Historical review: Peptidyl transfer, the Monro era

The peptide bond-forming reaction of protein synthesis, the peptidyl transfer reaction, takes place in a region of the 50S ribosomal subunit that consists entirely of RNA, the peptidyl transferase centre. Basic to the present knowledge of peptidyl transfer was the discovery by Robin Monro and his colleagues in the 1960s that the reaction is catalyzed by the 50S ribosome. The Monro experiments, and the historical context in which they were conceived, are described in this personal recollection. Monro's 'fragment reaction', the ribosome catalyzed reaction of a fragment of formylmethionyl-tRNA with puromycin, remains in use in work on peptidyl transfer.     

Novel post-translational oligomerization of peptidyl dehydrodopa model compound, 1,2-dehydro-N-acetyldopa methyl ester

Post-translational modification of peptidyl tyrosine to peptidyl dopa is widely observed in different marine organisms. While peptidyl dopas are oxidatively converted to dehydrodopa derivatives, nothing is known about the further fate of dehydrodopyl compounds. To fill this void, we studied the oxidation chemistry of a peptidyl dehydrodopa mimic, 1,2-dehydro-N-acetyldopa methyl ester with mushroom tyrosinase. We employed both routine biochemical studies and reversed phase liquid chromatography mass spectrometry to investigate the course of the reaction. Tyrosinase catalyzed the oxidation of 1,2-dehydro-N-acetyldopa methyl ester readily generating its typical o-quinone as the transient two-electron oxidation product. This quinone was extremely unstable and rapidly reacted with the parent compound forming benzodioxan type oligomeric products. Reaction mixture containing chemically made o-benzoquinone and 1,2-dehydro-N-acetyldopa methyl ester generated a mixed adduct of benzoquinone and 1,2-dehydro-N-acetyldopa methyl ester. Based on this finding, we propose that peptidyl dehydrodopa also exhibits a similar transformation accounting partially for the adhesive and cementing properties of dopyl proteins in nature.     

Investigation of the ribosomal peptidyl transferase center using a photoaffinity label

The synthesis of a peptidyl-tRNA photoaffinity analog, 2-nitro-4-azidophenoxy-4′-phenylacetyl-phenylalanyl-tRNA^Phe is described. Covalent attachment of this analog to Escherichia coli 70 S ribosomes requires poly(U)-stimulated binding prior to photolysis. Peptidyl site binding is indicated by the ability of puromycin to release the peptidyl moiety from non-photolyzed samples. Covalently attached 2-nitro-4-azidophenoxy-4-phenylacetyl-Phe-tRNA^Phe can subsequently participate in peptidyl transfer with [³H]Phe-tRNA^Phe bound at the aminoacyl site. This means that the covalent reaction does not produce sufficient distortion of the peptidyl site and its bound tRNA to inactivate the peptidyl transference. If peptidyl transfer with [³H]Phe-tRNA^Phe is allowed to proceed before photolysis, covalent reaction can still occur. In all cases, the main reaction products are two 50 S ribosomal proteins, L11 and L18. The results strongly indicate that these two proteins either form part of the peptidyl transferase center or are located adjacent to it. Presumably, α-halocarbonyl affinity reagents used previously failed to identify these two proteins because they lack easily accessible, reactive nucleophilic groups.     

Peptidyl transferase centres of rat and yeast ribosomes. Different response to modification of protein amino groups

Modification of rat liver ribosomes with dimethylmaleic anhydride, a reagent for protein amino groups, causes a large stimulation of peptidyl transferase activity assayed by the "fragment" reaction, as well as the inactivation of poly(U)-directed polyphenylalanine synthesis. In contrast to rat ribosomes, the peptidyl transferase of yeast ribosomes is little affected by modification. Although other interpretations are not excluded, these results might be due to differences between the peptidyl transferase centres of mammalian and yeast ribosomes.     

A new simpler photoaffinity analogue of peptidyl tRNA

The synthesis of the n-hydroxysuccinimide ester of N-(2-nitro-4-azidophenyl)glycine (NAG) is described. This reacts with E. coli phe-tRNA(Phe) to yield the photoaffinity label NAG-Phe-tRNA(Phe). This peptidyl tRNA analogue binds correctly to the peptidyl site of the E. coli ribosome. The only significant covalent products found after irradiation of a peptidyl site bound NAG-Phe-tRNA(Phe)-70S-poly(U) complex are 50S proteins L11 and L18. After irradiation the complex can still bind [(3)H]Phe-tRNA to the amino acyl site and participate in peptide bond formation with the covalently attached NAG-Phe moiety. Alternatively, one can allow peptide bond formation to occur first, prior to photolysis. The reaction products are still L11 and L18. Hence, both of these two proteins appear to be centrally located at the peptidyl transferase center.     

