A substrate binding model for the KEOPS tRNA modifying complex.
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ABSTRACT:
SUBMITTER: Beenstock J
PROVIDER: S-EPMC7718258 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:In Archaea and Eukaryotes, the synthesis of a universal tRNA modification, N6-threonyl-carbamoyl adenosine (t6A), is catalyzed by the KEOPS complex composed of Kae1, Bud32, Cgi121, and Pcc1. A fifth subunit, Gon7, is found only in Fungi and Metazoa. Here, we identify and characterize a fifth KEOPS subunit in Archaea. This protein, dubbed Pcc2, is a paralog of Pcc1 and is widely conserved in Archaea. Pcc1 and Pcc2 form a heterodimer in solution, and show modest sequence conservation but very high structural similarity. The five-subunit archaeal KEOPS does not form dimers but retains robust tRNA binding and t6A synthetic activity. Pcc2 can substitute for Pcc1 but the resulting KEOPS complex is inactive, suggesting a distinct function for the two paralogs. Comparative sequence and structure analyses point to a possible evolutionary link between archaeal Pcc2 and eukaryotic Gon7. Our work indicates that Pcc2 regulates the oligomeric state of the KEOPS complex, a feature that seems to be conserved from Archaea to Eukaryotes.
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Project description:N6-theronylcarbomyl adenosine (t6A) is a universal tRNA posttranscriptional modification that promotes translation fidelity and is therefore required for the fitness of virtually all living cells. Kae1, the t6A modifying enzyme in eukaryotes and archea, resides within the KEOPS complex with three auxiliary subunits of unknown function namely Cgi121, Bud32 and Pcc1. Through biochemical and X-ray crystallographic analyses we show that Cgi121 functions to recruit substrate tRNA to KEOPS via its universal 3’-CCA tail. Incorporation of the Cgi121-tRNA structure into a composite model of KEOPS reveals an extended tRNA contact surface that surprisingly spans all four subunits. Comprehensive mutational and functional analyses validate the relevance of the extended tRNA-binding surface in vivo and in vivo. Together this work provides insight into the t6A catalytic cycle and its regulation through the cooperative action of all four KEOPS subunit through direct contacts with tRNA.
2020-12-11 | PXD018007 | Pride
Project description:The highly conserved Kinase, Endopeptidase and Other Proteins of small Size (KEOPS)/Endopeptidase-like and Kinase associated to transcribed Chromatin (EKC) protein complex has been implicated in transcription, telomere maintenance and chromosome segregation, but its exact function remains unknown. The complex consists of five proteins, Kinase-Associated Endopeptidase (Kae1), a highly conserved protein present in bacteria, archaea and eukaryotes, a kinase (Bud32) and three additional small polypeptides. We showed that the complex is required for a universal tRNA modification, threonyl carbamoyl adenosine (t6A), found in all tRNAs that pair with ANN codons in mRNA. We also showed that the bacterial ortholog of Kae1, YgjD, is required for t6A modification of Escherichia coli tRNAs. The ATPase activity of Kae1 and the kinase activity of Bud32 are required for the modification. The yeast protein Sua5 has been reported previously to be required for t6A synthesis. Using yeast extracts, we established an in vitro system for the synthesis of t6A that requires Sua5, Kae1, threonine, bicarbonate and ATP. It remains to be determined whether all reported defects of KEOPS/EKC mutants can be attributed to the lack of t6A, or whether the complex has multiple functions.
| S-EPMC3049205 | biostudies-literature
Project description:N(6)-threonylcarbamoyladenosine (t(6)A) is a universal tRNA modification essential for normal cell growth and accurate translation. In Archaea and Eukarya, the universal protein Sua5 and the conserved KEOPS/EKC complex together catalyze t(6)A biosynthesis. The KEOPS/EKC complex is composed of Kae1, a universal metalloprotein belonging to the ASHKA superfamily of ATPases; Bud32, an atypical protein kinase and two small proteins, Cgi121 and Pcc1. In this study, we investigated the requirement and functional role of KEOPS/EKC subunits for biosynthesis of t(6)A. We demonstrated that Pcc1, Kae1 and Bud32 form a minimal functional unit, whereas Cgi121 acts as an allosteric regulator. We confirmed that Pcc1 promotes dimerization of the KEOPS/EKC complex and uncovered that together with Kae1, it forms the tRNA binding core of the complex. Kae1 binds l-threonyl-carbamoyl-AMP intermediate in a metal-dependent fashion and transfers the l-threonyl-carbamoyl moiety to substrate tRNA. Surprisingly, we found that Bud32 is regulated by Kae1 and does not function as a protein kinase but as a P-loop ATPase possibly involved in tRNA dissociation. Overall, our data support a mechanistic model in which the final step in the biosynthesis of t(6)A relies on a strictly catalytic component, Kae1, and three partner proteins necessary for dimerization, tRNA binding and regulation.
