Identification of unprecedented ATP-competitive choline kinase inhibitors.

In this article we describe the identification of unprecedented ATP-competitive ChoKα inhibitors starting from initial hit NMS-P830 that binds to ChoKα in an ATP concentration-dependent manner. This result is confirmed by the co-crystal structure of NMS-P830 in complex with Δ75-ChoKα. NMS-P830 is able to inhibit ChoKα in cells resulting in the reduction of intracellular phosphocholine formation. A structure-based medicinal chemistry program resulted in the identification of selective compounds that have good biochemical activity, solubility and metabolic stability and are suitable for further optimization. The ChoKα inhibitors disclosed in this article demonstrate for the first time the possibility to inhibit ChoKα with ATP-competitive compounds.

mRNAs and lncRNAs intrinsically form secondary structures with short end-to-end distances.

The 5' and 3' termini of RNA play important roles in many cellular processes. Using Förster resonance energy transfer (FRET), we show that mRNAs and lncRNAs have an intrinsic propensity to fold in the absence of proteins into structures in which the 5' end and 3' end are ≤7 nm apart irrespective of mRNA length. Computational estimates suggest that the inherent proximity of the ends is a universal property of most mRNA and lncRNA sequences. Only guanosine-depleted RNA sequences with low sequence complexity are unstructured and exhibit end-to-end distances expected for the random coil conformation of RNA. While the biological implications remain to be explored, short end-to-end distances could facilitate the binding of protein factors that regulate translation initiation by bridging mRNA 5' and 3' ends. Furthermore, our studies provide the basis for measuring, computing and manipulating end-to-end distances and secondary structure in RNA in research and biotechnology.

EF-G Activation by Phosphate Analogs.

Elongation factor G (EF-G) is a universally conserved translational GTPase that promotes the translocation of tRNA and mRNA through the ribosome. EF-G binds to the ribosome in a GTP-bound form and subsequently catalyzes GTP hydrolysis. The contribution of the ribosome-stimulated GTP hydrolysis by EF-G to tRNA/mRNA translocation remains debated. Here, we show that while EF-G•GDP does not stably bind to the ribosome and induce translocation, EF-G•GDP in complex with phosphate group analogs BeF3(-) and AlF4(-) promotes the translocation of tRNA and mRNA. Furthermore, the rates of mRNA translocation induced by EF-G in the presence of GTP and a non-hydrolyzable analog of GTP, GDP•BeF3(-) are similar. Our results are consistent with the model suggesting that GTP hydrolysis is not directly coupled to mRNA/tRNA translocation. Hence, GTP binding is required to induce the activated, translocation-competent conformation of EF-G while GTP hydrolysis triggers EF-G release from the ribosome.

An extended U2AF(65)-RNA-binding domain recognizes the 3' splice site signal.

How the essential pre-mRNA splicing factor U2AF(65) recognizes the polypyrimidine (Py) signals of the major class of 3' splice sites in human gene transcripts remains incompletely understood. We determined four structures of an extended U2AF(65)-RNA-binding domain bound to Py-tract oligonucleotides at resolutions between 2.0 and 1.5 Å. These structures together with RNA binding and splicing assays reveal unforeseen roles for U2AF(65) inter-domain residues in recognizing a contiguous, nine-nucleotide Py tract. The U2AF(65) linker residues between the dual RNA recognition motifs (RRMs) recognize the central nucleotide, whereas the N- and C-terminal RRM extensions recognize the 3' terminus and third nucleotide. Single-molecule FRET experiments suggest that conformational selection and induced fit of the U2AF(65) RRMs are complementary mechanisms for Py-tract association. Altogether, these results advance the mechanistic understanding of molecular recognition for a major class of splice site signals.

Movement of elongation factor G between compact and extended conformations.

Previous structural studies suggested that ribosomal translocation is accompanied by large interdomain rearrangements of elongation factor G (EF-G). Here, we follow the movement of domain IV of EF-G relative to domain II of EF-G using ensemble and single-molecule Förster resonance energy transfer. Our results indicate that ribosome-free EF-G predominantly adopts a compact conformation that can also, albeit infrequently, transition into a more extended conformation in which domain IV moves away from domain II. By contrast, ribosome-bound EF-G predominantly adopts an extended conformation regardless of whether it is interacting with pretranslocation ribosomes or with posttranslocation ribosomes. Our data suggest that ribosome-bound EF-G may also occasionally sample at least one more compact conformation. GTP hydrolysis catalyzed by EF-G does not affect the relative stability of the observed conformations in ribosome-free and ribosome-bound EF-G. Our data support a model suggesting that, upon binding to a pretranslocation ribosome, EF-G moves from a compact to a more extended conformation. This transition is not coupled to but likely precedes both GTP hydrolysis and mRNA/tRNA translocation.

