Non-canonical Binding Site for Bacterial Initiation Factor 3 on the Large Ribosomal Subunit

Genetic studies in yeast have shown that the translation initiation factor eIF5 plays an important role in the selection of the AUG start codon. In order to ensure translation fidelity, the hydrolysis of GTP bound to the 40S preinitiation complex (40S.Met-tRNA(i).eIF2.GTP), promoted by eIF5, must occur only when the complex has selected the AUG start codon. However, the mechanism that prevents the eIF5-promoted GTP hydrolysis, prior to AUG selection by the ribosomal machinery, is not known. In this work, we show that the presence of initiation factors eIF1, eIF1A and eIF3 in the 40S preinitiation complex (40S.eIF1.eIF1A.eIF3.Met-tRNA(i).eIF2.GTP) and the subsequent binding of the preinitiation complex to eIF4F bound at the 5'-cap structure of mRNA are necessary for preventing eIF5-promoted hydrolysis of GTP in the 40S preinitiation complex. This block in GTP hydrolysis is released upon AUG selection by the 40S preinitiation complex. These results, taken together, demonstrate the biochemical requirements for regulation of GTP hydrolysis and its coupling to the AUG selection process during translation initiation.

The bacteriophage λ's cI mRNA was utilized to examine the importance of the 5'-terminal phosphate on expression of leadered and leaderless mRNA in Escherichia coli. A hammerhead ribozyme was used to produce leadered and leaderless mRNAs, in vivo and in vitro, that contain a 5'-hydroxyl. Although these mRNAs may not occur naturally in the bacterial cell, they allow for the study of the importance of the 5'-phosphorylation state in ribosome binding and translation of leadered and leaderless mRNAs. Analyses with mRNAs containing either a 5'-phosphate or a 5'-hydroxyl indicate that leaderless cI mRNA requires a 5'-phosphate for stable ribosome binding in vitro as well as expression in vivo. Ribosome-binding assays show that 30S subunits and 70S ribosomes do not bind as strongly to 5'-hydroxyl as they do to 5'-phosphate containing leaderless mRNA and the tRNA-dependent ternary complex is less stable. Additionally, filter-binding assays revealed that the 70S ternary complex formed with a leaderless mRNA containing a 5'-hydroxyl has a dissociation rate (k(off)) that is 4.5-fold higher compared with the complex formed with a 5'-phosphate leaderless mRNA. Fusion to a lacZ reporter gene revealed that leaderless cI mRNA expression with a 5'-hydroxyl was >100-fold lower than the equivalent mRNA with a 5'-phosphate. These data indicate that a 5'-phosphate is an important feature of leaderless mRNA for stable ribosome binding and expression.

The binding of IF‐3 to 70‐S, 50‐S and 30‐S ribosomes and subunits of Escherichia coli and to MS2 RNA was studied employing Sephadex G‐200 chromatography and sucrose‐gradient centrifugation to recover the complex formed. In the latter case, reaction mixtures were fixed with 0.25% glutaraldehyde prior to centrifugation. Total reaction mixtures were also analyzed by polyacrylamide‐gel electrophoresis. Highly purified IF‐3, either unlabeled or labeled with 35S was employed and consistent results were obtained with both factor preparations. Binding of IF‐3 to 30‐S subunits was independent of the Mg2+ concentration in the range 1–14 mM. The binding to 50‐S subunits and MS2 RNA was counteracted by increasing the Mg2+ concentration in the same range. Binding at 0°C and at 37°C occurred to the same extent both with 30‐S and 50‐S ribosomal subunits. The number of IF‐3 molecules bound per 30‐S subunit approached unity, that bound per 50‐S subunit exceeded unity when the IF‐3 input was sufficiently raised. At low factor/ribosome input ratios IF‐3 binds preferentially to 30‐S particles. When the ratio is increased binding to 50‐S subunits and 70‐S ribosomes also occurs. The IF‐3‐mediated dissociation of 70‐S ribosomes is strongly inhibited by adding increasing amounts of 30‐S subunits. At all Mg2+ concentrations studied the relative extent of binding can be expressed as: 30‐S > 70‐S, 50‐S > MS2 RNA. Binding to all types of ribosomes and subunits and to MS2 RNA is not stimulated by the addition of IF‐1 and IF‐2. The latter factors stabilize the 30‐S · IF‐3 complexes, however.

