Competition between folding and glycosylation in the endoplasmic reticulum.

Using carboxypeptidase Y in Saccharomyces cerevisiae as a model system, the in vivo relationship between protein folding and N-glycosylation was studied. Seven new sites for N-glycosylation were introduced at positions buried in the folded protein structure. The level of glycosylation of such new acceptor sites was analysed by pulse-labelling under two sets of conditions that are known to reduce the rate of folding: (i) addition of dithiothreitol to the growth medium and (ii) introduction of deletions in the propeptide. A variety of effects was observed, depending on the position of the new acceptor sites. In some cases, all the newly synthesized mutant protein was modified at the novel site while in others no modification took place. In the most interesting category of mutants, the level of glycosylation was dependent on the conditions for folding. This shows that folding and glycosylation reactions can compete in vivo and that glycosylation does not necessarily precede folding. The approach described may be generally applicable for the analysis of protein folding in vivo.     

Crystal structure of Mycobacterium tuberculosis diaminopimelate decarboxylase,an essential enzyme in bacterial lysine biosynthesis

The Mycobacterium tuberculosis lysA gene encodes the enzyme meso-diaminopimelate decarboxylase (DAPDC), a pyridoxal-5'-phosphate (PLP)-dependent enzyme. The enzyme catalyzes the final step in the lysine biosynthetic pathway converting meso-diaminopimelic acid (DAP) to l-lysine. The lysA gene of M. tuberculosis H37Rv has been established as essential for bacterial survival in immunocompromised mice, demonstrating that de novo biosynthesis of lysine is essential for in vivo viability. Drugs targeted against DAPDC could be efficient anti-tuberculosis drugs, and the three-dimensional structure of DAPDC from M. tuberculosis complexed with reaction product lysine and the ternary complex with PLP and lysine in the active site has been determined. The first structure of a DAPDC confirms its classification as a fold type III PLP-dependent enzyme. The structure shows a stable 2-fold dimer in head-to-tail arrangement of a triose-phosphate isomerase (TIM) barrel-like alpha/beta domain and a C-terminal beta sheet domain, similar to the ornithine decarboxylase (ODC) fold family. PLP is covalently bound via an internal aldimine, and residues from both domains and both subunits contribute to the binding pocket. Comparison of the structure with eukaryotic ODCs, in particular with a di-fluoromethyl ornithine (DMFO)-bound ODC from Trypanosoma bruceii, indicates that corresponding DAP-analogues might be potential inhibitors for mycobacterial DAPDCs.     

In vitro folding and thermodynamic stability of an antibody fragment selected in vivo for high expression levels in Escherichia coli cytoplasm.

We recently isolated a mutant of a human anti-beta-galactosidase single chain antibody fragment (scFv) able to fold at high levels in Escherichia coli cytoplasm. When targeted to the periplasm, this mutant and the wild-type scFv are both expressed at comparable levels in a soluble, active and oxidized form. If a reducing agent is added to the growth medium, only the mutant scFv is still able to fold, showing that in vivo aggregation is a direct consequence of the lack of disulphide bond formation and not of the cellular localization. In vitro denaturation/renaturation experiments show that the mutant protein is more stable than the wild-type scFv. Furthermore, refolding kinetics under reducing conditions show that the mutant folds faster than the wild-type protein. Aggregation does not proceed from the native or unfolded conformation of the protein, but from a species only present during the unfolding/refolding transition. In conclusion, the in vivo properties of the mutant scFv can be explained by, first, an increase in the stability of the protein in order to tolerate the removal of the two disulphide bonds and, second, a modification of its folding properties that reduces the kinetic competition between folding and aggregation of a reduced folding intermediate.     

Mutation of the predicted ATP binding site inactivates both activities of isocitrate dehydrogenase kinase/phosphatase

