytically competent conformation that is nonetheless inactive in the absence of GlcN6P. Structural and analogue studies suggest that the amine of GlcN6P functions as a general acid-base catalyst, while its phosphate is important for binding affinity. However, the solution pK(a) of the amine is 8.06 ± 0.05, which is not optimal for proton transfer. Here we used Raman crystallography directly to determine the pK(a)'s of GlcN6P bound to the glmS ribozyme. Binding to the RNA lowers the pK(a) of the amine of GlcN6P to 7.26 ± 0.09 and raises the pK(a) of its phosphate to 6.35 ± 0.09. Remarkably, the pK(a)'s of these two functional groups are unchanged from their values for free GlcN6P (8.06 ± 0.05 and 5.98 ± 0.05, respectively) when GlcN6P binds to the catalytically inactive but structurally unperturbed G40A mutant of the ribozyme, thus implicating the ribozyme active site guanine in pK(a) tuning. This is the first demonstration that a ribozyme can tune the pK(a) of a small-molecule ligand. Moreover, the anionic glmS ribozyme in effect stabilizes the neutral amine of GlcN6P by lowering its pK(a). This is unprecedented and illustrates the chemical sophistication of ribozyme active sites.     

Effects of Temperature and Energy on Stability of Oligomeric Enzyme Probed on Electrospray Ionization Mass Spectrometry

Escherichia coli 3-Deoxy-D-manno-octulosonate 8-phosphate（KDO8P） synthase catalyzed the condensation reaction between D-arabinose 5-phosphate（A5P） and phosphoenolpyruvate（PEP） to form KDO8P and inorganic phosphate（Pi）. The noncovalent tetrameric association ofKDO8P synthase was observed and dissociated in gas phase by means of electrospray ionization mass spectrometry under the very ＂soft＂ conditions. The results indicate that PEP-bound enzyme generated abundant tetrameric species as well as monomeric species at the ＂soft＂ conditions, whereas, the unbound enzyme favored the formation of a dimeric species. The mass spectra of the mixture of the enzyme with one of substrates, PEP, and A5P or one of products, KDO8P and Pi show that the complex of the unbound enzyme with PEP or Pi was prone to the formation of a monomeric species, whereas, that of the unbound enzyme with A5P or KDO8P was similar to the unbound enzyme. The intensity of the dimeric species increased with the increase of temperature at a collision voltage of 10 V. Taken together, the results presented here suggest that mass spectrometry will be a powerful tool to explore subtile conformational changes and/or subunit-subunit interactions of multiprotein assembly induced by ligand-binding and/or the changes of environmental conditions.     

Identification of noncovalent dimeric complexes of the recombinant murine S100 protein CP10 by electrospray ionization mass spectrometry and chemical cross-linking

CP10 is a chemotactic S100 protein expressed by murine myeloid cells. A specific noncovalently linked dimeric complex of recombinant Ala₄₃ CP10 was identified after electrospray ionization mass spectrometry using a nondenaturing solvent of 5-mM ammonium acetate (pH 6. 5) and source temperature of 50°C. With a low cone voltage (75 V), major ions were observed at ～2075, 2305, and 2613 Da, which were attributed to partially desolvated multiply charged noncovalently linked dimeric species (+10, +9, and +8 charge states, respectively). Deconvolution produced a broad peak centered around 20750 Da corresponding to the approximate mass of dimeric recombinant Ala₄₃ CP10. Increasing the cone voltage, and collisionally activating the complex, gradually reduced the intensity of these dimeric ions, with a concomitant increase in higher and lower charge state monomeric ions. The intensities of these dimeric ions were greatly reduced in spectra recorded with a source temperature of 140°C and cone voltage of 75 V, indicating a thermally unstable noncovalent complex. Similar spectra were obtained using recombinant CP10. Specific noncovalent S100 dimeric complexes were confirmed by chemically cross-linking recombinant Ala₄₃ CP10 or CP10 with bis (sulfosuccinimidyl) suberate, followed by SDS/PAGE. The dominant silver-stained and CP10-immunoreactive component migrated at 20,000 suggesting that the complex represents the major isoform in solution.     