In recent years, many research groups have focused their interest on 5S rRNA, the smallest RNA component of the ribosome that is preserved in almost all organisms. Its precise role in the ribosome, the protein “biosynthetic machine”, has not been fully clarified. The results of structural studies place 5S rRNA in the large ribosomal subunit. Findings concerning relations between structure and function lead to the assumption that 5S rRNA stabilises the structure of the ribosome and acts as natural transmitter of information between functional centres. On the other hand, the ionic environment directly affects ribosome function. Monovalent and divalent ions as well as polycations, such as polyamines, influence the ribosomal conformation, and therefore are involved in the function of the ribosome. Preliminary studies in our laboratory have shown that polyamines, namely spermine, stabilize the secondary and tertiary structure of 5S rRNA, leading to a more cohesive configuration of the molecule. The aim of this thesis was to investigate the role of 5S rRNA configuration in protein synthesis of the enterobacterium E.coli. At the same time, the biological activity of polyamines in the structure and functioning of both 5S rRNA and the whole ribosomal complex was studied. Polyamine binding to 5S rRNA was investigated by photoaffinity labeling, using a photoreactive analogue of spermine, ABA-spermine, as a molecular probe. Kinetic study was performed and the number of cross-linking sites of ABA-spermine in free 5S rRNA, or 5S rRNA incorporated into 50S subunits or 70S ribosomal complexes from E.coli ribosomes was determined. The results revealed that in free 5S rRNA spermine binds mainly to nucleotides of helices III and V and loops A, C, D and E. Integration of 5S rRNA into 50S subunits or 70S poly (U) – programmed ribosomes results in a great reduction of the susceptibility of 5S rRNA to ABA-spermine. The impact of the ABA-spermine photo-incorporation into the 5S rRNA molecule was then investigated. Chemical modification experiments revealed that, regardless of whether 5S rRNA is free or assembled in 50S subunits or 70S ribosomal complexes, binding of ABA-spermine causes significant changes in the conformation of the molecule. Loop A adopts a “looser” structure, while loops C, D, E and helices III and V achieve a more compact folding. With the discovery of changes in the configuration of 5S rRNA, due to ABA-spermine’s binding, it was interesting to study whether these conformational changes influence fundamental functions of the ribosome. Initially, the effect of changing the configuration of 5S rRNA on 50S subunit and 70S ribosomal complex assembly was investigated. The results showed that binding of ABA-spermine to 5S rRNA does not eliminate the process of 50S subunit assembly, but impedes the necessary structural changes by stabilising a more coherent structure of 5S rRNA, thermodynamically less favourable for the process. Despite the fact that binding of ABA-spermine to 5S rRNA results in structural changes that reduce the assembly of 50S subunits, when the later are formed they acquire such a structure that allows their interaction with 30S subunits. Nevertheless, the presence of 5S rRNA seems to be necessary for the formation of active 50S subunits, since 5S RNA-depleted ribosomal subunits do not associate with 30S subunits to functional 70S ribosomes. Next, the effect of photolabeling 5S rRNA was studied, initially on the ability of ribosomes to bind substrate, and then on their catalytic activity. Photolabeling of 5S rRNA with ABA-spermine caused a small but obvious activation in AcPhe-tRNA binding to the P-site. In contrast, binding to the A-site did not appear to be affected. Of great importance was the effect on the activity of peptidyl-transferase; in the presence of spermine, an increase of up to 30% on the activity of PTase was observed. Interestedly, ribosomes that lack 5S rRNA maintained ~ 10% of PTase activity, which leads to the assumption that 5S rRNA is not absolutely necessary for the formation of peptide bonds. The study of the effect of 5S rRNA conformational changes on ribosome's ability to translocate substrates followed. Spontaneous (non-enzymatic) translocation was studied first, and then, EF-G-catalyzed translocation. The results confirmed the assumption that translocation of substrates is an inherent property of the ribosome itself, even though extremely slow. At the same time, it was shown that binding of spermine to 5S rRNA, stimulates non-enzymatic translocation, possibly through changes in the folding of ribosomal RNA, that are beneficial in translocating tRNAs. Further, the presence of spermine in 5S rRNA reduced the requirements for EF-G. This result can be explained either by a possible beneficial effect of spermine to the binding of EF-G to the ribosome, either through its impact in the process of hydrolysis of GTP. To clarify which of the two versions is accurate, we studied the effect of 5S rRNA conformational changes on the ribosomal ability to bind EF-G, and then on their ability to trigger EF-G-dependent GTP hydrolysis. It was found that the presence of spermine in 5S rRNA promotes EF-G binding to ribosomes. In contrast, the results of the study showed that the initial rate of GTP hydrolysis was not influenced by the presence of spermine. Ribosomes depleted of 5S rRNA retained some GTP activity, but were unable to effectively bind EF-G even in the presence of spermine. Taking into account that EF-G binds in domain II and VI of 23S rRNA, but direct contacts of 5S rRNA with these areas or with EF-G have not been found, we are led to the conclusion that 5S rRNA affects EF-G-GTP binding to ribosomes, through allosteric signals, without affecting the EF-G-mediated GTP hydrolysis. To confirm the above, we studied the effect of the ionic environment and the changes in the tertiary structure of 5S rRNA on the conformational state of fundamental functional centres in the ribosome. Specifically, the study was focused on the ability of free spermine or photolabeling 5S rRNA with ABA-spermine to affect the SRL and GAC regions of the ribosome as well as the A-and P-sites. It was found that interaction of free spermine with ribosomes intensifies the footprint of EF-G within the ribosome and induces the AcPhe-tRNA binding to the P-site, but not to the A-site. Similar, but of lower degree, was the effect 5S rRNA labelling by ABA-spermine. In summary, a comprehensive view of the 5S rRNA nucleotide residues involved in spermine binding is gained by the present study. The results further suggest that binding of spermine to 5S rRNA causes conformational changes in certain regions of the molecule. These changes, although not of the extreme amplitude, enabled us to investigate the functional role of 5S rRNA. In light of this role, it seems that PTase center and EF-G binding center are able to coordinate their functions by transmission of allosteric signals through 5S rRNA.