In Escherichia coli, the reversible phosphorylation of isocitrate dehydrogenase (IDH) is catalyzed by a bifunctional protein: IDH kinase/phosphatase. Although both IDH kinase and IDH phosphatase require ATP, the amino acid sequence of IDH kinase/phosphatase contains a single sequence that matches the consensus for ATP binding sites. A mutation that converted the "invariant" lysine (residue 336) of this consensus sequence to a methionine reduced the activities of both IDH kinase and IDH phosphatase by factors of greater than 500, to levels below the detection limits of the assays. The apparent elimination of both IDH kinase and IDH phosphatase by this mutation is consistent with the proposal that these activities share a common ATP binding site and that these reactions may occur at the same active site. Although conversion of Lys336 to a methionine eliminated detectable IDH kinase activity as measured in vitro, the mutant allele retained the ability to complement an aceK deletion mutation, restoring the ability of these cells to grow on minimal acetate medium. Complementation apparently resulted because the mutant protein retained sufficient activity to phosphorylate IDH in vivo. To determine whether the enzymatic assays performed in vitro had correctly reflected the activity of the mutant protein in vivo, we measured the rates at which mutant and wild-type cultures could incorporate [32P]inorganic phosphate into IDH. The wild-type culture achieved maximal incorporation in less than 3 min. In contrast, 32P incorporation was only barely detectable after 30 min in the mutant culture, indicating that the activity of the mutant protein is, indeed, greatly reduced in vivo. The ability of the mutant allele to complement an aceK null mutation thus suggests that IDH kinase/phosphatase levels in wild-type cells are in great excess over what is required for steady-state growth on acetate medium.     

Analysis of the factors that stabilize a designed two-stranded antiparallel beta-sheet

Autonomously folding beta-hairpins (two-strand antiparallel beta-sheets) have become increasingly valuable tools for probing the forces that control peptide and protein conformational preferences. We examine the effects of variations in sequence and solvent on the stability of a previously designed 12-residue peptide (1). This peptide adopts a beta-hairpin conformation containing a two-residue loop (D-Pro-Gly) and a four-residue interstrand sidechain cluster that is observed in the natural protein GB1. We show that the conformational propensity of the loop segment plays an important role in beta-hairpin stability by comparing 1 with (D)P--> N mutant 2. In addition, we show that the sidechain cluster contributes both to conformational stability and to folding cooperativity by comparing 1 with mutant 3, in which two of the four cluster residues have been changed to serine. Thermodynamic analysis suggests that the high loop-forming propensity of the (D)PG segment decreases the entropic cost of beta-hairpin formation relative to the more flexible NG segment, but that the conformational rigidity of (D)PG may prevent optimal contacts between the sidechains of the GB1-derived cluster. The enthalpic favorability of folding in these designed beta-hairpins suggests that they are excellent scaffolds for studying the fundamental mechanisms by which amino acid sidechains interact with one another in folded proteins.     

Aspartate kinase-independent lysine synthesis in an extremely thermophilic bacterium, Thermus thermophilus: lysine is synthesized via alpha-aminoadipic acid not via diaminopimelic acid

An aspartate kinase-deficient mutant of Thermus thermophilus, AK001, was constructed. The mutant strain did not grow in a minimal medium, suggesting that T. thermophilus contains a single aspartate kinase. Growth of the mutant strain was restored by addition of both threonine and methionine, while addition of lysine had no detectable effect on growth. To further elucidate the lysine biosynthetic pathway in T. thermophilus, lysine auxotrophic mutants of T. thermophilus were obtained by chemical mutagenesis. For all lysine auxotrophic mutants, growth in a minimal medium was not restored by addition of diaminopimelic acid, whereas growth of two mutants was restored by addition of alpha-aminoadipic acid, a precursor of lysine in biosynthetic pathways of yeast and fungi. A BamHI fragment of 4.34 kb which complemented the lysine auxotrophy of a mutant was cloned. Determination of the nucleotide sequence suggested the presence of homoaconitate hydratase genes, termed hacA and hacB, which could encode large and small subunits of homoaconitate hydratase, in the cloned fragment. Disruption of the chromosomal copy of hacA yielded mutants showing lysine auxotrophy which was restored by addition of alpha-aminoadipic acid or alpha-ketoadipic acid. All of these results indicated that in T. thermophilus, lysine was not synthesized via the diaminopimelic acid pathway, believed to be common to all bacteria, but via a pathway using alpha-aminoadipic acid as a biosynthetic intermediate.     

Growth of an Escherichia coli mutant deficient in respiration

An Escherichia coli mutant deficient in genes for heme biosynthesis grew in medium of initial pH 8 containing 1% tryptone and glucose under aerobic growth conditions, and its doubling time was approximately 60 min at 37°C. The growth rate was not increased under O₂-limiting conditions. When the mutant was grown in medium of initial pH 6, growth stopped at the middle of the exponential growth phase. This could be overcome and the growth yield increased by the addition of 20 mM lysine to the growth medium. Lysine did not prevent the decrease in the medium pH as growth proceeded, making it unlikely that lysine decarboxylation stimulates growth by the alkalinization of the medium. These results indicate that respiration is not obligatory for growth under aerobic conditions, but growth without respiration at low pH requires a large amount of lysine.     