Protein-metal interactions of calmodulin and α-synuclein monitored by selective noncovalent adduct protein probing mass spectrometry

The metal binding properties of proteins are biologically significant, particularly in relationship to the molecular origins of disease and the discovery of therapeutic pharmaceutical treatments. Herein, we demonstrate that selective noncovalent adduct protein probing mass spectrometry (SNAPP-MS) is a sensitive technique to investigate the structural effects of protein-metal interactions. We utilize specific, noncovalent interactions between 18-crown-6 ether (18C6) and lysine to probe protein structure in the presence and absence of metal ions. Application of SNAPP-MS to the calmodulin-Ca²⁺ system demonstrates that changes in protein structure are reflected in a substantial change in the number and intensity of 18C6s, which bind to the protein as observed by MS. In this manner, SNAPP is demonstrated to be a sensitive technique for monitoring ligand-induced conformational rearrangements in proteins. In addition, SNAPP is well-suited to examine the properties of natively unfolded proteins, where structural changes are more difficult to detect by other methods. For example, α-synuclein is a protein associated in the pathology of Parkinson’s disease, which is known to aggregate more rapidly in the presence of Al³⁺ and Cu²⁺. The 18C6 SNAPP distributions for α-synuclein change dramatically in the presence of 3 μM Al³⁺, revealing that Al³⁺ binding causes a significant change in the conformational dynamics of the monomeric form of this disordered protein. In contrast, binding of Cu²⁺ does not induce a significant shift in 18C6 binding, suggesting that noteworthy structural reorganizations at the monomeric level are minimal. These results are consistent with the idea that the metal-induced aggregation caused by Al³⁺ and Cu²⁺ proceed by independent pathways.     

Effect of the Substituents of the Neighboring Ring in the Conformational Equilibrium of Iduronate in Heparin‐like Trisaccharides

Based on the structure of the regular heparin, we have prepared a smart library of heparin‐like trisaccharides by incorporating some sulfate groups in the sequence α‐D ‐GlcNS‐ (1‐4)‐α‐L ‐Ido2S‐(1‐4)‐α‐D ‐GlcN. According to the 3D structure of heparin, which features one helix turn every four residues, this fragment corresponds to the minimum binding motif. We have performed a complete NMR study and found that the trisaccharides have a similar 3D structure to regular heparin itself, but their spectral properties are such that allow to extract very detailed information about distances and coupling constants as they are isotropic molecules. The characteristic conformational equilibrium of the central iduronate ring has been analyzed combining NMR and molecular dynamics and the populations of the conformers of the central iduronate ring have been calculated. We have found that in those compounds lacking the sulfate group at position 6 of the reducing end glucosamine, the population of ²S₀ of the central iduronate residue is sensitive to the temperature decreasing to 19 % at 278 K. On the contrary, the trisaccharides with 6‐O‐sulfate in the reducing end glucosamine keep the level of population constant with temperature circa 40 % of ²S₀ similar to that observed at room temperature. Another structural feature that has been revealed through this analysis is the larger flexibility of the L ‐IdoAS‐ D ‐GlcN glycosidic linkage, compared with the D ‐GlcNS‐L ‐IdoA. We propose that this is the point where the heparin chain is bended to form structures far from the regular helix known as kink that have been proposed to play an important role in the specificity of the heparin–protein interaction.     

Monomer-Dimer Control and Crystal Engineering in TASPs

We have reported a template assembled synthetic protein (cavitein?Q4) as an unexpected dimer in the solid state and as a monomer-dimer equilibrium in solution. We have since reported an ability to bias a cavitein's monomer-dimer equilibrium in solution by sequence design involving histidine metal chelation or disulfide incorporation. However, little remains known about the forces contributing to dimeric cavitein crystal nucleation and lattice stabilization. We, therefore, designed glutamine variants to probe factors involved in dimeric cavitein crystallization. It was found that a key glutamate hydrogen-bonding interaction between dimers is integral to crystal formation and stabilization. Additionally, we obtained a crystal structure of a cavitein (Q4-E3H) designed to bias the dimeric structure via histidine metal coordination. The resolved structure indicates a histidine cluster interaction that likely accounts for the biased dimeric form observed in solution.     

Monomer–Dimer Control and Crystal Engineering in TASPs

We have reported a template assembled synthetic protein (cavitein Q4) as an unexpected dimer in the solid state and as a monomer–dimer equilibrium in solution. We have since reported an ability to bias a cavitein’s monomer–dimer equilibrium in solution by sequence design involving histidine metal chelation or disulfide incorporation. However, little remains known about the forces contributing to dimeric cavitein crystal nucleation and lattice stabilization. We, therefore, designed glutamine variants to probe factors involved in dimeric cavitein crystallization. It was found that a key glutamate hydrogen-bonding interaction between dimers is integral to crystal formation and stabilization. Additionally, we obtained a crystal structure of a cavitein (Q4-E3H) designed to bias the dimeric structure via histidine metal coordination. The resolved structure indicates a histidine cluster interaction that likely accounts for the biased dimeric form observed in solution.     