Initiation factor (IF) 2 controls the fidelity of translation initiation by selectively increasing the rate of 50S ribosomal subunit joining to 30S initiation complexes (ICs) that carry an N-formyl-methionyl-tRNA (fMet-tRNAfMet). Previous studies suggest that rapid 50S subunit joining involves a GTP- and fMet-tRNAfMet-dependent “activation” of IF2, but a lack of data on the structure and conformational dynamics of 30S IC-bound IF2 has precluded a mechanistic understanding of this process. Here, using an IF2-tRNA single-molecule fluorescence resonance energy transfer signal, we directly observe the conformational switch that is associated with IF2 activation within 30S ICs that lack IF3. Based on these results, we propose a model of IF2 activation that reveals how GTP, fMet-tRNAfMet, and specific structural elements of IF2 drive and regulate this conformational switch. Notably, we find that domain III of IF2 plays a pivotal, allosteric, role in IF2 activation, suggesting that this domain can be targeted for the development of novel antibiotics.

The present work shows that lead(II) can be used as a convenient structure probe to map the conformation of large RNA's and to follow discrete conformational changes at different functional states. We have investigated the conformation of the 3' domain of the E. coli 16S rRNA (nucleotides 1295-1542) in its naked form, in the 30S subunit and in the 70S ribosome. Our study clearly shows a preferential affinity of Pb(II) for interhelical and loop regions and suggests a high sensitivity for dynamic and flexible regions. Within 30S subunits, some cleavages are strongly decreased as the result of protein-induced protection, while others are enhanced suggesting local conformational adjustments. These rearrangements occur at functionally strategic regions of the RNA centered around nucleotides 1337, 1400, 1500 and near the 3' end of the RNA. The association of 30S and 50S subunits causes further protections at several nucleotides and some enhanced reactivities that can be interpreted in terms of subunits interface and allosteric transitions. The binding of E. coli tRNA-Phe to the 70S ribosome results in message-independent (positions 1337 and 1397) and message-dependent (1399-1400, 1491-1492 and 1505) protections. A third class of protection (1344-1345, 1393-1395, 1403-1409, 1412-1414, 1504, 1506-1507 and 1517-1519) is observed in message-directed 30S subunits, which are induced by both tRNA binding and 50S subunit association. This extensive reduction of reactivity most probably reflects an allosteric transition rather than a direct shielding.