Active Site Mutations in Yeast Protein Disulfide Isomerase Cause Dithiothreitol Sensitivity and a Reduced Rate of Protein Folding in the Endoplasmic Reticulum

Aspects of protein disulfide isomerase (PDI) function have been studied in yeast in vivo. PDI contains two thioredoxin-like domains, a and a′, each of which contains an active-site CXXC motif. The relative importance of the two domains was analyzed by rendering each one inactive by mutation to SGAS. Such mutations had no significant effect on growth. The domains however, were not equivalent since the rate of folding of carboxypeptidase Y (CPY) in vivo was reduced by inactivation of the a domain but not the a′ domain. To investigate the relevance of PDI redox potential, the G and H positions of each CGHC active site were randomly mutagenized. The resulting mutant PDIs were ranked by their growth phenotype on medium containing increasing concentrations of DTT. The rate of CPY folding in the mutants showed the same ranking as the DTT sensitivity, suggesting that the oxidative power of PDI is an important factor in folding in vivo. Mutants with a PDI that cannot perform oxidation reactions on its own (CGHS) had a strongly reduced growth rate. The growth rates, however, did not correlate with CPY folding, suggesting that the protein(s) required for optimal growth are dependent on PDI for oxidation. pdi1-deleted strains overexpressing the yeast PDI homologue EUG1 are viable. Exchanging the wild-type Eug1p C(L/I)HS active site sequences for C(L/I)HC increased the growth rate significantly, however, further highlighting the importance of the oxidizing function for optimal growth.     

Lysine propionylation and butyrylation are novel post-translational modifications in histones

The positively charged lysine residue plays an important role in protein folding and functions. Neutralization of the charge often has a profound impact on the substrate proteins. Accordingly all the known post-translational modifications at lysine have pivotal roles in cell physiology and pathology. Here we report the discovery of two novel, in vivo lysine modifications in histones, lysine propionylation and butyrylation. We confirmed, by in vitro labeling and peptide mapping by mass spectrometry, that two previously known acetyltransferases, p300 and CREB-binding protein, could catalyze lysine propionylation and lysine butyrylation in histones. Finally p300 and CREB-binding protein could carry out autopropionylation and autobutyrylation in vitro. Taken together, our results conclusively establish that lysine propionylation and lysine butyrylation are novel post-translational modifications. Given the unique roles of propionyl-CoA and butyryl-CoA in energy metabolism and the significant structural changes induced by the modifications, the two modifications are likely to have important but distinct functions in the regulation of biological processes.     

Thermostability of temperature-sensitive folding mutants of the P22 tailspike protein

Temperature-sensitive folding mutations (tsf) of the thermostable P22 tailspike protein prevent the mutant polypeptide chain from reaching the native state at the higher end of the temperature range of bacterial growth (37-42 degrees C). At lower temperatures the mutant polypeptide chains fold and associate into native proteins. The melting temperatures of the purified native forms of seven different tsf mutant proteins have been determined by differential scanning calorimetry. Under conditions in which the wild type protein had a melting temperature of 88.4 degrees C, the melting temperatures of the mutant proteins were all above 82 degrees C, more than 40 degrees C higher than the temperature for expression of the folding defect. Because the folding defects were observed in vivo, the thermostability of the native protein was also examined with infected cells. Once matured at 28 degrees C, intracellular tsf mutant tailspikes remained native when the cells were transferred to 42 degrees C, a temperature that prevents newly synthesized tsf chains from folding correctly. These results confirm that the failure of tsf polypeptide chains to reach their native state is not due to a lowered stability of the native state. Such mutants differ from the class of ts mutations which render the native state thermolabile. The intracellular folding defects must reflect decreased stabilities of folding intermediates or alteration in the off-pathway steps leading to aggregation and inclusion body formation. These results indicate that the stability of a native protein within the cells is not sufficient to insure the successful folding of the newly synthesized chains into the native state.     

A lysine in the ATP-binding site of P130gag-fps is essential for protein-tyrosine kinase activity.