Non-stressful death of 23S rRNA mutant G2061C defective in puromycin reaction

Catalysis of peptide bond formation in the peptidyl transferase center is a major enzymatic activity of the ribosome. Mutations limiting peptidyl transferase activity are mostly lethal. However, cellular processes triggered by peptidyl transferase deficiency in the bacterial cell are largely unknown. Here we report a study of the lethal G2061C mutant of Escherichia coli 23S ribosomal RNA (rRNA). The G2061C mutation completely impaired the puromycin reaction and abolished formation of the active firefly luciferase in an in vitro translation system, while poly(U)- and short synthetic mRNA-directed peptidyl transferase reaction with aminoacylated tRNAs in vitro was seemingly unaffected. Study of the cellular proteome upon expression of the 23S rRNA gene carrying the G2061C mutation compared to cells expressing wild-type 23S rRNA gene revealed substantial differences. Most of the observed effects in the mutant were associated with reduced expression of stress response proteins and particularly proteins associated with the ppGpp-mediated stringent response.     

Immunological studies of the fragments of bovine cartilage proteoglycan produced by chondroitinase-trypsin digestion.

The peptidyl transferase activity of polysomes from Escherichia coli, rabbit reticulocytes and chick embryos, assayed in the fragment reaction, is 3- to 10-fold lower than the corresponding activity of single ribosomes. The polysomal peptidyl transferase activity is restored in full under conditions of in vitro protein synthesis that result in conversion of polysomes to single ribosomes. Thus, the peptidyl transferase center is masked in translating ribosomes. Unmasking of peptidyl transferase, however, does not require the release of ribosomes from messenger RNA: it is also seen upon treatment of polysomes with puromycin, under conditions in which polysomes remain intact. Apparently, release of nascent polypeptide chains is sufficient to allow access of formylmethionyl hexanucleotide substrate to the peptidyl transferase site.     

The pleuromutilin drugs tiamulin and valnemulin bind to the RNA at the peptidyl transferase centre on the ribosome

The pleuromutilin antibiotic derivatives, tiamulin and valnemulin, inhibit protein synthesis by binding to the 50S ribosomal subunit of bacteria. The action and binding site of tiamulin and valnemulin was further characterized on Escherichia coli ribosomes. It was revealed that these drugs are strong inhibitors of peptidyl transferase and interact with domain V of 23S RNA, giving clear chemical footprints at nucleotides A2058-9, U2506 and U2584-5. Most of these nucleotides are highly conserved phylogenetically and functionally important, and all of them are at or near the peptidyl transferase centre and have been associated with binding of several antibiotics. Competitive footprinting shows that tiamulin and valnemulin can bind concurrently with the macrolide erythromycin but compete with the macrolide carbomycin, which is a peptidyl transferase inhibitor. We infer from these and previous results that tiamulin and valnemulin interact with the rRNA in the peptidyl transferase slot on the ribosomes in which they prevent the correct positioning of the CCA-ends of tRNAs for peptide transfer.     

Identification of proteins involved in the peptidyl transferase activity of ribosomes by chemical modification.

Photochemical oxidation of Escherichia coli 50 S ribosomal subunits in the presence of methylene blue or Rose Bengal causes rapid loss of peptidyl transferase activity. Reconstitution experiments using mixtures of components from modified and unmodified ribosomes reveal that both RNA and proteins are affected, and that among the proteins responsible for inactivation there are both LiCl-split and core proteins. The proteins L2 and L16 from the split fraction and L4 from the core fraction of unmodified ribosomes were together nearly as effective as total unmodified proteins in restoring peptidyl transferase activity to reconstituted ribosomes when added with proteins from modified ribosomes. These three proteins are therefore the most important targets identified as responsible for loss of peptidyl transferase activity on photo-oxidation of 50 S ribosomal subunits.     

Engineering of an industrial polyoxin producer for the rational production of hybrid peptidyl nucleoside antibiotics

Polyoxins and nikkomycins are potent antifungal peptidyl nucleoside antibiotics, which inhibit fungal cell wall biosynthesis. They consist of a nucleoside core and one or two independent peptidyl moieties attached to the core at different sites. Making mutations and introducing heterologous genes into an industrial Streptomyces aureochromogenes polyoxin producer, resulted in the production of four polyoxin-nikkomycin hybrid antibiotics designated as polyoxin N and nikkoxin B-D, whose structures were confirmed using high resolution MS and NMR. Two of the hybrid antibiotics, polyoxin N and nikkoxin D, were significantly more potent against some human or plant fungal pathogens than their parents. The data provides an example for rational generation of novel peptidyl nucleoside antibiotics in an industrial producer.     