Ultrafast quenching of tryptophan fluorescence in proteins: Interresidue and intrahelical electron transfer

Quenching of tryptophan fluorescence in proteins has been critical to the understanding of protein dynamics and enzyme reactions using tryptophan as a molecular optical probe. We report here our systematic examinations of potential quenching residues with more than 40 proteins. With site-directed mutation, we placed tryptophan to desired positions or altered its neighboring residues to screen quenching groups among 20 amino acid residues and of peptide backbones. With femtosecond resolution, we observed the ultrafast quenching dynamics within 100 ps and identified two ultrafast quenching groups, the carbonyl- and sulfur-containing residues. The former is glutamine and glutamate residues and the later is disulfide bond and cysteine residue. The quenching by the peptide-bond carbonyl group as well as other potential residues mostly occurs in longer than 100 ps. These ultrafast quenching dynamics occur at van der Waals distances through intraprotein electron transfer with high directionality. Following optimal molecular orbital overlap, electron jumps from the benzene ring of the indole moiety in a vertical orientation to the LUMO of acceptor quenching residues. Molecular dynamics simulations were invoked to elucidate various correlations of quenching dynamics with separation distances, relative orientations, local fluctuations and reaction heterogeneity. These unique ultrafast quenching pairs, as recently found to extensively occur in high-resolution protein structures, may have significant biological implications.     

A snapshot of enzyme catalysis using electrospray ionization mass spectrometry

Insights into the early molecular events involving protein-ligand/substrate interactions such as protein signaling and enzyme catalysis can be obtained by examining these processes on a very short, millisecond time scale. We have used time-resolved electrospray mass spectrometry to delineate the catalytic mechanism of a key enzyme in bacterial lipopolysaccharide biosynthesis, 3-deoxy-d-manno-2-octulosonate-8-phosphate synthase (KDO8PS). Direct real-time monitoring of the catalytic reaction under single enzyme turnover conditions reveals a novel hemiketal phosphate intermediate bound to the enzyme in a noncovalent complex that establishes the reaction pathway. This study illustrates the successful application of mass spectrometry to reveal transient biochemical processes and opens a new time domain that can provide detailed structural information of short-lived protein-ligand complexes.     

Collision-Induced Release,Ion Mobility Separation,and Amino Acid Sequence Analysis of Subunits from Mass-Selected Noncovalent Protein Complexes

In recent years, mass spectrometry has become a valuable tool for detecting and characterizing protein–protein interactions and for measuring the masses and subunit stoichiometries of noncovalent protein complexes. The gas-phase dissociation of noncovalent protein assemblies via tandem mass spectrometry can be useful in confirming subunit masses and stoichiometries; however, dissociation experiments that are able to yield subunit sequence information must usually be conducted separately. Here, we furnish proof of concept for a method that allows subunit sequence information to be directly obtained from a protein aggregate in a single gas-phase analysis. The experiments were carried out using a quadrupole time-of-flight mass spectrometer equipped with a traveling-wave ion mobility separator. This instrument configuration allows for a noncovalent protein assembly to be quadrupole selected, then subjected to two successive rounds of collision-induced dissociation with an intervening stage of ion mobility separation. This approach was applied to four model proteins as their corresponding homodimers: glucagon, ubiquitin, cytochrome c, and β-lactoglobulin. In each case, b- and y-type fragment ions were obtained upon further collisional activation of the collisionally-released subunits, resulting in up to 50% sequence coverage. Owing to the incorporation of an ion mobility separation, these results also suggest the intriguing possibility of measuring complex mass, complex collisional cross section, subunit masses, subunit collisional cross sections, and sequence information for the subunits in a single gas-phase experiment. Overall, these findings represent a significant contribution towards the realization of protein interactomic analyses, which begin with native complexes and directly yield subunit identities. Figure

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Probing the interaction of the potassium channel modulating KCNE1 in lipid bilayers via solid‐state NMR spectroscopy

KCNE1 is known to modulate the voltage‐gated potassium channel α subunit KCNQ1 to generate slowly activating potassium currents. This potassium channel is essential for the cardiac action potential that mediates a heartbeat as well as the potassium ion homeostasis in the inner ear. Therefore, it is important to know the structure and dynamics of KCNE1 to better understand its modulatory role. Previously, the Sanders group solved the three‐dimensional structure of KCNE1 in LMPG micelles, which yielded a better understanding of this KCNQ1/KCNE1 channel activity. However, research in the Lorigan group showed different structural properties of KCNE1 when incorporated into POPC/POPG lipid bilayers as opposed to LMPG micelles. It is hence necessary to study the structure of KCNE1 in a more native‐like environment such as multi‐lamellar vesicles. In this study, the dynamics of lipid bilayers upon incorporation of the membrane protein KCNE1 were investigated using ³¹P solid‐state nuclear magnetic resonance (NMR) spectroscopy. Specifically, the protein/lipid interaction was studied at varying molar ratios of protein to lipid content. The static ³¹P NMR and T₁ relaxation time were investigated. The ³¹P NMR powder spectra indicated significant perturbations of KCNE1 on the phospholipid headgroups of multi‐lamellar vesicles as shown from the changes in the ³¹P spectral line shape and the chemical shift anisotropy line width. ³¹P T₁ relaxation times were shown to be reversely proportional to the molar ratios of KCNE1 incorporated. The ³¹P NMR data clearly indicate that KCNE1 interacts with the membrane. Copyright © 2017 John Wiley & Sons, Ltd.     

