Induced fit of a peptide loop of methionyl-tRNA formyltransferase triggered by the initiator tRNA substrate

A 16-aa insertion loop present in eubacterial methionyl-tRNA formyltransferases (MTF) is critical for specific recognition of the initiator tRNA in Escherichia coli. We have studied the interactions between this region of the E. coli enzyme and initiator methionyl-tRNA (Met-tRNA) by using two complementary protection experiments: protection of MTF against proteolytic cleavage by tRNA and protection of tRNA against nucleolytic cleavage by MTF. The insertion loop in MTF is uniquely sensitive to cleavage by trypsin. We show that the substrate initiator Met-tRNA protects MTF against trypsin cleavage, whereas a formylation-defective mutant initiator Met-tRNA, which binds to MTF with approximately the same affinity, does not. Also, mutants of MTF within the insertion loop (which are defective in formylation) are not protected by the initiator Met-tRNA. Thus, a functional enzyme-substrate complex is necessary for protection of MTF against trypsin cleavage. Along with other data, these results strongly suggest that a segment of the insertion loop, which is exposed and unstructured in MTF, undergoes an induced fit in the functional MTF.Met-tRNA complex but not in the nonfunctional one. Footprinting experiments show that MTF specifically protects the acceptor stem and the 3'-end region of the initiator Met-tRNA against cleavage by double and single strand-specific nucleases. This protection also depends on formation of a functional MTF.Met-tRNA complex. Thus, the insertion loop interacts mostly with the acceptor stem of the initiator Met-tRNA, which contains the critical determinants for formylation.     

Suppressor mutations in Escherichia coli methionyl-tRNA formyltransferase: role of a 16-amino acid insertion module in initiator tRNA recognition

The specific formylation of initiator methionyl-tRNA by methionyl-tRNA formyltransferase (MTF; EC 2.1.2.9) is important for the initiation of protein synthesis in eubacteria and in eukaryotic organelles. The determinants for formylation in the tRNA are clustered mostly in the acceptor stem. As part of studies on the molecular mechanism of recognition of the initiator tRNA by MTF, we report here on the isolation and characterization of suppressor mutations in Escherichia coli MTF, which compensate for the formylation defect of a mutant initiator tRNA, lacking a critical determinant in the acceptor stem. We show that the suppressor mutant in MTF has a glycine-41 to arginine change within a 16-amino acid insertion found in MTF from many sources. A mutant with glycine-41 changed to lysine also acts as a suppressor, whereas mutants with changes to aspartic acid, glutamine, and leucine do not. The kinetic parameters of the purified wild-type and mutant Arg-41 and Lys-41 enzymes, determined by using the wild-type and mutant tRNAs as substrates, show that the Arg-41 and Lys-41 mutant enzymes compensate specifically for the strong negative effect of the acceptor stem mutation on formylation. These and other considerations suggest that the 16-amino acid insertion in MTF plays an important role in the specific recognition of the determinants for formylation in the acceptor stem of the initiator tRNA.     

Functional interaction of an arginine conserved in the sixteen amino acid insertion module of Escherichia coli methionyl-tRNA formyltransferase with determinants for formylation in the initiator tRNA

Formylation of initiator methionyl-tRNA by methionyl-tRNA formyltransferase (MTF) is important for initiation of protein synthesis in eubacteria. The determinants for formylation are clustered mostly in the acceptor stem of the initiator tRNA. Previous studies suggested that a 16 amino acid insertion loop, present in all eubacterial MTF's (residues 34-49 in the E. coli enzyme), plays an important role in specific recognition of the initiator tRNA. Here, we have analyzed the effect of site-specific mutations of amino acids within this region. We show that an invariant arginine at position 42 within the loop plays a very important role both in the steps of substrate binding and in catalysis. The kinetic parameters of the R42K and R42L mutant enzymes using acceptor stem mutant initiator tRNAs as substrates suggest that arginine 42 makes functional contacts with the determinants at the 3:70 and possibly also the 2:71 base pairs in the acceptor stem of the initiator tRNA. The kinetic parameters of the G41R/R42L double mutant enzyme are essentially the same as those of R42L mutant, suggesting that the requirement for arginine at position 42 cannot be fulfilled by an arginine at position 41. Along with other data, this result suggests that the insertion loop, which is normally unstructured and flexible, adopts a defined conformation upon binding to the tRNA.     