The P130gag-fps transforming protein of Fujinami sarcoma virus (FSV) possesses tyrosine-specific protein kinase activity and autophosphorylates at Tyr-1073. Within the kinase domain of P130gag-fps is a putative ATP-binding site containing a lysine (Lys-950) homologous to lysine residues in cAMP-dependent protein kinase and p60v-src which bind the ATP analogue p-fluorosulfonylbenzoyl-5' adenosine. FSV mutants in which the codon for Lys-950 has been changed to codons for arginine or glycine encode metabolically stable but enzymatically defective proteins which are unable to effect neoplastic transformation. Kinase-defective P130gag-fps containing arginine at residue 950 was normally phosphorylated at serine residues in vivo suggesting that this amino acid substitution has a minimal effect on protein folding and processing. The inability of arginine to substitute for lysine at residue 950 suggests that the side chain of Lys-950 is essential for P130gag-fps catalytic activity, probably by virtue of a specific interaction with ATP at the phosphotransfer active site. Tyr-1073 of the Arg-950 P130gag-fps mutant protein was not significantly autophosphorylated either in vitro or in vivo, but could be phosphorylated in trans by enzymatically active P140gag-fps. These data indicate that Tyr-1073 can be modified by intermolecular autophosphorylation.     

Analysis of the effect of potato carboxypeptidase inhibitor pro-sequence on the folding of the mature protein.

Protein folding can be modulated in vivo by many factors. While chaperones act as folding catalysts and show broad substrate specificity, some pro-peptides specifically facilitate the folding of the mature protein to which they are bound. Potato carboxypeptidase inhibitor (PCI), a 39-residue protein carboxypeptidase inhibitor, is synthesized in vivo as a precursor protein that includes a 27-residue N-terminal and a seven-residue C-terminal pro-regions. In this work the disulfide-coupled folding of mature PCI in vitro has been compared with that of the same protein extended with either the N-terminal pro-sequence (ProNtPCI) or both N- and C-terminal pro-sequences (ProPCI), and also with the N-terminal pro-sequence in trans (ProNt + PCI). No significant differences can be observed in the folding kinetics or efficiencies of all these molecules. In addition, in vivo folding studies in Escherichia coli have been performed using wild-type PCI and three PCI mutant forms with and without the N-terminal pro-sequence, the mutations had been previously reported to affect folding of the PCI mature form. The extent to which the 'native-like' form was secreted to the media by each construction was not affected by the presence of the N-terminal pro-sequence. These results indicate that PCI does not depend on the N-terminal pro-sequence for its folding in both, in vitro and in vivo in E. coli. However, structural analysis by spectroscopy, hydrogen exchange and limited proteolysis by mass spectrometry, indicate the capability of such N-terminal pro-sequence to fold within the precursor form.     

Antibiotic amendment for suppression of indigenous microflora in feed sources for an Escherichia coli auxotroph lysine assay

Growth responses of lysine auxotrophic mutants of Escherichia coli have been used as a measurement of bioavailable lysine in protein sources and animal feeds. Sterilizing feed samples by autoclaving to eliminate non-specific background growth of indigenous feed micro-organisms prior to conducting the bacterial assay may introduce chemical and physical alterations to the feeds, influencing the estimation of available feed lysine. In this study, an antibiotic- and antifungal-supplemented medium was constructed to support growth of an E. coli lysine auxotroph assay organism, and was tested for its ability to repress indigenous bacterial and fungal growth in feed samples. To determine which antibiotics to include, an ampicillin-sensitive E. coli lysine mutant strain (ATCC no. 23812) was screened for antibiotic resistance and transformed with a plasmid carrying an ampicillin resistance gene. Maximum optical density quantitative response of the E. coli auxotroph to lysine was not altered by the antibiotic medium amendments (ampicillin, novobiocin and cycloheximide). Indigenous microfloral growth in a variety of typical animal feeds was suppressed in the presence of the antistatic agents. The estimated lysine recovery was 91.6% and 98.1% when the medium was used in an assay of available lysine in a lysine-supplemented feed. This indicates that the antibiotic-amended basal medium can be used for the E. coli-determined lysine availability of a variety of animal feeds without prior sterilization of the feed sources.     

Characterization of a lysK gene as an argE homolog in Thermus thermophilus HB27

We conducted a chromosome walk to obtain a DNA fragment downstream of lysJ and found an argE homolog in a putative operon composed of lysJ-orfC-orfD-argE homologs. A knockout mutant of the argE homolog showed significantly slow growth on a minimal medium, and the growth was markedly improved by addition of lysine. We therefore termed this gene lysK. Purified LysK protein has deacetylating activities for both N(2)-acetyllysine and N(2)-acetylornithine at almost equal efficiency. These results suggest that lysK which may share an ancestor with argE functions not only for the lysine biosynthesis, but also for arginine biosynthesis in Thermus thermophilus.     