Inhibition of the peptidyl transferase A-site function by 2'-O-aminoacyloligonucleotides

New “non-isomerizable” analogs of the 3′-terminus of AA-tRNA, C-A(2′Phe)H, C-A(2′Phe)Me, C-A(2′H)Phe and C-A(2′Me)Phe, were tested as acceptor substrates of ribosomal peptidyl transferase and inhibitors of the peptidyl transferase A-site function. The 3′-O-AA-derivatives were active acceptors of Ac-Phe in the peptidyl transferase reaction, while the 2′-O-AA-derivatives were completely inactive. Both 2′- and 3′-O-AA-derivatives were potent inhibitors of peptidyl transferase catalyzed Ac-Phe transfer to puromycin. The results indicate that although peptidyl transferase exclusively utilizes 3′-O-esters of tRNA as acceptor substrates, its A-site can also recognize the 3′-terminus of 2′-O-AA-tRNA.     

An approach for differentiating isozymes. Construction of libraries containing short aromatic peptides as part of a method to design selective inhibitors against lipases

Through synthesis and assays of peptidyl substrates, we could select substrates having peptidyl complementary against lipases. The best substrate showed 20-fold improved K(m) relative to non-peptidyl substrate. Using this information, we generated selective inhibitors. Lipase activities with peptidyl substrates were represented as fingerprints. Differences in fingerprints reflect different structures near active site of lipases, could be used for generating selective inhibitors.     

Sequence analysis of mitochondrial chloramphenicol resistance mutations in Chinese hamster cells

A series of mitochondrially inherited chloramphenicol-resistant (CAP-R) mutants were isolated in Chinese hamster cells. To determine whether the Chinese hamster CAP-R mutations were homologous to those isolated in mouse and human cell culture systems, we determined the nucleotide sequence of the region of the mitochondrial 16S rRNA gene spanning the peptidyl transferase-encoding region for eight CAP-R mutant lines in addition to the parental wildtype line. Three main conclusions are drawn from these studies. (1) Although the region of the gene encoding the peptidyl transferase domain is highly conserved relative to that of mice and rats, the contiguous sequences show less conservation. This sequence divergence not only includes the accumulation of single base pair replacements, but also the presence of small insertions or deletions. (2) For six of the CAP-R mutants, heteroplasmic single base pair changes were detected. These mapped to the same sites within the peptidyl transferase domain as the mutations found previously in mouse and human CAP-R mutants. (3) Two Chinese hamster CAP-R mutants, both with an unusual drug resistance phenotype, did not carry any mutations within the CAP-R peptidyl transferase domain. However, both carried a heteroplasmic mutation at the position corresponding to nucleotide 2505 of the mouse 16S rRNA gene, a site predicted to map within a stem/loop structure attached to this key domain of the ribosome. This is the first evidence for mitochondrial CAP-R mutations that map outside the peptidyl transferase region.     

Using peptidyl aldehydes in activity-based proteomics

The broad inhibitory spectrum of aldehydes and the possibility that amino acid residues modulate their specificity point to the potential of using peptidyl aldehydes as activity-based probes. Here, we establish the potential of peptidyl aldehydes in activity-based proteomics by synthesizing different probes and using them to specifically label a well-known serine protease in an activity-dependent manner. From our results, peptidyl aldehydes emerge as promising activity-based probes that enable multiple enzymatic-class detection by substrate recognition and can be used in diverse activity-based proteomics applications like protein identification and activity profiling.     

Involvement of mitochondrial ribosomal proteins in ribosomal RNA-mediated protein folding

The peptidyl transferase center of the domain V of large ribosomal RNA in the prokaryotic and eukaryotic cytosolic ribosomes acts as general protein folding modulator. We showed earlier that one part of the domain V (RNA1 containing the peptidyl transferase loop) binds unfolded protein and directs it to a folding competent state (FCS) that is released by the other part (RNA2) to attain the folded native state by itself. Here we show that the peptidyl transferase loop of the mitochondrial ribosome releases unfolded proteins in FCS extremely slowly despite its lack of the rRNA segment analogous to RNA2. The release of FCS can be hastened by the equivalent activity of RNA2 or the large subunit proteins of the mitochondrial ribosome. The RNA2 or large subunit proteins probably introduce some allosteric change in the peptidyl transferase loop to enable it to release proteins in FCS.     

