PylSn and the Homologous N-terminal Domain of Pyrrolysyl-tRNA Synthetase Bind the tRNA That Is Essential for the Genetic Encoding of Pyrrolysine

Pyrrolysine is represented by an amber codon in genes encoding proteins such as the methylamine methyltransferases present in some Archaea and Bacteria. Pyrrolysyl-tRNA synthetase (PylRS) attaches pyrrolysine to the amber-suppressing tRNA^Pyl. Archaeal PylRS, encoded by pylS, has a catalytic C-terminal domain but an N-terminal region of unknown function and structure. In Bacteria, homologs of the N- and C-terminal regions of archaeal PylRS are respectively encoded by pylSn and pylSc. We show here that wild type PylS from Methanosarcina barkeri and PylSn from Desulfitobacterium hafniense bind tRNA^Pyl in EMSA with apparent K_d values of 0.12 and 0.13 μm, respectively. Truncation of the N-terminal region of PylS eliminated detectable tRNA^Pyl binding as measured by EMSA, but not catalytic activity. A chimeric protein with PylSn fused to the N terminus of truncated PylS regained EMSA-detectable tRNA^Pyl binding. PylSn did not bind other D. hafniense tRNAs, nor did the competition by the Escherichia coli tRNA pool interfere with tRNA^Pyl binding. Further indicating the specificity of PylSn interaction with tRNA^Pyl, substitutions of conserved residues in tRNA^Pyl in the variable loop, D stem, and T stem and loop had significant impact in binding, whereas those having base changes in the acceptor stem or anticodon stem and loop still retained the ability to complex with PylSn. PylSn and the N terminus of PylS comprise the protein superfamily TIGR03129. The members of this family are not similar to any known RNA-binding protein, but our results suggest their common function involves specific binding of tRNA^Pyl.     

Pyrrolysyl-tRNA synthetase: An ordinary enzyme but an outstanding genetic code expansion tool

The genetic incorporation of the 22nd proteinogenic amino acid, pyrrolysine (Pyl) at amber codon is achieved by the action of pyrrolysyl-tRNA synthetase (PylRS) together with its cognate tRNA^Pyl. Unlike most aminoacyl-tRNA synthetases, PylRS displays high substrate side chain promiscuity, low selectivity toward its substrate α-amine, and low selectivity toward the anticodon of tRNA^Pyl. These unique but ordinary features of PylRS as an aminoacyl-tRNA synthetase allow the Pyl incorporation machinery to be easily engineered for the genetic incorporation of more than 100 non-canonical amino acids (NCAAs) or α-hydroxy acids into proteins at amber codon and the reassignment of other codons such as ochre UAA, opal UGA, and four-base AGGA codons to code NCAAs.     

The tRNA discriminator base defines the mutual orthogonality of two distinct pyrrolysyl-tRNA synthetase/tRNAPyl pairs in the same organism

Site-specific incorporation of distinct non-canonical amino acids into proteins via genetic code expansion requires mutually orthogonal aminoacyl-tRNA synthetase/tRNA pairs. Pyrrolysyl-tRNA synthetase (PylRS)/tRNA^Pyl pairs are ideal for genetic code expansion and have been extensively engineered for developing mutually orthogonal pairs. Here, we identify two novel wild-type PylRS/tRNA^Pyl pairs simultaneously present in the deep-rooted extremely halophilic euryarchaeal methanogen Candidatus Methanohalarchaeum thermophilum HMET1, and show that both pairs are functional in the model halophilic archaeon Haloferax volcanii. These pairs consist of two different PylRS enzymes and two distinct tRNAs with dissimilar discriminator bases. Surprisingly, these two PylRS/tRNA^Pyl pairs display mutual orthogonality enabled by two unique features, the A73 discriminator base of tRNA^Pyl2 and a shorter motif 2 loop in PylRS2. In vivo translation experiments show that tRNA^Pyl2 charging by PylRS2 is defined by the enzyme''s shortened motif 2 loop. Finally, we demonstrate that the two HMET1 PylRS/tRNA^Pyl pairs can simultaneously decode UAG and UAA codons for incorporation of two distinct noncanonical amino acids into protein. This example of a single base change in a tRNA leading to additional coding capacity suggests that the growth of the genetic code is not yet limited by the number of identity elements fitting into the tRNA structure.     