Following movement of domain IV of elongation factor G during ribosomal translocation.

Translocation of mRNA and tRNAs through the ribosome is catalyzed by a universally conserved elongation factor (EF-G in prokaryotes and EF-2 in eukaryotes). Previous studies have suggested that ribosome-bound EF-G undergoes significant structural rearrangements. Here, we follow the movement of domain IV of EF-G, which is critical for the catalysis of translocation, relative to protein S12 of the small ribosomal subunit using single-molecule FRET. We show that ribosome-bound EF-G adopts distinct conformations corresponding to the pre- and posttranslocation states of the ribosome. Our results suggest that, upon ribosomal translocation, domain IV of EF-G moves toward the A site of the small ribosomal subunit and facilitates the movement of peptidyl-tRNA from the A to the P site. We found no evidence of direct coupling between the observed movement of domain IV of EF-G and GTP hydrolysis. In addition, our results suggest that the pretranslocation conformation of the EF-G-ribosome complex is significantly less stable than the posttranslocation conformation. Hence, the structural rearrangement of EF-G makes a considerable energetic contribution to promoting tRNA translocation.

Isozyme-specific ligands for O-acetylserine sulfhydrylase, a novel antibiotic target.

The last step of cysteine biosynthesis in bacteria and plants is catalyzed by O-acetylserine sulfhydrylase. In bacteria, two isozymes, O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B, have been identified that share similar binding sites, although the respective specific functions are still debated. O-acetylserine sulfhydrylase plays a key role in the adaptation of bacteria to the host environment, in the defense mechanisms to oxidative stress and in antibiotic resistance. Because mammals synthesize cysteine from methionine and lack O-acetylserine sulfhydrylase, the enzyme is a potential target for antimicrobials. With this aim, we first identified potential inhibitors of the two isozymes via a ligand- and structure-based in silico screening of a subset of the ZINC library using FLAP. The binding affinities of the most promising candidates were measured in vitro on purified O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B from Salmonella typhimurium by a direct method that exploits the change in the cofactor fluorescence. Two molecules were identified with dissociation constants of 3.7 and 33 µM for O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B, respectively. Because GRID analysis of the two isoenzymes indicates the presence of a few common pharmacophoric features, cross binding titrations were carried out. It was found that the best binder for O-acetylserine sulfhydrylase-B exhibits a dissociation constant of 29 µM for O-acetylserine sulfhydrylase-A, thus displaying a limited selectivity, whereas the best binder for O-acetylserine sulfhydrylase-A exhibits a dissociation constant of 50 µM for O-acetylserine sulfhydrylase-B and is thus 8-fold selective towards the former isozyme. Therefore, isoform-specific and isoform-independent ligands allow to either selectively target the isozyme that predominantly supports bacteria during infection and long-term survival or to completely block bacterial cysteine biosynthesis.

Fine tuning of the active site modulates specificity in the interaction of O-acetylserine sulfhydrylase isozymes with serine acetyltransferase.

O-acetylserine sulfhydrylase (OASS) catalyzes the synthesis of l-cysteine in the last step of the reductive sulfate assimilation pathway in microorganisms. Its activity is inhibited by the interaction with serine acetyltransferase (SAT), the preceding enzyme in the metabolic pathway. Inhibition is exerted by the insertion of SAT C-terminal peptide into the OASS active site. This action is effective only on the A isozyme, the prevalent form in enteric bacteria under aerobic conditions, but not on the B-isozyme, the form expressed under anaerobic conditions. We have investigated the active site determinants that modulate the interaction specificity by comparing the binding affinity of thirteen pentapeptides, derived from the C-terminal sequences of SAT of the closely related species Haemophilus influenzae and Salmonella typhimurium, towards the corresponding OASS-A, and towards S. typhimurium OASS-B. We have found that subtle changes in protein active sites have profound effects on protein-peptide recognition. Furthermore, affinity is strongly dependent on the pentapeptide sequence, signaling the relevance of P3-P4-P5 for the strength of binding, and P1-P2 mainly for specificity. The presence of an aromatic residue at P3 results in high affinity peptides with K(diss) in the micromolar and submicromolar range, regardless of the species. An acidic residue, like aspartate at P4, further strengthens the interaction and results in the higher affinity ligand of S. typhimurium OASS-A described to date. Since OASS knocked-out bacteria exhibit a significantly decreased fitness, this investigation provides key information for the development of selective OASS inhibitors, potentially useful as novel antibiotic agents.