Protein tyrosine phosphatases (PTPs) are important enzymes that are involved in the regulation of cellular signaling. Evidence accumulated over the years has indicated that PTPs present exciting opportunities for drug discovery against diseases such as diabetes, cancer, autoimmune diseases, and tuberculosis. However, the highly conserved and partially positive charge of the catalytic sites of PTPs is a major challenge in the development of potent and highly selective PTP inhibitors. Here, we examine the strategy of developing bidentate inhibitors for selective inhibition of PTPs. Bidentate inhibitors are small-molecular-weight compounds with the ability to bind to both the active site and a non-conserved secondary phosphate binding site. This secondary phosphate binding site was initially discovered in protein tyrosine phosphatase 1B (PTP1B), and, hence, most of the bidentate inhibitors reported in this review are PTP1B inhibitors. Although bidentate inhibition is a good strategy for developing potent and selective inhibitors, the cell membrane permeability and pharmacokinetic properties of the inhibitors are also important for successful drug development. In this review, we will also summarize the various efforts made toward the development of phosphotyrosine (pTyr) mimetics for increasing cellular permeability. Even though the secondary phosphate binding site was initially found in PTP1B, structural data have shown that a secondary binding site can also be found in other PTPs, albeit with varying degrees of accessibility. Along with improvements in pTyr mimetics, we believe that the future will see an increase in the number of orally bioavailable bidentate inhibitors against the various classes of PTPs.

Bile salt aggregates are supramolecular systems containing two different binding sites. The effect of the addition of acetonitrile on the specificity and dynamics of guest binding to the two binding sites of cholate aggregates was studied. The protection of guests included in the aggregate from interaction with ions in the aqueous phase was evaluated from quenching of the singlet and triplet excited states of guest molecules bound to the cholate aggregates. The dynamics of guest binding to the primary and secondary binding sites of the cholate aggregates were determined at increasing acetonitrile mole fractions. The structure of the aggregates was not significantly altered provided the cholate concentrations were higher than 20 mM and the acetonitrile mole fraction did not exceed 0.033 (9.1% v/v). These results show that acetonitrile can be used to modulate the solubility of guests in the aggregates and to manipulate the residence time of guests in the primary and secondary binding sites.

Aminopeptidase N (APN, CD13) is a transmembrane ectopeptidase involved in many crucial cellular functions. Besides its role as a peptidase, APN also mediates signal transduction and is involved in the activation of matrix metalloproteinases (MMPs). MMPs function in tissue remodeling within the extracellular space and are therefore involved in many human diseases, such as fibrosis, rheumatoid arthritis, tumor angiogenesis, and metastasis, as well as viral infections. However, the exact mechanism that leads to APN-driven MMP activation is unclear. It was previously shown that extracellular 14-3-3 adapter proteins bind to APN and thereby induce the transcription of MMPs. As a first step, we sought to identify potential 14-3-3–binding sites in the APN sequence. We constructed a set of phosphorylated peptides derived from APN to probe for interactions. We identified and characterized a canonical 14-3-3–binding site (site 1) within the flexible, structurally unresolved N-terminal APN region using direct binding fluorescence polarization assays and thermodynamic analysis. In addition, we identified a secondary, noncanonical binding site (site 2), which enhances the binding affinity in combination with site 1 by many orders of magnitude. Finally, we solved crystal structures of 14-3-3σ bound to mono- and bis-phosphorylated APN-derived peptides, which revealed atomic details of the binding mode of mono- and bivalent 14-3-3 interactions. Therefore, our findings shed some light on the first steps of APN-mediated MMP activation and open the field for further investigation of this important signaling pathway.