Rationally evolving tRNAPyl for efficient incorporation of noncanonical amino acids

Genetic encoding of noncanonical amino acids (ncAAs) into proteins is a powerful approach to study protein functions. Pyrrolysyl-tRNA synthetase (PylRS), a polyspecific aminoacyl-tRNA synthetase in wide use, has facilitated incorporation of a large number of different ncAAs into proteins to date. To make this process more efficient, we rationally evolved tRNA^Pyl to create tRNA^Pyl-opt with six nucleotide changes. This improved tRNA was tested as substrate for wild-type PylRS as well as three characterized PylRS variants (N^ϵ-acetyllysyl-tRNA synthetase [AcKRS], 3-iodo-phenylalanyl-tRNA synthetase [IFRS], a broad specific PylRS variant [PylRS-AA]) to incorporate ncAAs at UAG codons in super-folder green fluorescence protein (sfGFP). tRNA^Pyl-opt facilitated a 5-fold increase in AcK incorporation into two positions of sfGFP simultaneously. In addition, AcK incorporation into two target proteins (Escherichia coli malate dehydrogenase and human histone H3) caused homogenous acetylation at multiple lysine residues in high yield. Using tRNA^Pyl-opt with PylRS and various PylRS variants facilitated efficient incorporation of six other ncAAs into sfGFP. Kinetic analyses revealed that the mutations in tRNA^Pyl-opt had no significant effect on the catalytic efficiency and substrate binding of PylRS enzymes. Thus tRNA^Pyl-opt should be an excellent replacement of wild-type tRNA^Pyl for future ncAA incorporation by PylRS enzymes.     

Recognition of Non-α-amino Substrates by Pyrrolysyl-tRNA Synthetase

Pyrrolysyl-tRNA synthetase (PylRS), an aminoacyl-tRNA synthetase (aaRS) recently found in some methanogenic archaea and bacteria, recognizes an unusually large lysine derivative, l-pyrrolysine, as the substrate, and attaches it to the cognate tRNA (tRNA^Pyl). The PylRS-tRNA^Pyl pair interacts with none of the endogenous aaRS-tRNA pairs in Escherichia coli, and thus can be used as a novel aaRS-tRNA pair for genetic code expansion. The crystal structures of the Methanosarcina mazei PylRS revealed that it has a unique, large pocket for amino acid binding, and the wild type M. mazei PylRS recognizes the natural lysine derivative as well as many lysine analogs, including N^?-(tert-butoxycarbonyl)-l-lysine (Boc-lysine), with diverse side chain sizes and structures. Moreover, the PylRS only loosely recognizes the α-amino group of the substrate, whereas most aaRSs, including the structurally and genetically related phenylalanyl-tRNA synthetase (PheRS), strictly recognize the main chain groups of the substrate. We report here that wild type PylRS can recognize substrates with a variety of main-chain α-groups: α-hydroxyacid, non-α-amino-carboxylic acid, N_α-methyl-amino acid, and d-amino acid, each with the same side chain as that of Boc-lysine. In contrast, PheRS recognizes none of these amino acid analogs. By expressing the wild type PylRS and its cognate tRNA^Pyl in E. coli in the presence of the α-hydroxyacid analog of Boc-lysine (Boc-LysOH), the amber codon (UAG) was recoded successfully as Boc-LysOH, and thus an ester bond was site-specifically incorporated into a protein molecule. This PylRS-tRNA^Pyl pair is expected to expand the backbone diversity of protein molecules produced by both in vivo and in vitro ribosomal translation.     

吡咯赖氨酸：第 22 种参与蛋白质生物合成的氨基酸

吡咯赖氨酸在产甲烷菌的甲胺甲基转移酶中发现,是目前已知的第 22 种参与蛋白质生物合成的氨基酸,与标准氨基酸不同的是,它由终止密码子 UAG 的有义编码形成 . 与之对应的在产甲烷菌中也含有特异的吡咯赖氨酰 -tRNA 合成酶 (PylRS) 和吡咯赖氨酸 tRNA (tRNA^Pyl). tRNA^Pyl具有不同于经典 tRNA 的特殊结构 . 产甲烷菌通过直接途径和间接途径这两种途径生成吡咯赖氨酰 -tRNA^Pyl(Pyl-tRNA^Pyl) ,它还可能通过 mRNA 上的特殊结构以及其他还未发现的机制,控制 UAG 编码成为终止密码子或者吡咯赖氨酸 . 比较了吡咯赖氨酸与另一种非标准氨基酸,第 21 种氨基酸———硒代半胱氨酸的相似点与不同点 .     