The multifaceted pyridoxal 5'-phosphate-dependent O-acetylserine sulfhydrylase.

Cysteine is the final product of the reductive sulfate assimilation pathway in bacteria and plants and serves as the precursor for all sulfur-containing biological compounds, such as methionine, S-adenosyl methionine, iron-sulfur clusters and glutathione. Moreover, in several microorganisms cysteine plays a role as a reducing agent, eventually counteracting host oxidative defense strategies. Cysteine is synthesized by the PLP-dependent O-acetylserine sulfhydrylase, a dimeric enzyme belonging to the fold type II, catalyzing a beta-replacement reaction. In this review, the spectroscopic properties, catalytic mechanism, three-dimensional structure, conformational changes accompanying catalysis, determinants of enzyme stability, role of selected amino acids in catalysis, and the regulation of enzyme activity by ligands and interaction with serine acetyltransferase, the preceding enzyme in the biosynthetic pathway, are described. Given the key biological role played by O-acetylserine sulfhydrylase in bacteria, inhibitors with potential antibiotic activity have been developed. This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology.

Exploring O-acetylserine sulfhydrylase-B isoenzyme from Salmonella typhimurium by fluorescence spectroscopy.

The pyridoxal 5'-phosphate (PLP)-dependent enzyme O-acetylserine sulfhydrylase (OASS) catalyzes the synthesis of cysteine in bacteria and plants. In bacteria two isoenzymes are present, OASS-A and OASS-B, with distinct structural, functional, and regulatory properties. In order to gain a deeper insight into OASS-B dynamic and functional properties, single and double mutants of the three tryptophan residues, Trp28, Trp159, and Trp212, were prepared and their fluorescence emission properties were characterized in the absence and presence of substrate and ligands by steady-state and time-resolved spectrofluorimetry. Residue Trp28 was found to be mainly responsible for Trp fluorescence emission, whereas Trp212, located in a highly flexible region near the active site, is mainly responsible for an energy-transfer to PLP leading to an emission at 500 nm. Not surprisingly, mutation of Trp212 affects OASS-B activity. Trp159 slightly contributes to both direct emission and energy transfer to PLP. Time-resolved fluorescence measurements confirmed these findings, observing a third longer tryptophan lifetime for apo-OASS-B, in addition to the two lifetimes that are present in the holo-enzyme and mutants. A comparison with the emissions previously determined for OASS-A indicates that OASS-B active site is likely to be more polar and flexible, in agreement with a broader substrate specificity and higher catalytic efficiency.

Identification of the structural determinants for the stability of substrate and aminoacrylate external Schiff bases in O-acetylserine sulfhydrylase-A.

O-Acetylserine sulfhydrylase is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the final step in the cysteine biosynthetic pathway in enteric bacteria and plants, the replacement of the beta-acetoxy group of O-acetyl-L-serine (OAS) by a thiol to give L-cysteine. Previous studies of the K41A mutant enzyme showed L-methionine bound in an external Schiff base (ESB) linkage to PLP as the enzyme was isolated. The mutant enzyme exists in a closed form, optimizing the orientation of the cofactor PLP and properly positioning active site functional groups for reaction. The trigger for closing the active site upon formation of the ESB is thought to be interaction of the substrate alpha-carboxylate with the substrate-binding loop comprised of T68, S69, G70, and N71, and Q142, which is positioned above the cofactor as one looks into the active site. To probe the contribution of these residues to the active site closing and orientation of PLP in the ESB, T68, S69, N71, and Q142 were changed to alanine. Absorbance, fluorescence, near UV-visible CD, and (31)P NMR spectral studies and pre-steady state kinetic studies were used to characterize the mutant enzymes. All of the mutations affect closure of the active site, but to differing extents. In addition, the site appears to be more hydrophilic given that the ESBs do not exhibit a significant amount of the enolimine tautomer. No buildup of the alpha-aminoacrylate intermediate (AA) is observed for the T68A and Q142A mutant enzymes. However, pyruvate is produced at a rate much greater than that of the wild-type enzyme. Data suggest that T68 and Q142 play a role in stabilizing the AA. Both residues donate a hydrogen bond to one of the carboxylate oxygens of the methionine ESB and may also be responsible for the proper orientation of the ESB to generate the AA. The S69A and N71A mutants exhibit formation of the AA, but the rate constant for its formation from the ESB is decreased by 1 order of magnitude compared to that of the wild type. S69 donates a hydrogen bond to the substrate carboxylate in the ESB, while N71 donates hydrogen bonds to O3' of the cofactor and the carboxylate of the ESB; these side chains may also affect the orientation of the ESB. Data suggest that interaction of intermediates with the substrate-binding loop and Q142 gives a properly aligned Michaelis complex and facilitates the beta-elimination reaction.

