Liron Klipcan

Eukaryotic cytosolic and mitochondrial phenylalanyl-tRNA synthetases catalyze the charging of tRNA with the meta-tyrosine

The accumulation of proteins damaged by reactive oxygen species (ROS), conventionally regarded as having pathological potentials, is associated with age-related diseases such as Alzheimers, atherosclerosis, and cataractogenesis. Exposure of the aromatic amino acid phenylalanine to ROS-generating systems produces multiple isomers of tyrosine: m-tyrosine (m-Tyr), o-tyrosine (o-Tyr), and the standard p-tyrosine (Tyr). Previously it was demonstrated that exogenously supplied, oxidized amino acids could be incorporated into bacterial and eukaryotic proteins. It is, therefore, likely that in many cases, in vivo-damaged amino acids are available for de novo synthesis of proteins. Although the involvement of aminoacyl-tRNA synthetases in this process has been hypothesized, the specific pathway by which ROS-damaged amino acids are incorporated into proteins remains unclear. We provide herein evidence that mitochondrial and cytoplasmic phenylalanyl-tRNA synthetases (HsmtPheRS and HsctPheRS, respectively) catalyze direct attachment of m-Tyr to tRNAPhe, thereby opening the way for delivery of the misacylated tRNA to the ribosome and incorporation of ROS-damaged amino acid into eukaryotic proteins. Crystal complexes of mitochondrial and bacterial PheRSs with m-Tyr reveal the net of highly specific interactions within the synthetic and editing sites.

The tRNA-Induced Conformational Activation of Human Mitochondrial Phenylalanyl-tRNA Synthetase.

All class II aminoacyl-tRNA synthetases (aaRSs) are known to be active as functional homodimers, homotetramers, or heterotetramers. However, multimeric organization is not a prerequisite for phenylalanylation activity, as monomeric mitochondrial phenylalanyl-tRNA synthetase (PheRS) is also active. We herein report the structure, at 2.2 A resolution, of a human monomeric mitPheRS complexed with Phe-AMP. The smallest known aaRS, which is, in fact, 1/5 of a cytoplasmic analog, is a chimera of the catalytic module of the alpha and anticodon binding domain (ABD) of the bacterial beta subunit of (alphabeta)2 PheRS. We demonstrate that the ABD located at the C terminus of mitPheRS overlaps with the acceptor stem of phenylalanine transfer RNA (tRNAPhe) if the substrate is positioned in a manner similar to that seen in the binary Thermus thermophilus complex. Thus, formation of the PheRS-tRNAPhe complex in human mitochondria must be accompanied by considerable rearrangement (hinge-type rotation through approximately 160 degrees) of the ABD upon tRNA binding.

Structure of Human Cytosolic Phenylalanyl-tRNA Synthetase: Evidence for Kingdom-Specific Design of the Active Sites and tRNA Binding Patterns

The existence of three types of phenylalanyl-tRNA synthetase (PheRS), bacterial (alphabeta)(2), eukaryotic/archaeal cytosolic (alphabeta)(2), and mitochondrial alpha, is a prominent example of structural diversity within the aaRS family. PheRSs have considerably diverged in primary sequences, domain compositions, and subunit organizations. Loss of the anticodon-binding domain B8 in human cytosolic PheRS (hcPheRS) is indicative of variations in the tRNA(Phe) binding and recognition as compared to bacterial PheRSs. We report herein the crystal structure of hcPheRS in complex with phenylalanine at 3.3 A resolution. A novel structural module has been revealed at the N terminus of the alpha subunit. It stretches out into the solvent of approximately 80 A and is made up of three structural domains (DBDs) possessing DNA-binding fold. The dramatic reduction of aminoacylation activity for truncated N terminus variants coupled with structural data and tRNA-docking model testify that DBDs play crucial role in hcPheRS activity.

Idiosyncrasy and identity in the prokaryotic phe-system: Crystal structure of E. coli phenylalanyl-tRNA synthetase complexed with phenylalanine and AMP

The crystal structure of Phenylalanyl‐tRNA synthetase from E. coli (EcPheRS), a class II aminoacyl‐tRNA synthetase, complexed with phenylalanine and AMP was determined at 3.05 Å resolution. EcPheRS is a (αβ)2 heterotetramer: the αβ heterodimer of EcPheRS consists of 11 structural domains. Three of them: the N‐terminus, A1 and A2 belong to the α‐subunit and B1‐B8 domains to the β subunit. The structure of EcPheRS revealed that architecture of four helix‐bundle interface, characteristic of class IIc heterotetrameric aaRSs, is changed: each of the two long helices belonging to CLM transformed into the coil‐short helix structural fragments. The N‐terminal domain of the α‐subunit in EcPheRS forms compact triple helix domain. This observation is contradictory to the structure of the apo form of TtPheRS, where N‐terminal domain was not detected in the electron density map. Comparison of EcPheRS structure with TtPheRS has uncovered significant rearrangements of the structural domains involved in tRNAPhe binding/translocation. As it follows from modeling experiments, to achieve a tighter fit with anticodon loop of tRNA, a shift of ∼5 Å is required for C‐terminal domain B8, and of ∼6 to 7 Å for the whole N terminus. EcPheRSs have emerged as an important target for the incorporation of novel amino acids into genetic code. Further progress in design of novel compounds is anticipated based on the structural data of EcPheRS.