Adding l-lysine derivatives to the genetic code of mammalian cells with engineered pyrrolysyl-tRNA synthetases

We report a method for site-specifically incorporating l-lysine derivatives into proteins in mammalian cells, based on the expression of the pyrrolysyl-tRNA synthetase (PylRS)-tRNA^Pyl pair from Methanosarcina mazei. Different types of external promoters were tested for the expression of tRNA^Pyl in Chinese hamster ovary cells. When tRNA^Pyl was expressed from a gene cluster under the control of the U6 promoter, the wild-type PylRS-tRNA^Pyl pair facilitated the most efficient incorporation of a pyrrolysine analog, N^ε-tert-butyloxycarbonyl-l-lysine (Boc-lysine), into proteins at the amber position. This PylRS-tRNA^Pyl system yielded the Boc-lysine-containing protein in an amount accounting for 1% of the total protein in human embryonic kidney (HEK) 293 cells. We also created a PylRS variant specific to N^ε-benzyloxycarbonyl-l-lysine, to incorporate this long, bulky, non-natural lysine derivative into proteins in HEK293. The recently reported variant specific to N^ε-acetyllysine was also expressed, resulting in the genetic encoding of this naturally-occurring lysine modification in mammalian cells.     

Specificity of Pyrrolysyl-tRNA Synthetase for Pyrrolysine and Pyrrolysine Analogs

Pyrrolysine, the 22nd amino acid, is encoded by amber (TAG = UAG) codons in certain methanogenic archaea and bacteria. PylS, the pyrrolysyl-tRNA synthetase, ligates pyrrolysine to tRNA^Pyl for amber decoding as pyrrolysine. PylS and tRNA^Pyl have potential utility in making tailored recombinant proteins. Here, we probed interactions necessary for recognition of substrates by archaeal PylS via synthesis of close pyrrolysine analogs and testing their reactivity in amino acid activation assays. Replacement of the methylpyrroline ring of pyrrolysine with cyclopentane indicated that solely hydrophobic interactions with the ring-binding pocket of PylS are sufficient for substrate recognition. However, a 100-fold increase in the specificity constant of PylS was observed with an analog, 2-amino-6-((R)-tetrahydrofuran-2-carboxamido)hexanoic acid (2Thf-lys), in which tetrahydrofuran replaced the pyrrolysine methylpyrroline ring. Other analogs in which the electronegative atom was moved to different positions suggested PylS preference for a hydrogen-bond-accepting group at the imine nitrogen position in pyrrolysine. 2Thf-lys was a preferred substrate over a commonly employed pyrrolysine analog, but the specificity constant for 2Thf-lys was 10-fold lower than for pyrrolysine itself, largely due to the change in K_m. The in vivo activity of the analogs in supporting UAG suppression in Escherichia coli bearing genes for PylS and tRNA^Pyl was similar to in vitro results, with l-pyrrolysine and 2Thf-lys supporting the highest amounts of UAG translation. Increasing concentrations of either PylS substrate resulted in a linear increase in UAG suppression, providing a facile method to assay bioactive pyrrolysine analogs. These results illustrate the relative importance of the H-bonding and hydrophobic interactions in the recognition of the methylpyrroline ring of pyrrolysine and provide a promising new series of easily synthesized pyrrolysine analogs that can serve as scaffolds for the introduction of novel functional groups into recombinant proteins.     

Distinct genetic code expansion strategies for selenocysteine and pyrrolysine are reflected in different aminoacyl-tRNA formation systems

Selenocysteine and pyrrolysine, known as the 21st and 22nd amino acids, are directly inserted into growing polypeptides during translation. Selenocysteine is synthesized via a tRNA-dependent pathway and decodes UGA (opal) codons. The incorporation of selenocysteine requires the concerted action of specific RNA and protein elements. In contrast, pyrrolysine is ligated directly to tRNA^Pyl and inserted into proteins in response to UAG (amber) codons without the need for complex re-coding machinery. Here we review the latest updates on the structure and mechanisms of molecules involved in Sec-tRNA^Sec and Pyl-tRNA^Pyl formation as well as the distribution of the Pyl-decoding trait.     