A two-step process controls the formation of the bienzyme cysteine synthase complex.

The regulation of enzyme activity through the transient formation of multiprotein assemblies plays an important role in the control of biosynthetic pathways. One of the first regulatory complexes to be discovered was cysteine synthase (CS), formed by the pyridoxal 5'-phosphate-dependent enzyme O-acetylserine sulfhydrylase (OASS) and serine acetyltransferase (SAT). These enzymes are at the branch point of the sulfur, carbon, and nitrogen assimilation pathways. Understanding the mechanism of complex formation helps to clarify the role played by CS in the regulation of sulfur assimilation in bacteria and plants. To this goal, stopped-flow fluorescence spectroscopy was used to characterize the interaction of SAT with OASS, at different temperatures and pH values, and in the presence of the physiological regulators cysteine and bisulfide. Results shed light on the mechanism of complex formation and regulation, so far poorly understood. Cysteine synthase assembly occurs via a two-step mechanism involving rapid formation of an encounter complex between the two enzymes, followed by a slow conformational change. The conformational change likely results from the closure of the active site of OASS upon binding of the SAT C-terminal peptide. Bisulfide, the second substrate and a feedback inhibitor of OASS, stabilizes the CS complex mainly by decreasing the back rate of the isomerization step. Cysteine, the product of the OASS reaction and a SAT inhibitor, slightly affects the kinetics of CS formation leading to destabilization of the complex.

Design of O-acetylserine sulfhydrylase inhibitors by mimicking nature.

The inhibition of cysteine biosynthesis in prokaryotes and protozoa has been proposed to be relevant for the development of antibiotics. Haemophilus influenzae O-acetylserine sulfhydrylase (OASS), catalyzing l-cysteine formation, is inhibited by the insertion of the C-terminal pentapeptide (MNLNI) of serine acetyltransferase into the active site. Four-hundred MNXXI pentapeptides were generated in silico, docked into OASS active site using GOLD, and scored with HINT. The terminal P5 Ile accounts for about 50% of the binding energy. Glu or Asp at position P4 and, to a lesser extent, at position P3 also significantly contribute to the binding interaction. The predicted affinity of 14 selected pentapeptides correlated well with the experimentally determined dissociation constants. The X-ray structure of three high affinity pentapeptide-OASS complexes were compared with the docked poses. These results, combined with a GRID analysis of the active site, allowed us to define a pharmacophoric scaffold for the design of peptidomimetic inhibitors.

Interaction of serine acetyltransferase with O-acetylserine sulfhydrylase active site: evidence from fluorescence spectroscopy.

Serine acetyltransferase is a key enzyme in the sulfur assimilation pathway of bacteria and plants, and is known to form a bienzyme complex with O-acetylserine sulfhydrylase, the last enzyme in the cysteine biosynthetic pathway. The biological function of the complex and the mechanism of reciprocal regulation of the constituent enzymes are still poorly understood. In this work the effect of complex formation on the O-acetylserine sulfhydrylase active site has been investigated exploiting the fluorescence properties of pyridoxal 5'-phosphate, which are sensitive to the cofactor microenvironment and to conformational changes within the protein matrix. The results indicate that both serine acetyltransferase and its C-terminal decapeptide bind to the alpha-carboxyl subsite of O-acetylserine sulfhydrylase, triggering a transition from an open to a closed conformation. This finding suggests that serine acetyltransferase can inhibit O-acetylserine sulfhydrylase catalytic activity with a double mechanism, the competition with O-acetylserine for binding to the enzyme active site and the stabilization of a closed conformation that is less accessible to the natural substrate.