Human neutrophil elastase (hNE) is a serine protease that plays a major role in defending the bacterial infection. However, elevated expression of hNE is reported in lung and breast cancer, among others. Moreover, hNE is a target for the treatment of cardiopulmonary diseases. Ecotin (ET) is a serine protease inhibitor present in many Gram-negative bacteria, and it plays a physiological role in inhibiting host proteases, including hNE. Despite this known interaction, the structure of the hNE-ET complex has not been reported, and the mechanism of ecotin inhibition is not available. We determined the structure of the hNE-ET complex by molecular replacement method. The structure of the hNE-ET complex revealed the presence of six interface regions comprising 50s, 60s, and 80s loops, between the ET dimer and two independent hNE monomers, which explains the high affinity of ecotin for hNE (12 pM). Notably, we observed a secondary binding site of hNE located 24 Å from the primary binding site. Comparison of the closely related trypsin-ecotin complex with our hNE-ET complex shows movement of the backbone atoms of the 80s and 50s loops by 4.6 Å, suggesting the flexibility of these loops in inhibiting a range of proteases. Through a detailed structural analysis, we demonstrate the flexibility of the hNE subsites to dock various side chains concomitant with inhibition, indicating the broad specificity of hNE against various inhibitors. These findings will aid in the design of chimeric inhibitors that target both sites of hNE and in the development of therapeutics for controlling hNE-mediated pathogenesis.

By exploiting (125)I-polyubiquitin chain formation as a functional readout of enzyme activity, we have quantitatively examined the mechanism of human E6AP/UBE3A for the first time. Initial rate studies identify UbcH7 as the cognate E2 carrier protein for E6AP, although related Ubc5 isoforms and the ISG15-specific UbcH8 paralog also support E6AP with reduced efficacy due to impaired binding and catalytic competence. Initial rates of polyubiquitin chain formation displayed hyperbolic kinetics with respect to UbcH7 concentration (K(m) = 57.6 ± 5.7 nM and kcat = 0.032 ± 0.001 s(-1)) and substrate inhibition above 2 μM. Competitive inhibition by an isosteric UbcH7C86S-ubiquitin oxyester substrate analog (K(i) = 64 ± 18 nM) demonstrates that Km reflects intrinsic substrate affinity. In contrast, noncompetitive inhibition by a UbcH7C86A product analog (K(i) = 7 ± 0.7 μM) and substrate inhibition at high concentrations require two functionally distinct E2∼ubiquitin substrate binding sites. The kinetics of polyubiquitin chain formation reflect binding at a cryptic Site 1 not previously recognized that catalyzes E6AP∼ubiquitin thioester formation. Subsequent binding of E2∼ubiquitin at the canonical Site 2 present in the extant crystal structure is responsible for polyubiquitin chain elongation. Other rate studies show that the conserved -4 Phe(849) residue is required for polyubiquitin chain formation rather than target protein conjugation as originally suggested. The present studies unambiguously preclude earlier models for the mechanism of Hect domain-catalyzed conjugation through the canonical binding site suggested by the crystal structure and define a novel two-step mechanism for formation of the polyubiquitin degradation signal.

Aminoacyl-tRNA synthetases mediate the activation and transfer of amino acids to their cognate tRNA, which constitutes one of the initial events in protein biosynthesis. Even though different mechanisms of action have been proposed for the catalysis of these enzymes, their entire catalytic cycle remains elusive. Here, we used electron paramagnetic resonance spectroscopy in vitro and in cells in combination with molecular dynamics simulations to study the role of amino acid interactions in the catalytic cycle of pyrrolysyl-tRNA synthetases (PylRS), a widely used tool for genetic code expansion. Experiments using the paramagnetic non-canonical amino acid SLK-1 revealed the presence and occupation of secondary amino acid binding sites in PylRS located at the intermonomer interface, distant from the catalytic binding site. Based on our results, we propose a model that assumes an alternating mode of action of the two PylRS monomers for the catalytic cycle of PylRS.