Role of the ribosomal protein L27 revealed by single‐molecule FRET study

The ribosome is a ribozyme. However, in bacterial ribosomes, the N‐terminus of L27 is located within the peptidyl transfer center. The roles of this protein in real time remain unclear. We present single‐molecule fluorescence resonance energy transfer (FRET) studies of tRNA dynamics at the peptidyl transfer center in ribosomes containing either wild type (WT) L27, or L27 mutants with A2H3, A2H3K4 or nine N‐terminal residues removed. Removing L27's first three N‐terminal residues or mutating a single residue, K4, reduces the formation of a stable peptidyl tRNA after translocation. These results imply that L27 stabilizes the peptidyl tRNA and residue K4 contributes significantly to the stabilization.     

Peptidyl boronates inhibit Salmonella enterica serovar Typhimurium Lon protease by a competitive ATP-dependent mechanism

Lon is a homo-oligomeric ATP-dependent serine protease that functions in the degradation of damaged and certain regulatory proteins. This enzyme has emerged as a novel target in the development of antibiotics because of its importance in conferring bacterial virulence. In this study, we explored the mechanism by which the proteasome inhibitor MG262, a peptidyl boronate, inhibits the peptide hydrolysis activity of Salmonella enterica serovar Typhimurium Lon. In addition, we synthesized a fluorescent peptidyl boronate inhibitor based upon the amino acid sequence of a product of peptide hydrolysis by the enzyme. Using steady-state kinetic techniques, we have shown that two peptidyl boronate variants are competitive inhibitors of the peptide hydrolysis activity of Lon and follow the same two-step, time-dependent inhibition mechanism. The first step is rapid and involves binding of the inhibitor and formation of a covalent adduct with the active site serine. This is followed by a second slow step in which Lon undergoes a conformational change or isomerization to increase the interaction of the inhibitor with the proteolytic active site to yield an overall inhibition constant of 5-20 nM. Although inhibition of serine and threonine proteases by peptidyl boronates has been detected previously, Lon is the first protease that has required the binding of ATP in order to observe inhibition.     

HIV protease (HIV PR) inhibitor structure-activity-selectivity, and active site molecular modeling of high affinity Leu [CH(OH)CH2]Val modified viral and nonviral substrate analogs.

This report details the structure-activity relationships of the HIV gag substrate analog Val-Ser-Gln-Asn-Leu psi[CH(OH)CH2]Val-Ile-Val (U-85548E), an inhibitor exhibiting subnanomolar affinity towards HIV type-1 aspartic proteinase (HIV-1 PR). Our data show that the P1-P2' tripeptidyl sequence provides the minimal chemical determinant for HIV-1 PR binding. We describe the structure-activity properties of Leu psi[CH(OH)CH2]Val substitution in other peptidyl ligands of nonviral substrate origin (e.g., angiotensinogen, insulin and pepstatin). Furthermore, the aspartic proteinase selectivities of a few key compounds are summarized relative to evaluation against human renin, human pepsin, and the fungal enzyme, rhizopuspepsin. These studies have led to the rational design of nanomolar potent inhibitors of both HIV-1 and HIV-2 PR. Finally, a 2.5 A resolution X-ray crystallographic structure of U-85548E complexed to synthetic HIV-1 PR dimer (Jaskolski et al., Biochemistry 30, 1600 [1991]) provided a 3-D picture of the inhibitor bound to the enzyme active site, and we performed computer-assisted molecular modeling studies to explore the possible binding modes of the above series of Leu psi[CH(OH)CH2]Val substituted HIV-1 PR inhibitors.     

A molluscan peptide alpha-amidating enzyme precursor that generates five distinct enzymes.

Mechanisms underlying the specificity and efficiency of enzymes, which modify peptide messengers, especially with the variable requirements of synthesis in the neuronal secretory pathway, are poorly understood. Here, we examine the process of peptide alpha-amidation in individually identifiable Lymnaea neurons that synthesize multiple proproteins, yielding complex mixtures of structurally diverse peptide substrates. The alpha-amidation of these peptide substrates is efficiently controlled by a multifunctional Lymnaea peptidyl glycine alpha-amidating monooxygenase (LPAM), which contains four different copies of the rate-limiting Lymnaea peptidyl glycine alpha-hydroxylating monooxygenase (LPHM) and a single Lymnaea peptidyl alpha-hydroxyglycine alpha-amidating lyase. Endogenously, this zymogen is converted to yield a mixture of monofunctional isoenzymes. In vitro, each LPHM displays a unique combination of substrate affinity and reaction velocity, depending on the penultimate residue of the substrate. This suggests that the different isoenzymes are generated in order to efficiently amidate the many peptide substrates that are present in molluscan neurons. The cellular expression of the LPAM gene is restricted to neurons that synthesize amidated peptides, which underscores the critical importance of regulation of peptide alpha-amidation.