Expression and characterization of bovine mitochondrial methionyl-tRNA transformylase

Translational initiation in bacteria and some organelles such as mitochondria and chloroplasts requires formyl-methionyl-tRNA (fMet-tRNA). Methionyl-tRNA (Met-tRNA) undergoes formylation by methionyl-tRNA transformylase (MTF), and the resulting fMet-tRNA is utilized exclusively in the initiation process. The gene encoding mammalian mitochondrial MTF (MTFmt) was cloned recently. When the cDNA corresponding to mature MTFmt was cloned into an expression vector, no expression of MTFmt was observed. However, if the cDNA was fused with the histidine-tag sequence at the N-terminus, MTFmt could be expressed in Escherichia coli. The recombinant enzyme was purified by a single step on a histidine-binding metal affinity column. We previously found that native MTFmt is able to formylate E. coli elongator Met-tRNA as well as the initiator Met-tRNA. The specific formylation of the initiator Met-tRNA by E. coli MTF is quite important in bacterial translational initiation. The purified recombinant MTFmt with the histidine-tag showed almost identical kinetic parameters to those of native MTFmt. This expression system is suitable for the rapid, efficient production of MTFmt in amounts adequate for further biophysical studies, which will provide another approach for elucidating the formylation mechanism, in addition to studies on E. coli MTF.     

HLA-B27 (B*2701) specificity for peptides lacking Arg2 is determined by polymorphism outside the B pocket

B*2701 differs from B*2705-by three amino acid changes: D-->Y74, D-->N77, L-->A81, and from B*2702 only by two: D-->Y74 and T-->I80. Tyr74 is located in the C/F cavity of the peptide-binding site, and is unique to B*2701 among HLA-B27 subtypes. Binding of natural B*2705 and B*2702 ligands to B*2701, and to mutants mimicking subtype changes, was analyzed. In addition, sequencing of the peptides bound in vivo by B*2701 and the Y74 mutant was carried out. The main distinctive feature of B*2701 was its presentation of peptides with Gln2. Synthetic analogs bound in vitro similarly as the corresponding ligands with Arg2. Moreover, both Gln2 and Arg2 were dominant upon pool sequencing of B*2701-bound peptides, and 2 of 8 natural ligands contained Gln2. Suitability of Gln2 was largely determined by the Y74 change, as indicated by: 1) binding of Gln2 analogs to this mutant, and 2) detection of Gln2 by pool sequencing of Y74-bound peptides. B*2701 bound peptides with C-terminal aromatic or Leu residues, and interacted with these motifs more strongly than B*2702. The Y74 mutation alone was not responsible for poor binding of peptides with C-terminal basic residues to B*2701, since they bound efficiently and at least one was presented in vivo by this mutant. Most peptides bound to the A81 mutant worse than to B*2705, but frequently better than to B*2701 or B*2702, suggesting that other subtype changes were compensatory. The peptide specificity of B*2701 suggests that this subtype may determine susceptibility to spondyloarthropathy.     