YjeQ is a protein broadly conserved in bacteria containing an N-terminal oligonucleotide/oligosaccharide fold (OB-fold) domain, a central GTPase domain, and a C-terminal zinc-finger domain. YjeQ binds tightly and stoichiometrically to the 30S subunit, which stimulates its GTPase activity by 160-fold. Despite growing evidence for the involvement of the YjeQ protein in bacterial 30S subunit assembly, the specific function and mechanism of this protein remain unclear. Here, we report the costructure of YjeQ with the 30S subunit obtained by cryo-electron microscopy. The costructure revealed that YjeQ interacts simultaneously with helix 44, the head and the platform of the 30S subunit. This binding location of YjeQ in the 30S subunit suggests a chaperone role in processing of the 3' end of the rRNA as well as in mediating the correct orientation of the main domains of the 30S subunit. In addition, the YjeQ binding site partially overlaps with the interaction site of initiation factors 2 and 3, and upon binding, YjeQ covers three inter-subunit bridges that are important for the association of the 30S and 50S subunits. Hence, our structure suggests that YjeQ may assist in ribosome maturation by preventing premature formation of the translation initiation complex and association with the 50S subunit. Together, these results support a role for YjeQ in the late stages of 30S maturation.

Translation initiation is a complex process in which initiator tRNA, 40S, and 60S ribosomal subunits are assembled by eukaryotic initiation factors (eIFs) into an 80S ribosome at the initiation codon of mRNA. The cap-binding complex eIF4F and the factors eIF4A and eIF4B are required for binding of 43S complexes (comprising a 40S subunit, eIF2/GTP/Met-tRNAi and eIF3) to the 5' end of capped mRNA but are not sufficient to promote ribosomal scanning to the initiation codon. eIF1A enhances the ability of eIF1 to dissociate aberrantly assembled complexes from mRNA, and these factors synergistically mediate 48S complex assembly at the initiation codon. Joining of 48S complexes to 60S subunits to form 80S ribosomes requires eIF5B, which has an essential ribosome-dependent GTPase activity and hydrolysis of eIF2-bound GTP induced by eIF5. Initiation on a few mRNAs is cap-independent and occurs instead by internal ribosomal entry. Encephalomyocarditis virus (EMCV) and hepatitis C virus epitomize distinct mechanisms of internal ribosomal entry site (IRES)-mediated initiation. The eIF4A and eIF4G subunits of eIF4F bind immediately upstream of the EMCV initiation codon and promote binding of 43S complexes. EMCV initiation does not involve scanning and does not require eIF1, eIF1A, and the eIF4E subunit of eIF4F. Initiation on some EMCV-like IRESs requires additional noncanonical initiation factors, which alter IRES conformation and promote binding of eIF4A/4G. Initiation on the hepatitis C virus IRES is even simpler: 43S complexes containing only eIF2 and eIF3 bind directly to the initiation codon as a result of specific interaction of the IRES and the 40S subunit.

Protein L6 from the 50-S ribosomal subunit has been investigated using fluorimetric techniques. The intrinsic fluorophore Trp-61 and fluorescent labels (acetylaminoethyl-dansyl and acetylaminofluorescein) attached to the residue Cys-124 were used. It proved possible to incorporate fluorescence-labelled L6 into the 50-S ribosome. Trp-61 is exposed to solvent, as shown by its emission wavelength and by quenching experiments; the latter also show that it lies in a pocket with a high positive charge due to the basic residues in the N-terminal fragment. Cys-124 lies in a less strongly positive region. Upon incorporation into the 50-S subunit, the label on Cys-124 becomes less accessible for quenching but its positive potential rises, showing the absence of direct contact with 23-S RNA. Analysis of anisotropy data indicates a considerable degree of asphericity of free L6. Energy transfer between Trp-61 and the dansyl label on Cys-124, measured by donor quenching and acceptor enhancement, reveals a separation of 3.5 +/- 0.4 nm (35 +/- 4 A) between fluorophores.