The amino-terminal domain of pyrrolysyl-tRNA synthetase is dispensable in vitro but required for in vivo activity

Pyrrolysine (Pyl) is co-translationally inserted into a subset of proteins in the Methanosarcinaceae and in Desulfitobacterium hafniense programmed by an in-frame UAG stop codon. Suppression of this UAG codon is mediated by the Pyl amber suppressor tRNA, tRNA(Pyl), which is aminoacylated with Pyl by pyrrolysyl-tRNA synthetase (PylRS). We compared the behavior of several archaeal and bacterial PylRS enzymes towards tRNA(Pyl). Equilibrium binding analysis revealed that archaeal PylRS proteins bind tRNA(Pyl) with higher affinity (K(D)=0.1-1.0 microM) than D. hafniense PylRS (K(D)=5.3-6.9 microM). In aminoacylation the archaeal PylRS enzymes did not distinguish between archaeal and bacterial tRNA(Pyl) species, while the bacterial PylRS displays a clear preference for the homologous cognate tRNA. We also show that the amino-terminal extension present in archaeal PylRSs is dispensable for in vitro activity, but required for PylRS function in vivo.     

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.     

Crystal structure of glutamyl-queuosine tRNAAsp synthetase complexed with L-glutamate: structural elements mediating tRNA-independent activation of glutamate and glutamylation of tRNAAsp anticodon

Glutamyl-queuosine tRNA^Asp synthetase (Glu-Q-RS) from Escherichia coli is a paralog of the catalytic core of glutamyl-tRNA synthetase (GluRS) that catalyzes glutamylation of queuosine in the wobble position of tRNA^Asp. Despite important structural similarities, Glu-Q-RS and GluRS diverge strongly by their functional properties. The only feature common to both enzymes consists in the activation of Glu to form Glu-AMP, the intermediate of transfer RNA (tRNA) aminoacylation. However, both enzymes differ by the mechanism of selection of the cognate amino acid and by the mechanism of its activation. Whereas GluRS selects l-Glu and activates it only in the presence of the cognate tRNA^Glu, Glu-Q-RS forms Glu-AMP in the absence of tRNA. Moreover, while GluRS transfers the activated Glu to the 3′ accepting end of the cognate tRNA^Glu, Glu-Q-RS transfers the activated Glu to Q34 located in the anticodon loop of the noncognate tRNA^Asp. In order to gain insight into the structural elements leading to distinct mechanisms of amino acid activation, we solved the three-dimensional structure of Glu-Q-RS complexed to Glu and compared it to the structure of the GluRS·Glu complex. Comparison of the catalytic site of Glu-Q-RS with that of GluRS, combined with binding experiments of amino acids, shows that a restricted number of residues determine distinct catalytic properties of amino acid recognition and activation by the two enzymes. Furthermore, to explore the structural basis of the distinct aminoacylation properties of the two enzymes and to understand why Glu-Q-RS glutamylates only tRNA^Asp among the tRNAs possessing queuosine in position 34, we performed a tRNA mutational analysis to search for the elements of tRNA^Asp that determine recognition by Glu-Q-RS. The analyses made on tRNA^Asp and tRNA^Asn show that the presence of a C in position 38 is crucial for glutamylation of Q34. The results are discussed in the context of the evolution and adaptation of the tRNA glutamylation system.     

Structural states of the flexible catalytic loop of M. tuberculosis tyrosyl-tRNA synthetase in different enzyme–substrate complexes

Tyrosyl-tRNA synthetase from Mycobacterium tuberculosis (MtTyrRS) is an enzyme that belongs to class I of aminoacyl-tRNA synthetases, which catalyze the attachment of l-tyrosine to its cognate tRNATyr in the preribosomal step of protein synthesis. MtTyrRS is incapable of cross-recognition and aminoacylation of human cytoplasmic tRNATyr, so this enzyme may be a promising target for development of novel selective inhibitors as putative antituberculosis drugs. As a class I aminoacyl-tRNA synthetase, MtTyrRS contains the HIGH-like and KFGKS catalytic motifs that catalyze amino acid activation with ATP. In this study, the conformational mobility of MtTyrRS catalytic KFGKS loop was analyzed by 100-ns all-atoms molecular dynamics simulations of the free enzyme and its complexes with different substrates: tyrosine, ATP, and the tyrosyl–adenylate intermediate. It was shown that in the closed state of the active site, the KFGKS loop, readily adopts different stable conformations depending on the type of bound substrate. Molecular dynamics simulations revealed that the closed state of the loop is stabilized by dynamic formation of two antiparallel β-sheets at flanking ends which hold the KFGKS fragment inside the active center. Prevention of β-sheet formation by introducing point mutations in the loop sequence results in a rapid (<20 ns) transition of the loop from its functional “closed” M-like structure to an inactive “open” O-like structure, i.e. rapid diffusion of the catalytic loop outside the active site. The flexibility and rapid dynamics of the wild-type aaRS catalytic loop structure are crucial for formation of protein–substrate interactions and subsequently for overall enzyme functional activity.     