The reactivity of functional groups as a probe for investigating the topography of tobacco mosaic virus. The use of mutants with additional lysine residues in the coat protein

Several mutants of tobacco mosaic virus that contain additional lysine residues as a result of mutations in the coat protein were investigated. Mutant E66 has a lysine residue replacing asparagine at position 140 when compared with the wild-type vulgare and this lysine residue reacts readily in the intact virus with methyl picolinimidate. Mutant B13a has two new lysine residues in the coat protein, replacing a glutamine at position 9 and an asparagine at position 33, whereas mutant B13b has the single replacement of glutamine by lysine at position 9. The lysine residue at position 9 in mutants B13a and B13b also reacts readily with methyl picolinimidate in the intact virus but the lysine at position 33 in mutant B13a did not react under these conditions. However, when the isolated coat protein from mutant B13a was treated with methyl picolinimidate, the lysine residue at position 33 did become modified, showing that the loss in reactivity of this residue towards the imidoester in the intact virus is a result of the assembly of the protein subunit into the virus structure. These results are compatible with and extend previous studies on the sero-logical properties of mutants of tobacco mosaic virus and illustrate the value of methyl picolinimidate as a reagent for probing the accessibility of amino groups in proteins. When intact tobacco mosaic virus (vulgare) was treated with p-iodobenzenesulphonyl chloride, no reaction with the lysine residues at positions 33 or 68 in the virus subunit could be detected but complete modification of tyrosine-139 was achieved. This result also extends previous studies with other reagents. The usefulness of the differential reactivity of the lysine residues in tobacco mosaic virus and its mutants as a means of attaching heavy-atom labels at chemically defined positions for subsequent X-ray-diffraction analysis and the implications of these experiments for deciphering the folding of the peptide chain in the virus subunit are discussed.     

Thialysine and selenalysine utilization for protein synthesis by E. coli in comparison with lysine

Utilization of thialysine and selenalysine for protein synthesis by a lysine requiring E. coli mutant was studied. Incorporation into proteins of thialysine or selenalysine, added to culture medium together with lysine, becomes evident when the amount of available lysine in the medium is highly reduced, that is the mutant utilizes the isologs only after all the available natural aminoacid has been utilized. Compared to selenalysine, thialysine is better utilized; when both isologs are present in the medium at equal concentrations, up to 46% of protein lysine is substituted by thialysine and only 12% by selenalysine.     

Escherichia coli regulatory mutation affecting lysine transport and lysine decarboxylase

A spontaneous thiosine-resistant mutant of Escherichia coli was shown to have the following characteristics: lowered initial rate of lysine uptake and lowered plateau level of accumulation of exogenous lysine by both the lysine-specific and the general basic amino acid transport systems; altered repressibility of these two lysine transport systems; a derepressed level of lysine decarboxylase; normal growth rate; parental levels of lysyl-transfer ribonucleic acid synthetase and the inducible and constitutive arginine and ornithine decarboxylases. Both the mutant (lysP) and its parent (lysP+) feed a lysine auxotroph when they are plated in proximity on solid medium. However, the feeding response was observable after 1 day less of incubation when the mutant was the feeding strain. Despite the derepressed level of lysine decarboxylase in exponential cultures of the mutant extracts of these cultures had no detectable cadaverine pool. Conjugation experiments established the following gene order: gyrA (formerly nalA) lysP metG his. All thiosine-resistant recombinants assayed showed reduced lysine transport. In many of these recombinants the derepression of lysine decarboxylase was not expressed.     

Protein aggregation kinetics in an Escherichia coli strain overexpressing a Salmonella typhimurium CheY mutant gene.

The tendency of recombinant protein in bacteria to partition into soluble and insoluble forms is attributed, in general, to a kinetic competition between protein folding and aggregation. However, little experimental work has actually been performed in vivo on the kinetics and mechanisms of protein folding and aggregation. Results are presented here from radiolabeling experiments which monitored the kinetics of recombinant protein aggregation in actively growing cultures. The strain used was an Escherichia coli strain overexpressing a Salmonella typhimurium CheY mutant gene. The rate of CheY aggregation was found to be time dependent in that the tendency of CheY to aggregate was greater for newly translated molecules, i.e., those translated within the previous several minutes, than for molecules translated less recently. CheY protein molecules that were translated less recently continued to aggregate for several hours but at a lower rate. The movement of soluble CheY to the insoluble form was enhanced at elevated growth temperatures and inhibited by the presence of chloramphenicol. The latter observation suggests that ongoing translation facilitates the movement of soluble CheY to the insoluble form. The implications of these results for the mechanism of protein aggregation in vivo, i.e., inclusion body formation, are discussed.