Reaction of aspirin with cysteinyl residues of lens γ-crystallins: a mechanism for the proposed anti-cataract effect of aspirin

Incubation of lens crystallins with aspirin inhibits the development of opacities caused by cyanate. Cyanate-induced opacities are thought to be due to carbamylation of the lysyl residues which causes a decrease in the protein charge and subsequent conformational changes that permit disulfide bonding. Because aspirin can also react with lysyl residues, it has been proposed that the aspiring inhibition of cataractogonesis is due to acetylation of the lysyl which would block their reaction with cyanata. However, acetylation oflysyl residues also lowers the protein charge and would be expected to effect changes in protein conformation similar to those caused by carbamylation. Therefore, acetylation of the lysyl residues is not a satisfactory explanation for the inhibitory effect of aspirin on lens opacification. Our investigations of the reactions of cyanate and aspirin with bovine γ_II-crystallins show that the cysteinyl residues are also carbamylated and acetylated at pH 7.4. At this pH, the carbamylation at the cysteinyl residues is reversible, leading to regeneration of the thiol group and disulfide bonding. In contrast, the acetylation at cysteinyl residues is stable at pH 7.4 and can prevent disulfide bonding. This difference in stability explains how cyanate promotes, and aspirin inhibits, cataractogenesis.     

Residues located in the primase domain of the bacteriophage T7 primase-helicase are essential for loading the hexameric complex onto DNA

The T7 primase-helicase plays a pivotal role in the replication of T7 DNA. Using affinity isolation of peptide–nucleic acid crosslinks and mass spectrometry, we identify protein regions in the primase-helicase and T7 DNA polymerase that form contacts with the RNA primer and DNA template. The contacts between nucleic acids and the primase domain of the primase-helicase are centered in the RNA polymerase subdomain of the primase domain, in a cleft between the N-terminal subdomain and the topoisomerase-primase fold. We demonstrate that residues along a beta sheet in the N-terminal subdomain that contacts the RNA primer are essential for phage growth and primase activity in vitro. Surprisingly, we found mutations in the primase domain that had a dramatic effect on the helicase. Substitution of a residue conserved in other DnaG-like enzymes, R84A, abrogates both primase and helicase enzymatic activities of the T7 primase-helicase. Alterations in this residue also decrease binding of the primase-helicase to ssDNA. However, mass photometry measurements show that these mutations do not interfere with the ability of the protein to form the active hexamer.     

Dissection of the DNA Mimicry of the Bacteriophage T7 Ocr Protein using Chemical Modification

The homodimeric Ocr (overcome classical restriction) protein of bacteriophage T7 is a molecular mimic of double-stranded DNA and a highly effective competitive inhibitor of the bacterial type I restriction/modification system. The surface of Ocr is replete with acidic residues that mimic the phosphate backbone of DNA. In addition, Ocr also mimics the overall dimensions of a bent 24-bp DNA molecule. In this study, we attempted to delineate these two mechanisms of DNA mimicry by chemically modifying the negative charges on the Ocr surface. Our analysis reveals that removal of about 46% of the carboxylate groups per Ocr monomer results in an ∼ 50-fold reduction in binding affinity for a methyltransferase from a model type I restriction/modification system. The reduced affinity between Ocr with this degree of modification and the methyltransferase is comparable with the affinity of DNA for the methyltransferase. Additional modification to remove ∼ 86% of the carboxylate groups further reduces its binding affinity, although the modified Ocr still binds to the methyltransferase via a mechanism attributable to the shape mimicry of a bent DNA molecule. Our results show that the electrostatic mimicry of Ocr increases the binding affinity for its target enzyme by up to ∼ 800-fold.     