Classical molecular dynamics simulation of seryl tRNA synthetase and threonyl tRNA synthetase bound with tRNA and aminoacyl adenylate

Lacunae of understanding exist concerning the active site organization during the charging step of the aminoacylation reaction. We present here a molecular dynamics simulation study of the dynamics of the active site organization during charging step of subclass IIa dimeric SerRS from Thermus thermophilus (^ttSerRS) bound with ^tttRNA^Ser and dimeric ThrRS from Escherichia coli (^ecThrRS) bound with ^ectRNA^Thr. The interactions between the catalytically important loops and tRNA contribute to the change in dynamics of tRNA in free and bound states, respectively. These interactions help in the development of catalytically effective organization of the active site. The A76 end of the ^tttRNA^Ser exhibits fast dynamics in free State, which is significantly slowed down within the active site bound with adenylate. The loops change their conformation via multimodal dynamics (a slow diffusive mode of nanosecond time scale and fast librational mode of dynamics in picosecond time scale). The active site residues of the motif 2 loop approach the proximal bases of tRNA and adenylate by slow diffusive motion (in nanosecond time scale) and make conformational changes of the respective side chains via ultrafast librational motion to develop precise hydrogen bond geometry. Presence of bound Mg²⁺ ions around tRNA and dynamically slow bound water are other common features of both aaRSs. The presence of dynamically rigid Zinc ion coordination sphere and bipartite mode of recognition of ^ectRNA^Thr are observed.     

Human Tyrosine tRNA Is Also Internally Cleavable by E. coli Ribonuclease P RNA Ribozyme in Vitro

Transfer RNA is an essential molecule for biological system, and each tRNA molecule commonly has a cloverleaf structure. Previously, we experimentally showed that some Drosophila tRNA (tRNA^Ala, tRNA^His, and tRNA_i ^Met) molecules fit to form another, non-cloverleaf, structure in which the 3'-half of the tRNA molecules forms an alternative hairpin, and that the tRNA molecules are internally cleaved by the catalytic RNA of bacterial ribonuclease P (RNase P). Until now, the hyperprocessing reaction of tRNA has only been reported with Drosophila tRNAs. This time, we applied the hyperprocessing reaction to one of human tRNAs, human tyrosine tRNA, and we showed that this tRNA was also hyperprocessed by E. coli RNase P RNA. This tRNA is the first example for hyperprocessed non-Drosophila tRNAs. The results suggest that the hyperprocessing reaction can be a useful tool to detect destablized tRNA molecules from any species.     

Comparative Analysis of Membrane-Associated Fusion Peptide Secondary Structure and Lipid Mixing Function of HIV gp41 Constructs that Model the Early Pre-Hairpin Intermediate and Final Hairpin Conformations

Fusion between viral and host cell membranes is the initial step of human immunodeficiency virus infection and is mediated by the gp41 protein, which is embedded in the viral membrane. The ∼ 20-residue N-terminal fusion peptide (FP) region of gp41 binds to the host cell membrane and plays a critical role in fusion catalysis. Key gp41 fusion conformations include an early pre-hairpin intermediate (PHI) characterized by extended coiled-coil structure in the region C-terminal of the FP and a final hairpin state with compact six-helix bundle structure. The large “N70” (gp41 1-70) and “FP-Hairpin” constructs of the present study contained the FP and respectively modeled the PHI and hairpin conformations. Comparison was also made to the shorter “FP34” (gp41 1-34) fragment. Studies were done in membranes with physiologically relevant cholesterol content and in membranes without cholesterol. In either membrane type, there were large differences in fusion function among the constructs with little fusion induced by FP-Hairpin, moderate fusion for FP34, and very rapid fusion for N70. Overall, our findings support acceleration of gp41-induced membrane fusion by early PHI conformation and fusion arrest after folding to the final six-helix bundle structure. FP secondary structure at Leu7 of the membrane-associated constructs was probed by solid-state nuclear magnetic resonance and showed populations of molecules with either β-sheet or helical structure with greater β-sheet population observed for FP34 than for N70 or FP-Hairpin. The large differences in fusion function among the constructs were not obviously correlated with FP secondary structure. Observation of cholesterol-dependent FP structure for fusogenic FP34 and N70 and cholesterol-independent structure for non-fusogenic FP-Hairpin was consistent with membrane insertion of the FP for FP34 and N70 and with lack of insertion for FP-Hairpin. Membrane insertion of the FP may therefore be associated with the early PHI conformation and FP withdrawal with the final hairpin conformation.     