Hydroxyl radical-induced cross-linking of thymine and lysine: identification of the primary structure and mechanism

Hydroxyl radical-induced formation of a cross-link of thymine (Thy) and lysine (Lys) in the gamma-radiolysis of N2O-saturated aqueous solution was studied. A Thy-Lys cross-link (I) of the formal structure that OH radical and 4-carbon-centered Lys radical added respectively to C(5) and C(6) positions of Thy was isolated by a preparative HPLC and identified by a FAB-HRMS. The primary cross-link I was dehydrated by treatment with HCl at 120 degrees C to yield the secondary structure (II) possessing a C(5)-C(6) double bond in the Thy moiety: the latter structure II was reported previously (Dizdaroglu, M.; Gajewski, E. Cancer Res. 1989, 49, 3463-3467). A pulse radiolysis study with a redox titration method indicated that 4-carbon centered Lys radical intermediate was of neutral redox reactivity in contrast to reducing reactivity of 5-hydroxy-5,6-dihydrothymin-6-yl radical intermediate. The cross-link I could be formed by a conventional radical recombination mechanism, but not by an ionic recombination mechanism involving a redox reaction between the radical intermediates.     

Glutaminyl-tRNA synthetase

Among the twenty aminoacyl-tRNA synthetases glutaminyl-tRNA synthetase occupies a special position: it is one of only two enzymes of this family which is not found in all organisms, being mainly absent from gram positive eubacteria, archaebacteria and organelles. The E. coli GlnRS is relatively small with 553 amino acids and a molecular mass of 64.4 kDa and functions as a monomer. The mammalian enzymes are somewhat larger and can be parts of multienzyme complexes. Crystal structures were solved of E. coli GlnRS complexed with tRNA(Gln) and ATP, of this complex containing tRNA(Gln) replaced by unmodified tRNA(Gln), and of three complexes with mutated GlnRS enzymes. The GlnRS molecule consists of four domains, the catalytic site is located in the Rossman fold, typical for class I synthetases, and the reaction mechanism follows the normal adenylate pathway. The enzyme shows many similarities with glutamyl-tRNA synthetase; a common ancestor of both molecules is well established. In the E. coli system recognition of the cognate tRNA has been studied in many details using both natural and artificial mutants of tRNA(Gln) and of the enzyme: GlnRS recognizes mainly conventional parts of the tRNA molecule, namely some bases of the anticodon loop and parts of the acceptor stem.     

CCA addition by tRNA nucleotidyltransferase: polymerization without translocation?

The CCA-adding enzyme repairs the 3'-terminal CCA sequence of all tRNAs. To determine how the enzyme recognizes tRNA, we probed critical contacts between tRNA substrates and the archaeal Sulfolobus shibatae class I and the eubacterial Escherichia coli class II CCA-adding enzymes. Both CTP addition to tRNA-C and ATP addition to tRNA-CC were dramatically inhibited by alkylation of the same tRNA phosphates in the acceptor stem and TPsiC stem-loop. Both enzymes also protected the same tRNA phosphates in tRNA-C and tRNA-CC. Thus the tRNA substrate must remain fixed on the enzyme surface during CA addition. Indeed, tRNA-C cross-linked to the S. shibatae enzyme remains fully active for addition of CTP and ATP. We propose that the growing 3'-terminus of the tRNA progressively refolds to allow the solitary active site to reuse a single CTP binding site. The ATP binding site would then be created collaboratively by the refolded CC terminus and the enzyme, and nucleotide addition would cease when the nucleotide binding pocket is full. The template for CCA addition would be a dynamic ribonucleoprotein structure.     