| S-EPMC3814370 | biostudies-literature
Project description:KEOPS complex is one of the most conserved protein complexes in eukaryotes. It plays important roles in both telomere uncapping and tRNA N6-threonylcarbamoyladenosine (t6A) modification in budding yeast. But whether KEOPS complex plays any roles in DNA repair remains unknown. Here, we show that KEOPS complex plays positive roles in both DNA damage response and homologous recombination-mediated DNA repair independently of its t6A synthesis function. Additionally, KEOPS displays DNA binding activity in vitro, and is recruited to the chromatin at DNA breaks in vivo, suggesting a direct role of KEOPS in DSB repair. Mechanistically, KEOPS complex appears to promote DNA end resection through facilitating the association of Exo1 and Dna2 with DNA breaks. Interestingly, inactivation of both KEOPS and Mre11/Rad50/Xrs2 (MRX) complexes results in synergistic defect in DNA resection, revealing that KEOPS and MRX have some redundant functions in DNA resection. Thus we uncover a t6A-independent role of KEOPS complex in DNA resection, and propose that KEOPS might be a DSB sensor to assist cells in maintaining chromosome stability.
| S-EPMC6582355 | biostudies-literature
Project description:The accuracy of in vivo incorporation of amino acids during protein biosynthesis is controlled to a significant extent by aminoacyl-tRNA synthetases (aaRS). This paper describes the application of the HierDock computational method to study the molecular basis of amino acid binding to the Escherichia coli methionyl tRNA synthetase (MetRS). Starting with the protein structure from the MetRS cocrystal, the HierDock calculations predict the binding site of methionine in MetRS to a root mean square deviation in coordinates (CRMS) of 0.55 A for all the atoms, compared with the crystal structure. The MetRS conformation in the cocrystal structure shows good discrimination between cognate and the 19 noncognate amino acids. In addition, the calculated binding energies of a set of five methionine analogs show a good correlation (R(2) = 0.86) to the relative free energies of binding derived from the measured in vitro kinetic parameters, K(m) and k(cat). Starting with the crystal structure of MetRS without the methionine (apo-MetRS), the putative binding site of methionine was predicted. We demonstrate that even the apo-MetRS structure shows a preference for binding methionine compared with the 19 other natural amino acids. On comparing the calculated binding energies of the 20 natural amino acids for apo-MetRS with those for the cocrystal structure, we observe that the discrimination against the noncognate substrate increases dramatically in the second step of the physical binding process associated with the conformation change in the protein.
| S-EPMC2286561 | biostudies-literature
Project description:An important aspect of molecular mechanics simulations of a protein structure and ligand binding often involves the generation of reliable force fields for nonstandard residues and ligands. We consider the aminoacyl-tRNA synthetase (AaRS) system that involves nucleic acid and amino acid derivatives, obtaining force field atomic charges using the restrained electrostatic potential (RESP) approach. These charges are shown to predict observed properties of the post-transfer editing reaction in this system, in contrast to simulations performed using approximate charges conceived based upon standard charges for related systems present in force field databases. In particular, the simulations predicted key properties induced by mutation. The approach taken for generating the RESP charges retains established charges for known fragments, defining new charges only for the novel chemical features present in the modified residues. This approach is of general relevance for the design of force fields for pharmacological applications, and indeed the AaRS target system is itself relevant to antibiotics development.