Crystal structure of the passenger domain of the Escherichia coli autotransporter EspP

Autotransporters represent a large superfamily of known and putative virulence factors produced by Gram-negative bacteria. They consist of an N-terminal “passenger domain” responsible for the specific effector functions of the molecule and a C-terminal “β-domain” responsible for translocation of the passenger across the bacterial outer membrane. Here, we present the 2.5-Å crystal structure of the passenger domain of the extracellular serine protease EspP, produced by the pathogen Escherichia coli O157:H7 and a member of the serine protease autotransporters of Enterobacteriaceae (SPATEs). Like the previously structurally characterized SPATE passenger domains, the EspP passenger domain contains an extended right-handed parallel β-helix preceded by an N-terminal globular domain housing the catalytic function of the protease. Of note, however, is the absence of a second globular domain protruding from this β-helix. We describe the structure of the EspP passenger domain in the context of previous results and provide an alternative hypothesis for the function of the β-helix within SPATEs.     

Structure of the human protein kinase CK2 catalytic subunit CK2α' and interaction thermodynamics with the regulatory subunit CK2β

Protein kinase CK2 (formerly “casein kinase 2”) is composed of a central dimer of noncatalytic subunits (CK2β) binding two catalytic subunits. In humans, there are two isoforms of the catalytic subunit (and an additional splicing variant), one of which (CK2α) is well characterized. To supplement the limited biochemical knowledge about the second paralog (CK2α′), we developed a well-soluble catalytically active full-length mutant of human CK2α′, characterized it by Michaelis-Menten kinetics and isothermal titration calorimetry, and determined its crystal structure to a resolution of 2 Å. The affinity of CK2α′ for CK2β is about 12 times lower than that of CK2α and is less driven by enthalpy. This result fits the observation that the β4/β5 loop, a key element of the CK2α/CK2β interface, adopts an open conformation in CK2α′, while in CK2α, it opens only after assembly with CK2β. The open β4/β5 loop in CK2α′ is stabilized by two elements that are absent in CK2α: (1) the extension of the N-terminal β-sheet by an additional β-strand, and (2) the filling of a conserved hydrophobic cavity between the β4/β5 loop and helix αC by a tryptophan residue. Moreover, the interdomain hinge region of CK2α′ adopts a fully functional conformation, while unbound CK2α is often found with a nonproductive hinge conformation that is overcome only by CK2β binding. Taken together, CK2α′ exhibits a significantly lower affinity for CK2β than CK2α; moreover, in functionally critical regions, it is less dependent on CK2β to obtain a fully functional conformation.     

A bridge between the aminoacylation and editing domains of leucyl-tRNA synthetase is crucial for its synthetic activity

Leucyl-tRNA synthetases (LeuRSs) catalyze the linkage of leucine with tRNA^Leu. LeuRS contains a catalysis domain (aminoacylation) and a CP1 domain (editing). CP1 is inserted 35 Å from the aminoacylation domain. Aminoacylation and editing require CP1 to swing to the coordinated conformation. The neck between the CP1 domain and the aminoacylation domain is defined as the CP1 hairpin. The location of the CP1 hairpin suggests a crucial role in the CP1 swing and domain–domain interaction. Here, the CP1 hairpin of Homo sapiens cytoplasmic LeuRS (hcLeuRS) was deleted or substituted by those from other representative species. Lack of a CP1 hairpin led to complete loss of aminoacylation, amino acid activation, and tRNA binding; however, the mutants retained post-transfer editing. Only the CP1 hairpin from Saccharomyces cerevisiae LeuRS (ScLeuRS) could partly rescue the hcLeuRS functions. Further site-directed mutagenesis indicated that the flexibility of small residues and the charge of polar residues in the CP1 hairpin are crucial for the function of LeuRS.