Identification of transglutaminase-reactive residues in S100A11

The recent finding that S100A11 is a component of the keratinocyte cornified envelope (CE) (Robinson, N. A., Lapic, S., Welter, J. F., and Eckert, R. L. (1997) J. Biol. Chem. 272, 12035-12046) suggests that S100A11 is a transglutaminase (TG) substrate. In the present study we show that S100A11 forms multimers when cultured keratinocytes are challenged by increased levels of intracellular calcium and that multimer formation is inhibited by the TG inhibitor, cystamine. These S100A11 multimers appear to be incorporated into the CE, as immunoreactive S100A11 is detected in purified envelopes prepared from cultured cells and from foreskin epidermis. To study S100A11 as a transglutaminase substrate, recombinant human S100A11 (rhS100A11) was used in a cell-free cross-linking system. [14C]Putrescine, a primary amine, labels rhS100A11 in a TG-dependent manner. Trypsin digestion of [14C]putrescine-labeled rhS100A11 releases one radiolabeled peptide, Ala98-Lys103. The glutamine residue in this segment, Gln102, is the site of radiolabel incorporation indicating that Gln102 functions as an amine acceptor. The ability of S100A11 to form multimers indicates that it also has a reactive lysine residue that functions as an amine donor. To identify the reactive residue, we compared the high pressure liquid chromatography profile of trypsin-digested rhS100A11 monomer to that of cross-linked rhS100A11. A unique cross-linked peptide was purified and identified as Met-Ala-Lys3-Ilu-Ser-Ser-Pro-Thr-Glu-Thr-Glu-Arg cross-linked via an Lys3-Gln102 isopeptide bond to Ala-Val-Pro-Ser-Gln102-Lys. These studies show that S100A11 is post-translationally modified by transglutaminase, that it can be cross-linked to form multimers, that it is present in CEs from cultured keratinocytes and in vivo epidermis, and that Lys3 and Gln102 are specific sites of cross-link formation.     

Ligand interactions with eukaryotic translation initiation factor 2: role of the gamma-subunit

Eukaryotic translation initiation factor 2 (eIF-2) comprises three non-identical subunits alpha, beta and gamma. In vitro, eIF-2 binds the initiator methionyl-tRNA in a GTP-dependent fashion. Based on similarities between eukaryotic eIF-2gamma proteins and eubacterial EF-Tu proteins, we previously proposed a major role for the gamma-subunit in binding guanine nucleotide and tRNA. We have tested this hypothesis by examining the biochemical activities of yeast eIF-2 purified from wild-type strains and strains harboring mutations in the eIF-2gamma structural gene (GCD11) predicted to alter ligand binding by eIF-2. The alteration of tyrosine 142 in yeast eIF-2gamma, corresponding to histidine 66 in Escherichia coli EF-Tu, dramatically reduced the affinity of eIF-2 for Met-tRNAi(Met) without affecting the k(off) value for guanine nucleotides. In contrast, non-lethal substitutions at a conserved lysine residue (K250) in the putative guanine ring-binding loop increased the off-rate for GDP, thereby mimicking the function of the guanine nucleotide exchange factor eIF-2B, without altering the apparent dissociation constant for Met-tRNAi(Met). For eIF-2[gamma-K250R], the increased off-rate also seen for GTP was masked by the presence of Met-tRNAi(Met) in vitro. In vivo, increasing the dose of the yeast initiator tRNA gene suppressed the slow-growth phenotype and reduced GCN4 expression in gcd11-K250R and gcd11-Y142H strains. These studies indicate that the gamma-subunit of eIF-2 does indeed provide EF-Tu-like function to the eIF-2 complex, and further suggest that the level of Met-tRNAi(Met) is critical for maintaining wild-type rates of initiation in vivo.     

Editing function of Escherichia coli cysteinyl-tRNA synthetase: cyclization of cysteine to cysteine thiolactone

A cyclic sulfur compound, identified as cysteine thiolactone by several chemical and enzymatic tests, is formed from cysteine during in vitro tRNA(Cys) aminoacylation catalyzed by Escherichia coli cysteinyl-tRNA synthetase. The mechanism of cysteine thiolactone formation involves enzymatic deacylation of Cys-tRNA(Cys) (k = 0.017 s-1) in which nucleophilic sulfur of the side chain of cysteine in Cys-tRNA(Cys) attacks its carboxyl carbon to yield cysteine thiolactone. Nonenzymatic deacylation of Cys-tRNA(Cys) (k = 0.0006 s-1) yields cysteine, as expected. Inhibition of enzymatic deacylation of Cys-tRNA(Cys) by cysteine and Cys-AMP, but not by ATP, indicates that both synthesis of Cys-tRNA(Cys) and cyclization of cysteine to the thiolactone occur in a single active site of the enzyme. The cyclization of cysteine is mechanistically similar to the editing reactions of methionyl-tRNA synthetase. However, in contrast to methionyl-tRNA synthetase which needs the editing function to reject misactivated homocysteine, cysteinyl-tRNA synthetase is highly selective and is not faced with a problem in rejecting noncognate amino acids. Despite this, the present day cysteinyl-tRNA synthetase, like methionyl-tRNA synthetase, still retains an editing activity toward the cognate product, the charged tRNA. This function may be a remnant of a chemistry used by an ancestral cysteinyl-tRNA synthetase.     