Cleavage Specificity of Enterococcus faecalis EnpA (EF1473), a Peptidoglycan Endopeptidase Related to the LytM/Lysostaphin Family of Metallopeptidases

Enterococcus faecalis EnpA (EF1473) is a 1721-residue predicted protein encoded by prophage 03 that displays similarity to the staphylolytic glycyl-glycyl endopeptidases lysostaphin and LytM. We purified a catalytically active fragment of the protein, EnpA_C, comprising residues 1374-1505 and showed that the recombinant polypeptide efficiently cleaved cross-linked muropeptides generated by muramidases, but was poorly active in intact sacculi. Analysis of the products of digestion of purified dimers by mass spectrometry indicated that EnpA_C cleaves the d-Ala-l-Ala bond formed by the d,d-transpeptidase activity of penicillin-binding proteins in the last cross-linking step of peptidoglycan synthesis. Synthetic d was identified as the minimum substrate of EnpA_C indicating that interaction of the enzyme with the donor peptide stem of cross-linked dimers is sufficient for its activity. Peptidoglycan was purified from various bacterial species and digested with mutanolysin and EnpA_C to assess enzyme specificity. EnpA_C did not cleave direct cross-links, but tolerated extensive variation in cross-bridges with respect to both their length (one to five residues) and their amino acid sequence. Recognition of the donor stem of cross-linked dimers could account for the substrate specificity of EnpA_C, which is significantly broader in comparison to endopeptidases belonging to the lysostaphin family.     

Detection of RNA nucleoside modifications with the uridine-specific ribonuclease MC1 from Momordica charantia

A codon-optimized recombinant ribonuclease, MC1 is characterized for its uridine-specific cleavage ability to map nucleoside modifications in RNA. The published MC1 amino acid sequence, as noted in a previous study, was used as a template to construct a synthetic gene with a natural codon bias favoring expression in Escherichia coli. Following optimization of various expression conditions, the active recombinant ribonuclease was successfully purified as a C-terminal His-tag fusion protein from E. coli [Rosetta 2(DE3)] cells. The isolated protein was tested for its ribonuclease activity against oligoribonucleotides and commercially available E. coli tRNA^{Tyr I}. Analysis of MC1 digestion products by ion-pairing reverse phase liquid-chromatography coupled with mass spectrometry (IP-RP-LC-MS) revealed enzymatic cleavage of RNA at the 5′-termini of uridine and pseudouridine, but cleavage was absent if the uridine was chemically modified or preceded by a nucleoside with a bulky modification. Furthermore, the utility of this enzyme to generate complementary digestion products to other common endonucleases, such as RNase T1, which enables the unambiguous mapping of modified residues in RNA is demonstrated.     

Pyruvate Formate-Lyase Interacts Directly with the Formate Channel FocA to Regulate Formate Translocation

The FNT (formate-nitrite transporters) form a superfamily of pentameric membrane channels that translocate monovalent anions across biological membranes. FocA (formate channel A) translocates formate bidirectionally but the mechanism underlying how translocation of formate is controlled and what governs substrate specificity remains unclear. Here we demonstrate that the normally soluble dimeric enzyme pyruvate formate-lyase (PflB), which is responsible for intracellular formate generation in enterobacteria and other microbes, interacts specifically with FocA. Association of PflB with the cytoplasmic membrane was shown to be FocA dependent and purified, Strep-tagged FocA specifically retrieved PflB from Escherichia coli crude extracts. Using a bacterial two-hybrid system, it could be shown that the N-terminus of FocA and the central domain of PflB were involved in the interaction. This finding was confirmed by chemical cross-linking experiments. Using constraints imposed by the amino acid residues identified in the cross-linking study, we provide for the first time a model for the FocA–PflB complex. The model suggests that the N-terminus of FocA is important for interaction with PflB. An in vivo assay developed to monitor changes in formate levels in the cytoplasm revealed the importance of the interaction with PflB for optimal translocation of formate by FocA. This system represents a paradigm for the control of activity of FNT channel proteins.     

Acetylation of L12 increases interactions in the Escherichia coli ribosomal stalk complex