Characterization of the Molecular Basis of Group II Intron RNA Recognition by CRS1-CRM Domains

CRM (chloroplast RNA splicing and ribosome maturation) is a recently recognized RNA-binding domain of ancient origin that has been retained in eukaryotic genomes only within the plant lineage. Whereas in bacteria CRM domains exist as single domain proteins involved in ribosome maturation, in plants they are found in a family of proteins that contain between one and four repeats. Several members of this family with multiple CRM domains have been shown to be required for the splicing of specific plastidic group II introns. Detailed biochemical analysis of one of these factors in maize, CRS1, demonstrated its high affinity and specific binding to the single group II intron whose splicing it facilitates, the plastid-encoded atpF intron RNA. Through its association with two intronic regions, CRS1 guides the folding of atpF intron RNA into its predicted “catalytically active” form. To understand how multiple CRM domains cooperate to achieve high affinity sequence-specific binding to RNA, we analyzed the RNA binding affinity and specificity associated with each individual CRM domain in CRS1; whereas CRM3 bound tightly to the RNA, CRM1 associated specifically with a unique region found within atpF intron domain I. CRM2, which demonstrated only low binding affinity, also seems to form specific interactions with regions localized to domains I, III, and IV. We further show that CRM domains share structural similarities and RNA binding characteristics with the well known RNA recognition motif domain.

The Reverse Transcriptase/RNA Maturase Protein MatR Is Required for the Splicing of Various Group II Introns in Brassicaceae Mitochondria

MatR, a highly conserved, essential mitochondrial protein, functions in the processing and maturation of various pre-RNAs in plant mitochondria, as revealed by in vivo analyses. Group II introns are large catalytic RNAs that are ancestrally related to nuclear spliceosomal introns. Sequences corresponding to group II RNAs are found in many prokaryotes and are particularly prevalent within plants organellar genomes. Proteins encoded within the introns themselves (maturases) facilitate the splicing of their own host pre-RNAs. Mitochondrial introns in plants have diverged considerably in sequence and have lost their maturases. In angiosperms, only a single maturase has been retained in the mitochondrial DNA: the matR gene found within NADH dehydrogenase 1 (nad1) intron 4. Its conservation across land plants and RNA editing events, which restore conserved amino acids, indicates that matR encodes a functional protein. However, the biological role of MatR remains unclear. Here, we performed an in vivo investigation of the roles of MatR in Brassicaceae. Directed knockdown of matR expression via synthetically designed ribozymes altered the processing of various introns, including nad1 i4. Pull-down experiments further indicated that MatR is associated with nad1 i4 and several other intron-containing pre-mRNAs. MatR may thus represent an intermediate link in the gradual evolutionary transition from the intron-specific maturases in bacteria into their versatile spliceosomal descendants in the nucleus. The similarity between maturases and the core spliceosomal Prp8 protein further supports this intriguing theory.

Presence of tRNA-dependent pathways correlates with high cysteine content in methanogenic Archaea

Archeal proteomes can be clustered into two groups based on their cysteine content. One group of proteomes displays a low cysteine content ( approximately 0.7% of the entire proteome), whereas the second group contains twice as many cysteines as the first ( approximately 1.3%). All cysteine-rich organisms belong to the methanogenic Archaea, which generates special cysteine clusters associated with primitive metabolic reactions. Our findings suggest that cysteine plays an important role in early forms of life.

Large‐scale movement of functional domains facilitates aminoacylation by human mitochondrial phenylalanyl‐tRNA synthetase

Structural studies suggest rearrangement of the RNA‐binding and catalytic domains of human mitochondrial PheRS (mtPheRS) is required for aminoacylation. Crosslinking the catalytic and RNA‐binding domains resulted in a “closed” form of mtPheRS that still catalyzed ATP‐dependent Phe activation, but was no longer able to transfer Phe to tRNA and complete the aminoacylation reaction. SAXS experiments indicated the presence of both the closed and open forms of mtPheRS in solution. Together, these results indicate that conformational flexibility of the two functional modules in mtPheRS is essential for its phenylalanylation activity. This is consistent with the evolution of the aminoacyl‐tRNA synthetases as modular enzymes consisting of separate domains that display independent activities.

Bacterial and Eukaryotic Phenylalanyl-tRNA Synthetases Catalyze Misaminoacylation of tRNAPhe with 3,4-Dihydroxy-L-Phenylalanine

Aminoacyl-tRNA synthetases exert control over the accuracy of translation by selective pairing the correct amino acids with their cognate tRNAs, and proofreading the misacylated products. Here we show that three existing, structurally different phenylalanyl-tRNA synthetases-human mitochondrial (HsmtPheRS), human cytoplasmic (HsctPheRS), and eubacterial from Thermus thermophilus (TtPheRS), catalyze mischarging of tRNA(Phe) with an oxidized analog of tyrosine-L-dopa. The lowest level of L-dopa discrimination over the cognate amino acid, exhibited by HsmtPheRS, is comparable to that of tyrosyl-tRNA synthetase. HsmtPheRS and TtPheRS complexes with L-dopa revealed in the active sites an electron density shaping this ligand. HsctPheRS and TtPheRS possessing editing activity are capable of hydrolyzing the exogenous L-dopa-tRNA(Phe) as efficiently as Tyr-tRNA(Phe). However, editing activity of PheRS does not guarantee reduction of the aminoacylation error rate to escape misincorporation of L-dopa into polypeptide chains.

Optimal growth temperature of prokaryotes correlates with class II amino acid composition

Partitioning of aminoacyl‐tRNA synthetases and their associated amino acids into two classes allows us to distinguish between thermophilic and mesophilic species based only on amino acids composition. The CLASSDB program has been developed for amino acid content analysis in organisms treated individually or pooled together to form a pattern of characteristic properties. A strong correlation has been observed between optimal growth temperature (OGT) of organisms and class II amino acids content. Amino acid composition in organisms closely related phylogenetically but dissimilar in their OGT testifies that thermo‐adaptation happens rather rapidly on the time scale of evolution.