Overlapping peptide-binding specificities of HLA-B27 and B39: evidence for a role of peptide supermotif in the pathogenesis of spondylarthropathies

OBJECTIVE: Previous studies indicated the increase of HLA-B39 among HLA-B27 negative patients with spondylarthropathies (SpA). This study was performed to examine whether the natural ligands of HLA-B27 are capable of binding to HLA-B39. METHODS: Peptides were synthesized according to the sequences of known natural ligands of HLA-B27 or B39 and were tested for their binding to HLA-B*3901 and B*2705 by quantitative peptide binding assay, using a TAP-deficient RMA-S cell line transfected with human beta2-microglobulin and HLA class I heavy chain genes. RESULTS: Four of the 10 HLA-B27 binding peptides significantly bound to HLA-B*3901. All 4 peptides had hydrophobic/aromatic amino acids (Leu or Phe) at the C-terminus. In contrast, peptides with basic residues (Lys, Arg) or Tyr at the C-terminus did not bind to B*3901. In parallel experiments, 1 of the 2 natural ligands of HLA-B*3901 was found to bind to B*2705. CONCLUSION: A subset of natural HLA-B27 ligands was capable of binding to B*3901. In addition to Arg at position 2 (Arg2), hydrophobic/aromatic C-terminal residues, such as Leu or Phe, seemed to be crucial for the cross-specificity. These results suggested that HLA-B27 and B39 recognize overlapping peptide repertoires, supporting the hypothesis that the peptides presented by both of these class I antigens play a role in the pathogenesis of SpA.     

tRNA anticodon recognition and specification within subclass IIb aminoacyl-tRNA synthetases

Subclass IIb aminoacyl-tRNA synthetases (Asn-, Asp- and LysRS) recognize the anticodon triplet of their cognate tRNA (GUU, GUC and UUU, respectively) through an OB-folded N-terminal extension. In the present study, the specificity of constitutive lysyl-tRNA synthetase (LysS) from Escherichia coli was analyzed by cross-mutagenesis of the tRNA(Lys) anticodon, on the one hand, and of the amino acid residues composing the anticodon binding site on the other. From this analysis, a tentative model is deduced for both the recognition of the cognate anticodon and the rejection of non-cognate anticodons. In this model, the enzyme offers a rigid scaffold of amino acid residues along the beta-strands of the OB-fold for tRNA binding. Phe85 and Gln96 play a critical role in this spatial organization. This scaffold can recognize directly U35 at the center of the anticodon. Specification of the correct enzyme:tRNA complex is further achieved through the accommodation of U34 and U36. The binding of these bases triggers the conformationnal change of a flexible seven-residue loop between strands 4 and 5 of the OB-fold (L45). Additional free energy of binding is recovered from the resulting network of cooperative interactions. Such a mechanism would not depend on the modifications of the anticodon loop of tRNA(Lys) (mnm5s2U34 and t6A37). In the model, exclusion by the synthetase of non-cognate anticodons can be accounted for by a hindrance to the positioning of the L45 loop. In addition, Glu135 would repulse a cytosine base at position 35. Sequence comparisons show that the composition and length of the L45 loop are markedly conserved in each of the families composing subclass IIb aminoacyl-tRNA synthetases. The possible role of the loop is discussed for each case, including that of archaebacterial aspartyl-tRNA synthetases.     