The ribosomal stalk complex in Escherichia coli consists of L10 and four copies of L7/L12, and is largely responsible for binding and recruiting translation factors. Structural characterisation of this stalk complex is difficult, primarily due to its dynamics. Here, we apply mass spectrometry to follow post-translational modifications and their effect on structural changes of the stalk proteins on intact ribosomes. Our results show that increased acetylation of L12 occurs during the stationary phase on ribosomes harvested from cells grown under optimal conditions. For cells grown in minimal medium, L12 acetylation and processing is altered, resulting in deficient removal of N-terminal methionine in ∼ 50% of the L12 population, while processed L12 is almost 100% acetylated. Our results show also that N-acetylation of L12 correlates with an increased stability of the stalk complex in the gas phase. To investigate further the basis of this increased stability, we applied a solution phase hydrogen deuterium exchange protocol to compare the rate of deuterium incorporation in the proteins L9, L10, L11 and L12 as well as the acetylated form of L12 (L7), in situ on the ribosome. Results show that deuterium incorporation is consistently slower for L7 relative to L12 and for L10 when L7 is predominant. Our results imply a tightening of the interaction between L7 and L10 relative to that between L12 and L10. Since acetylation is predominant when cells are grown in minimal medium, we propose that these modifications form part of the cell's strategy to increase stability of the stalk complex under conditions of stress. More generally, our results demonstrate that it is possible to discern the influence of a 42 Da post-translational modification by mass spectrometry and to record subtle changes in hydrogen/deuterium exchange within the context of an intact 2.5 MDa particle.     

Escherichia coli peptide binding protein OppA has a preference for positively charged peptides

The Escherichia coli peptide binding protein OppA is an essential component of the oligopeptide transporter Opp. Based on studies on its orthologue from Salmonella typhimurium, it has been proposed that OppA binds peptides between two and five amino acids long, with no apparent sequence selectivity. Here, we studied peptide binding to E. coli OppA directly and show that the protein has an unexpected preference for basic peptides. OppA was expressed in the periplasm, where it bound to available peptides. The protein was purified in complex with tightly bound peptides. The crystal structure (up to 2.0 Å) of OppA liganded with the peptides indicated that the protein has a preference for peptides containing a lysine. Mass spectrometry analysis of the bound peptides showed that peptides between two and five amino acids long bind to the protein and indeed hinted at a preference for positively charged peptides. The preference of OppA for peptides with basic residues, in particular lysines, was corroborated by binding studies with peptides of defined sequence using isothermal titration calorimetry and intrinsic protein fluorescence titration. The protein bound tripeptides and tetrapeptides containing positively charged residues with high affinity, whereas related peptides without lysines/arginines were bound with low affinity. A structure of OppA in an open conformation in the absence of ligands was also determined to 2.0 Å, revealing that the initial binding site displays a negative surface charge, consistent with the observed preference for positively charged peptides. Taken together, E. coli OppA appears to have a preference for basic peptides.     

Response of a DNA-binding protein to radiation-induced oxidative stress

The DNA-binding protein MC1 is a chromosomal protein extracted from the archaebacterium Methanosarcina sp. CHTI55. It binds any DNA, and exhibits an enhanced affinity for some short sequences and structures (circles, cruciform DNA). Moreover, the protein bends DNA strongly at the binding site. MC1 was submitted to oxidative stress through gamma-ray irradiation. In our experimental conditions, damage is essentially due to hydroxyl radicals issued from water radiolysis.Upon irradiation, the regular complex between MC1 and DNA disappears, while a new complex appears. In the new complex, the protein loses its ability to recognise preferential sequences and DNA circles, and bends DNA less strongly than in the regular one. The new complex disappears and the protein becomes totally inactivated by high doses.A model has been proposed to explain these experimental results. Two targets, R(1) and R(2), are concomitantly destroyed in the protein, with different kinetics. R(2) oxidation has no effect on the regular binding, whereas R(1) oxidation modifies the functioning of MC1: loss of preferential site and structure recognition, weaker bending. The destruction of both R(1) and R(2) targets leads to a total inactivation of the protein. This model accounts for the data obtained by titrations of DNA with irradiated proteins.When the protein is irradiated in the complex with DNA, bound DNA protects its binding site on the protein very efficiently.The highly oxidisable tryptophan and methionine could be the amino acid residues implicated in the inactivation process.     

Structural basis for acyl acceptor specificity in the achromobactin biosynthetic enzyme AcsD