| S-EPMC7906584 | biostudies-literature
Project description:KEOPS is an important cellular complex conserved in Eukarya, with some subunits conserved in Archaea and Bacteria. This complex was recently found to play an essential role in formation of the tRNA modification threonylcarbamoyladenosine (t(6)A), and was previously associated with telomere length maintenance and transcription. KEOPS subunits are conserved in Archaea, especially in the Euryarchaea, where they had been studied in vitro. Here we attempted to delete the genes encoding the four conserved subunits of the KEOPS complex in the euryarchaeote Haloferax volcanii and study their phenotypes in vivo. The fused kae1-bud32 gene was shown to be essential as was cgi121, which is dispensable in yeast. In contrast, pcc1 (encoding the putative dimerizing unit of KEOPS) was not essential in H. volcanii. Deletion of pcc1 led to pleiotropic phenotypes, including decreased growth rate, reduced levels of t(6)A modification, and elevated levels of intra-cellular glycation products.
| S-EPMC3426518 | biostudies-literature
Project description:The N6-threonylcarbamoyl adenosine (t6A) tRNA modification is critical for ensuring translation fidelity across three domains of life. Our prior work highlighted the KEOPS complex, organized in a Pcc1-Kae1-Bud32-Cgi121 linear arrangement, not only serves an evolutionarily conserved role in t6A tRNA modification but also exerts diverse functional impacts on pathobiological characteristics in Cryptococcus neoformans, a leading cause of fungal meningitis worldwide. However, the extent to which the pleiotropic functions of the KEOPS complex are specifically tied to tRNA modification remains uncertain. To address this, we undertook a functional characterization of Sua5, responsible for generating the precursor threonylcarbamoyl-adenylate (TC-AMP) for t6A tRNA modification, using a reverse genetics approach. Comparative phenotypic analyses with KEOPS mutants revealed that Sua5 plays a vital role in multiple cellular processes, such as t6A tRNA modification, growth, sexual development, stress response, and virulence factor production, thus reflecting the multifaceted functions of the KEOPS complex. In support of this, sua5Δ bud32Δ double mutants showed phenotypes comparable to those of the corresponding single mutants. Intriguingly, a SUA5 allele lacking a mitochondria targeting sequence (SUA5MTSΔ) was sufficient to restore the wild-type phenotypes in the sua5Δ mutant, suggesting that Sua5's primary functional locus may be cytosolic, akin to the KEOPS complex. Further supporting this, the deletion of Qri7, a mitochondrial paralog of Kae1, had no discernible phenotypic impact on C. neoformans. We concluded that cytosolic t6A tRNA modifications, orchestrated by Sua5 and the KEOPS complex, are central to the regulation of diverse pathobiological functions in C. neoformans.IMPORTANCEUnderstanding cellular functions at the molecular level is crucial for advancing disease treatments. Our research reveals a critical connection between the KEOPS complex and Sua5 in Cryptococcus neoformans, a significant cause of fungal meningitis. While the KEOPS complex is known for its versatile roles in cellular processes, Sua5 is specialized in t6A tRNA modification. Our key finding is that the diverse roles of the KEOPS complex, ranging from cell growth and stress response to virulence, are fundamentally linked to its function in t6A tRNA modification. This conclusion is supported by the remarkable similarities between the impacts of Sua5 and KEOPS on these processes, despite their roles in different steps of the t6A modification pathway. This newfound understanding deepens our insight into fungal biology and opens new avenues for developing potential therapies against dangerous fungal diseases.
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Project description:RNase P is a universal enzyme that removes 5' leader sequences from tRNA precursors. The enzyme is therefore essential for maturation of functional tRNAs and mRNA translation. RNase P represents a unique example of an enzyme that can occur either as ribonucleoprotein or as protein alone. The latter form of the enzyme, called protein-only RNase P (PRORP), is widespread in eukaryotes in which it can provide organellar or nuclear RNase P activities. Here, we have focused on Arabidopsis nuclear PRORP2 and its interaction with tRNA substrates. Affinity measurements helped assess the respective importance of individual pentatricopeptide repeat motifs in PRORP2 for RNA binding. We characterized the PRORP2 structure by X-ray crystallography and by small-angle X-ray scattering in solution as well as that of its complex with a tRNA precursor by small-angle X-ray scattering. Of note, our study reports the first structural data of a PRORP-tRNA complex. Combined with complementary biochemical and biophysical analyses, our structural data suggest that PRORP2 undergoes conformational changes to accommodate its substrate. In particular, the catalytic domain and the RNA-binding domain can move around a central hinge. Altogether, this work provides a refined model of the PRORP-tRNA complex that illustrates how protein-only RNase P enzymes specifically bind tRNA and highlights the contribution of protein dynamics to achieve this specific interaction.
| S-EPMC5572917 | biostudies-literature