Accelerating the Association of the Most Stable Protein–Ligand Complex by More than Two Orders of Magnitude

The complex between the bacterial type 1 pilus subunit FimG and the peptide corresponding to the N‐terminal extension (termed donor strand, Ds) of the partner subunit FimF (DsF) shows the strongest reported noncovalent molecular interaction, with a dissociation constant (K_D) of 1.5×10^?20 m . However, the complex only exhibits a slow association rate of 330 m ^?1 s^?1 that limits technical applications, such as its use in affinity purification. Herein, a structure‐based approach was used to design pairs of FimGt (a FimG variant lacking its own N‐terminal extension) and DsF variants with enhanced electrostatic surface complementarity. Association of the best mutant FimGt/DsF pairs was accelerated by more than two orders of magnitude, while the dissociation rates and 3D structures of the improved complexes remained essentially unperturbed. A K_D value of 8.8×10^?22 m was obtained for the best mutant complex, which is the lowest value reported to date for a protein/ligand complex.     

Coherent Vibration and Femtosecond Dynamics of the Platinum Complex Oligomers upon Intermolecular Bond Formation in the Excited State

Femtosecond time-resolved absorption and picosecond time-resolved emission measurements were carried out for highly concentrated aqueous solutions of K₂[Pt(CN)₄] to investigate excited-state dynamics of the [Pt(CN)₄²⁻] oligomers formed with metallophilic interactions. Time-resolved absorption spectra exhibit complicated dynamics that are represented with five time constants. Among them, the 90-ps and 400-ps dynamics were assigned to the S₁ → T₁ intersystem crossing of the trimer and tetramer coexisting in the solution by comparison with the fluorescence decays. Clear oscillations of transient absorption were observed in the first few picoseconds, and the frequency-detected-wavelength 2D analysis revealed that the 135-cm⁻¹ and 65-cm⁻¹ oscillations arise from the Pt–Pt stretch motions of the S₁ trimer and S₁ tetramer, respectively. The obtained time-resolved spectroscopic data provide a clear view of the excited-state dynamics of the [Pt(CN)₄²⁻] oligomers in the femto-/picosecond time region.     

A single amino acid determines preference between phospholipids and reveals length restriction for activation ofthe S1P<Subscript>4</Subscript> receptor

Background

Sphingosine-1-phosphate and lysophosphatidic acid (LPA) are ligands for two related families of G protein-coupled receptors, the S1P and LPA receptors, respectively. The lysophospholipid ligands of these receptors are structurally similar, however recognition of these lipids by these receptors is highly selective. A single residue present within the third transmembrane domain (TM) of S1P receptors is thought to determine ligand selectivity; replacement of the naturally occurring glutamic acid with glutamine (present at this position in the LPA receptors) has previously been shown to be sufficient to change the specificity of S1P₁ from S1P to 18:1 LPA.

Results

We tested whether mutation of this "ligand selectivity" residue to glutamine could confer LPA-responsiveness to the related S1P receptor, S1P₄. This mutation severely affected the response of S1P₄ to S1P in a [³⁵S]GTPγS binding assay, and imparted sensitivity to LPA species in the order 14:0 LPA > 16:0 LPA > 18:1 LPA. These results indicate a length restriction for activation of this receptor and demonstrate the utility of using LPA-responsive S1P receptor mutants to probe binding pocket length using readily available LPA species. Computational modelling of the interactions between these ligands and both wild type and mutant S1P₄ receptors showed excellent agreement with experimental data, therefore confirming the fundamental role of this residue in ligand recognition by S1P receptors.

Conclusions

Glutamic acid in the third transmembrane domain of the S1P receptors is a general selectivity switch regulating response to S1P over the closely related phospholipids, LPA. Mutation of this residue to glutamine confers LPA responsiveness with preference for short-chain species. The preference for short-chain LPA species indicates a length restriction different from the closely related S1P₁ receptor.