Siderophores are known virulence factors, and their biosynthesis is a target for new antibacterial agents. A non-ribosomal peptide synthetase-independent siderophore biosynthetic pathway in Dickeya dadantii is responsible for production of the siderophore achromobactin. The D. dadantii achromobactin biosynthesis protein D (AcsD) enzyme has been shown to enantioselectively esterify citric acid with l-serine in the first committed step of achromobactin biosynthesis. The reaction occurs in two steps: stereospecific activation of citric acid by adenylation, followed by attack of the enzyme-bound citryl adenylate by l-serine to produce the homochiral ester. We now report a detailed characterization of the substrate profile and mechanism of the second (acyl transfer) step of AcsD enzyme. We demonstrate that the enzyme catalyzes formation of not only esters but also amides from the citryl-adenylate intermediate. We have rationalized the substrate utilization profile for the acylation reaction by determining the first X-ray crystal structure of a product complex for this enzyme class. We have identified the residues that are important for both recognition of l-serine and catalysis of ester formation. Our hypotheses were tested by biochemical analysis of various mutants, one of which shows a reversal of specificity from the wild type with respect to non-natural substrates. This change can be rationalized on the basis of our structural data. That this change in specificity is accompanied by no loss in activity suggests that AcsD and other members of the non-ribosomal peptide synthetase-independent siderophore superfamily may have biotransformation potential.     

Identification of Residues on Hsp70 and Hsp90 Ubiquitinated by the Cochaperone CHIP

Molecular chaperones Hsp70 and Hsp90 are in part responsible for maintaining the viability of cells by facilitating the folding and maturation process of many essential client proteins. The ubiquitin ligase C-terminus of Hsc70 interacting protein (CHIP) has been shown in vitro and in vivo to associate with Hsp70 and Hsp90 and ubiquitinate them, thus targeting them to the proteasome for degradation. Here, we study one facet of this CHIP-mediated turnover by determining the lysine residues on human Hsp70 and Hsp90 ubiquitinated by CHIP. We performed in vitro ubiquitination reactions of the chaperones using purified components and analyzed the samples by tandem mass spectrometry to identify modified lysine residues. Six such ubiquitination sites were identified on Hsp70 (K325, K451, K524, K526, K559, and K561) and 13 ubiquitinated lysine residues were found on Hsp90 (K107, K204, K219, K275, K284, K347, K399, K477, K481, K538, K550, K607, and K623). We mapped the ubiquitination sites on homology models of almost full-length human Hsp70 and Hsp90, which were found to cluster in certain regions of the structures. Furthermore, we determined that CHIP forms polyubiquitin chains on Hsp70 and Hsp90 linked via K6, K11, K48, and K63. These findings clarify the mode of ubiquitination of Hsp70 and Hsp90 by CHIP, which ultimately leads to their degradation.     

Archaeal N-terminal protein maturation commonly involves N-terminal acetylation: a large-scale proteomics survey

We present the first large-scale survey of N-terminal protein maturation in archaea based on 873 proteomically identified N-terminal peptides from the two haloarchaea Halobacterium salinarum and Natronomonas pharaonis. The observed protein maturation pattern can be attributed to the combined action of methionine aminopeptidase and N-terminal acetyltransferase and applies to cytosolic proteins as well as to a large fraction of integral membrane proteins. Both N-terminal maturation processes primarily depend on the amino acid in penultimate position, in which serine and threonine residues are over represented. Removal of the initiator methionine occurs in two-thirds of the haloarchaeal proteins and requires a small penultimate residue, indicating that methionine aminopeptidase specificity is conserved across all domains of life. While N-terminal acetylation is rare in bacteria, our proteomic data show that acetylated N termini are common in archaea affecting about 15% of the proteins and revealing a distinct archaeal N-terminal acetylation pattern. Haloarchaeal N-terminal acetyltransferase reveals narrow substrate specificity, which is limited to cleaved N termini starting with serine or alanine residues. A comparative analysis of 140 ortholog pairs with identified N-terminal peptide showed that acetylatable N-terminal residues are predominantly conserved amongst the two haloarchaea. Only few exceptions from the general N-terminal acetylation pattern were observed, which probably represent protein-specific modifications as they were confirmed by ortholog comparison.     