Sugar cross-linked gelatin for controlled release: microspheres and disks

The aim of the present paper was to find a 'biocompatible' means to cross-link gelatin-based pharmaceutical devices. In particular, we have studied the ability of native and oxidized mono- and di-saccharides to induce the cross-linking of gelatin. To this end, gelatin discs and gelatin microspheres were produced and their dissolution kinetics at 37 degrees C were examined. In order to find evidence of sugar-mediated cross-linking, DSC and FTIR experiments were performed. The obtained results indicated that both native and oxidized sugars resulted to different extents, in the formation of a cross-linked gelatin network able to reduce the dissolution of gelatin. These results suggest that oxidized mono- and disaccharides could be an interesting method by which to cross-link gelatin thereby reducing the risk of toxic side effects arising from the use of synthetic cross-linkers.     

Site-directed mutagenesis of surfactant protein A reveals dissociation of lipid aggregation and lipid uptake by alveolar type II cells

Surfactant protein A (SP-A) binds to dipalmitoylphosphatidylcholine (DPPC) and induces phospholipid vesicle aggregation. It also regulates the uptake and secretion of surfactant lipids by alveolar type II cells. We introduced the single mutations Glu195-->Gln (rE195Q), Lys201-->Ala (rK201A) and Lys203-->Ala (rK203A) for rat SP-A, Arg199-->Ala (hR199A) and Lys201-->Ala (hK201A) for human SP-A, and the triple mutations Arg197, Lys201 and Lys203-->Ala (rR197A/K201A/K203A) for rat SP-A, into cDNAs for SP-A, and expressed the recombinant proteins using baculovirus vectors. All recombinant proteins avidly bound to DPPC liposomes. rE195Q, rK201A, rK203A, hR199A and hK201A function with activity comparable to wild type SP-A. Although rR197A/K201A/K203A was a potent inducer of phospholipid vesicle aggregation, it failed to stimulate lipid uptake. rR197A/K201A/K203A was a weak inhibitor for lipid secretion and did not competed with rat [125I]SP-A for receptor occupancy. From these results, we conclude that Lys201 and Lys203 of rat SP-A, and Arg199 and Lys201 of human SP-A are not individually critical for the interaction with lipids and type II cells, and that Glu195 of rat SP-A can be replaced with Gln without loss of SP-A functions. This study also demonstrates that the SP-A-mediated lipid uptake is not directly correlated with phospholipid vesicle aggregation, and that specific interactions of SP-A with type II cells are involved in the lipid uptake process.     

Types I and II collagens in intervertebral disc. Interchanging radial distributions in annulus fibrosus

Intervertebral disc is a highly specialized cartilaginous tissue, containing two genetic types of collagen (I and II). Analysis of peptides from a CNBr digest of collagen showed that the proportions of I and II varied gradually and inversely across pig annulus fibrosus, with exclusively type I at the extreme outer edge and exclusively type II in the nucleus pulposus.     

Peptide therapy for treatment of allergic diseases

G.U wobble pairs are crucial to many examples of RNA-protein recognition. We previously concluded that the G.U wobble pair in the acceptor helix of Escherichia coli alanine tRNA (tRNA(Ala)) is recognized indirectly by alanyl-tRNA synthetase (AlaRS), although direct recognition may play some role. Our conclusion was based on the finding that amber suppressor tRNA Ala with G.U shifted to an adjacent helical site retained substantial but incomplete Ala acceptor function in vivo. Other researchers concluded that only direct recognition is operative. We report here a repeat of our original experiment using tRNA(Lys) instead of tRNA(Ala). We find, as in the original experiment, that a shifted G.U confers Ala acceptor activity. Moreover, the modified tRNA(Lys) was specific for Ala, corroborating our original conclusion and making it more compelling.