An alternative mechanism for the catalysis of peptide bond formation by L/F transferase: substrate binding and orientation

Eubacterial leucyl/phenylalanyl tRNA protein transferase (L/F transferase) catalyzes the transfer of a leucine or a phenylalanine from an aminoacyl-tRNA to the N-terminus of a protein substrate. This N-terminal addition of an amino acid is analogous to that of peptide synthesis by ribosomes. A previously proposed catalytic mechanism for Escherichia coli L/F transferase identified the conserved aspartate 186 (D186) and glutamine 188 (Q188) as key catalytic residues. We have reassessed the role of D186 and Q188 by investigating the enzymatic reactions and kinetics of enzymes possessing mutations to these active-site residues. Additionally three other amino acids proposed to be involved in aminoacyl-tRNA substrate binding are investigated for comparison. By quantitatively measuring product formation using a quantitative matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based assay, our results clearly demonstrate that, despite significant reduction in enzymatic activity as a result of different point mutations introduced into the active site of L/F transferase, the formation of product is still observed upon extended incubations. Our kinetic data and existing X-ray crystal structures result in a proposal that the critical roles of D186 and Q188, like the other amino acids in the active site, are for substrate binding and orientation and do not directly participate in the chemistry of peptide bond formation. Overall, we propose that L/F transferase does not directly participate in the chemistry of peptide bond formation but catalyzes the reaction by binding and orientating the substrates for reaction in an analogous mechanism that has been described for ribosomes.     

Protein folding stabilities are a major determinant of oxidation rates for buried methionine residues

The oxidation of protein-bound methionines to form methionine sulfoxides has a broad range of biological ramifications, making it important to delineate factors that influence methionine oxidation rates within a given protein. This is especially important for biopharmaceuticals, where oxidation can lead to deactivation and degradation. Previously, neighboring residue effects and solvent accessibility have been shown to impact the susceptibility of methionine residues to oxidation. In this study, we provide proteome-wide evidence that oxidation rates of buried methionine residues are also strongly influenced by the thermodynamic folding stability of proteins. We surveyed the Escherichia coli proteome using several proteomic methodologies and globally measured oxidation rates of methionine residues in the presence and absence of tertiary structure, as well as the folding stabilities of methionine-containing domains. These data indicated that buried methionines have a wide range of protection factors against oxidation that correlate strongly with folding stabilities. Consistent with this, we show that in comparison to E. coli, the proteome of the thermophile Thermus thermophilus is significantly more stable and thus more resistant to methionine oxidation. To demonstrate the utility of this correlation, we used native methionine oxidation rates to survey the folding stabilities of E. coli and T. thermophilus proteomes at various temperatures and propose a model that relates the temperature dependence of the folding stabilities of these two species to their optimal growth temperatures. Overall, these results indicate that oxidation rates of buried methionines from the native state of proteins can be used as a metric of folding stability.     

The functional molecular mass of the Pasteurella hyaluronan synthase is a monomer

Hyaluronan (HA), a linear polysaccharide composed of β1,3-GlcNAc-β1,4-GlcUA repeats, is found in the extracellular matrix of vertebrate tissues as well as the capsule of several pathogenic bacteria. All known HA synthases (HASs) are dual-action glycosyltransferases that catalyze the addition of two different sugars from UDP-linked precursors to the growing HA chain. The bacterial hyaluronan synthase, PmHAS from Gram-negative Pasteurella multocida, is a 972-residue membrane-associated protein. Previously, the Gram-positive Streptococcus pyogenes enzyme, SpHAS (419 residues), and the vertebrate enzyme, XlHAS1 (588 residues), were found to function as monomers of protein, but the PmHAS is not similar at the protein sequence level and has quite different enzymological properties. We have utilized radiation inactivation to measure the target size of recombinant full-length and truncated PmHAS. The target size of HAS activity was confirmed using internal enzyme standards of known molecular weight. We found that the Pasteurella HA synthase protein functions catalytically as a monomer. Functional truncated soluble PmHAS also behaves as a polypeptide monomer as assessed by gel filtration chromatography and light scattering.     

Evidence for involvement of the C-terminal domain in the dimerization of the CopY repressor protein from Enterococcus hirae

Metal binding to the C-terminal region of the copper-responsive repressor protein CopY is responsible for homodimerization and the regulation of the copper homeostasis pathway in Enterococcus hirae. Specific involvement of the 38 C-terminal residues of CopY in dimerization is indicated by zonal and frontal (large zone) size-exclusion chromatography studies. The studies demonstrate that the attachment of these CopY residues to the immunoglobulin-binding domain of streptococcal protein G (GB1) promotes dimerization of the monomeric protein. Although sensitivity of dimerization to removal of metal from the fusion protein is smaller than that found for CopY (as measured by ultracentrifugation studies), the demonstration that an unrelated protein (GB1) can be induced to dimerize by extending its sequence with the C-terminal portion of CopY confirms the involvement of this region in CopY homodimerization.     

Identification of pheromone-like compounds in male reproductive organs of the oriental locust Locusta migratoria

Despite the great economical interest of locusts in agriculture, knowledge on their chemoreception systems is still poor. Phenylacetonitrile is recognised as a pheromone of the desert locust Schistocerca gregaria, triggering gregarization, promoting aggregation and inhibiting courtship. However, in the other major locust species, Locusta migratoria, pheromones have not been reported. We have identified the two isomers of naphthylpropionitrile from the male reproductive organs of L. migratoria. Chemical synthesis has confirmed the identity of the two compounds. Both isomers show significant affinity to CSP91, a protein reported in the testis, but not to three other proteins of the same family (CSP180, CSP540 and CSP884) expressed in female accessory glands. The striking similarity of these compounds with phenylacetonitrile and the unusual nature of such chemicals strongly suggest that naphthylpropionitrile could be pheromones for L. migratoria, while their site of expression and binding activity indicate a role in communication between sexes.     

A new structural insight into XPA–DNA interactions

XPA (xeroderma pigmentosum group A) protein is an essential factor for NER (nucleotide excision repair) which is believed to be involved in DNA damage recognition/verification, NER factor recruiting and stabilization of repair intermediates. Past studies on the structure of XPA have focused primarily on XPA interaction with damaged DNA. However, how XPA interacts with other DNA structures remains unknown though recent evidence suggest that these structures could be important for its roles in both NER and non-NER activities. Previously, we reported that XPA recognizes undamaged DNA ds/ssDNA (double-strand/single-strandDNA) junctions with a binding affinity much higher than its ability to bind bulky DNA damage. To understand how this interaction occurs biochemically we implemented a structural determination of the interaction using a MS-based protein footprinting method and limited proteolysis. By monitoring surface accessibility of XPA lysines to NHS-biotin modification in the free protein and the DNA junction-bound complex we show that XPA physically interacts with the DNA junctions via two lysines, K168 and K179, located in the previously known XPA(98–219) DBD (DNA-binding domain). Importantly, we also uncovered new lysine residues, outside of the known DBD, involved in the binding. We found that residues K221, K222, K224 and K236 in the C-terminal domain are involved in DNA binding. Limited proteolysis analysis of XPA–DNA interactions further confirmed this observation. Structural modelling with these data suggests a clamp-like DBD for the XPA binding to ds/ssDNA junctions. Our results provide a novel structure-function view of XPA–DNA junction interactions.     

Structure and organisation of SinR, the master regulator of biofilm formation in Bacillus subtilis

sinR encodes a tetrameric repressor of genes required for biofilm formation in Bacillus subtilis. sinI, which is transcribed under Spo0A control, encodes a dimeric protein that binds to SinR to form a SinR-SinI heterodimer in which the DNA-binding functions of SinR are abrogated and repression of biofilm genes is relieved. The heterodimer-forming surface comprises residues conserved between SinR and SinI. Each forms a pair of α-helices that hook together to form an intermolecular four-helix bundle. Here, we are interested in the assembly of the SinR tetramer and its binding to DNA. Size-exclusion chromatography with multi-angle laser light scattering and crystallographic analysis reveal that a DNA-binding fragment of SinR (residues 1-69) is a monomer, while a SinI-binding fragment (residues 74-111) is a tetramer arranged as a dimer of dimers. The SinR(74-111) chain forms two α-helices with the organisation of the dimer similar to that observed in the SinR-SinI complex. The tetramer is formed through interactions of residues at the C-termini of the four chains. A model of the intact SinR tetramer in which the DNA binding domains surround the tetramerisation core was built. Fluorescence anisotropy and surface plasmon resonance experiments showed that SinR binds to an oligonucleotide duplex, 5′-TTTGTTCTCTAAAGAGAACTTA-3′, containing a pair of SinR consensus sequences in inverted orientation with a K_d of 300 nM. The implications of these data for promoter binding and the curious quaternary structural transitions of SinR upon binding to (i) SinI and (ii) the SinR-like protein SlrR, which “repurposes” SinR as a repressor of autolysin and motility genes, are discussed.