The Nep1 (Emg1) SPOUT-class methyltransferase is an essential ribosome assembly factor and the human Bowen–Conradi syndrome (BCS) is caused by a specific Nep1D86G mutation. We recently showed in vitro that Methanocaldococcus jannaschii Nep1 is a sequence-specific pseudouridine-N1-methyltransferase. Here, we show that in yeast the in vivo target site for Nep1-catalyzed methylation is located within loop 35 of the 18S rRNA that contains the unique hypermodification of U1191 to 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouri-dine (m1acp3Ψ). Specific 14C-methionine labelling of 18S rRNA in yeast mutants showed that Nep1 is not required for acp-modification but suggested a function in Ψ1191 methylation. ESI MS analysis of acp-modified Ψ-nucleosides in a Δnep1-mutant showed that Nep1 catalyzes the Ψ1191 methylation in vivo. Remarkably, the restored growth of a nep1-1ts mutant upon addition of S-adenosylmethionine was even observed after preventing U1191 methylation in a Δsnr35 mutant. This strongly suggests a dual Nep1 function, as Ψ1191-methyltransferase and ribosome assembly factor. Interestingly, the Nep1 methyltransferase activity is not affected upon introduction of the BCS mutation. Instead, the mutated protein shows enhanced dimerization propensity and increased affinity for its RNA-target in vitro. Furthermore, the BCS mutation prevents nucleolar accumulation of Nep1, which could be the reason for reduced growth in yeast and the Bowen-Conradi syndrome.

Crystal structures of enzymes are indispensable to understanding their mechanisms on a molecular level. It, however, remains challenging to determine which structures are adopted in solution, especially for dynamic complexes. Here, we study the bilobed decapping enzyme Dcp2 that removes the 5′ cap structure from eukaryotic mRNA and thereby efficiently terminates gene expression. The numerous Dcp2 structures can be grouped into six states where the domain orientation between the catalytic and regulatory domains significantly differs. Despite this wealth of structural information it is not possible to correlate these states with the catalytic cycle or the activity of the enzyme. Using methyl transverse relaxation-optimized NMR spectroscopy, we demonstrate that only three of the six domain orientations are present in solution, where Dcp2 adopts an open, a closed, or a catalytically active state. We show how mRNA substrate and the activator proteins Dcp1 and Edc1 influence the dynamic equilibria between these states and how this modulates catalytic activity. Importantly, the active state of the complex is only stably formed in the presence of both activators and the mRNA substrate or the m7GDP decapping product, which we rationalize based on a crystal structure of the Dcp1:Dcp2:Edc1:m7GDP complex. Interestingly, we find that the activating mechanisms in Dcp2 also result in a shift of the substrate specificity from bacterial to eukaryotic mRNA. mRNA decapping | Dcp2 | NMR spectroscopy | catalytic activity | protein dynamics

Nep1 (Emg1) is a highly conserved nucleolar protein with an essential function in ribosome biogenesis. A mutation in the human Nep1 homolog causes Bowen–Conradi syndrome—a severe developmental disorder. Structures of Nep1 revealed a dimer with a fold similar to the SPOUT-class of RNA-methyltransferases suggesting that Nep1 acts as a methyltransferase in ribosome biogenesis. The target for this putative methyltransferase activity has not been identified yet. We characterized the RNA-binding specificity of Methanocaldococcus jannaschii Nep1 by fluorescence- and NMR-spectroscopy as well as by yeast three-hybrid screening. Nep1 binds with high affinity to short RNA oligonucleotides corresponding to nt 910–921 of M. jannaschii 16S rRNA through a highly conserved basic surface cleft along the dimer interface. Nep1 only methylates RNAs containing a pseudouridine at a position corresponding to a previously identified hypermodified N1-methyl-N3-(3-amino-3-carboxypropyl) pseudouridine (m1acp3-Ψ) in eukaryotic 18S rRNAs. Analysis of the methylated nucleoside by MALDI-mass spectrometry, HPLC and NMR shows that the methyl group is transferred to the N1 of the pseudouridine. Thus, Nep1 is the first identified example of an N1-specific pseudouridine methyltransferase. This enzymatic activity is also conserved in human Nep1 suggesting that Nep1 is the methyltransferase in the biosynthesis of m1acp3-Ψ in eukaryotic 18S rRNAs.

Several peptides in clinical use are derived from non-ribosomal peptide synthetases (NRPS). In these systems multiple NRPS subunits interact with each other in a specific linear order mediated by specific docking domains (DDs), whose structures are not known yet, to synthesize well-defined peptide products. In contrast to classical NRPSs, single-module NRPS subunits responsible for the generation of rhabdopeptide/xenortide-like peptides (RXPs) can act in different order depending on subunit stoichiometry thereby producing peptide libraries. To define the basis for their unusual interaction patterns, we determine the structures of all N-terminal DDs (NDDs) as well as of an NDD-CDD complex and characterize all putative DD interactions thermodynamically for such a system. Key amino acid residues for DD interactions are identified that upon their exchange change the DD affinity and result in predictable changes in peptide production. Recognition rules for DD interactions are identified that also operate in other megasynthase complexes.

The chemically most complex modification in eukaryotic rRNA is the conserved hypermodified nucleotide N1-methyl-N3-aminocarboxypropyl-pseudouridine (m1acp3Ψ) located next to the P-site tRNA on the small subunit 18S rRNA. While S-adenosylmethionine was identified as the source of the aminocarboxypropyl (acp) group more than 40 years ago the enzyme catalyzing the acp transfer remained elusive. Here we identify the cytoplasmic ribosome biogenesis protein Tsr3 as the responsible enzyme in yeast and human cells. In functionally impaired Tsr3-mutants, a reduced level of acp modification directly correlates with increased 20S pre-rRNA accumulation. The crystal structure of archaeal Tsr3 homologs revealed the same fold as in SPOUT-class RNA-methyltransferases but a distinct SAM binding mode. This unique SAM binding mode explains why Tsr3 transfers the acp and not the methyl group of SAM to its substrate. Structurally, Tsr3 therefore represents a novel class of acp transferase enzymes.

Eukaryotic ribosome biogenesis requires the concerted action of numerous ribosome assembly factors, for most of which structural and functional information is currently lacking. Nob1, which can be identified in eukaryotes and archaea, is required for the final maturation of the small subunit ribosomal RNA in yeast by catalyzing cleavage at site D after export of the preribosomal subunit into the cytoplasm. Here, we show that this also holds true for Nob1 from the archaeon Pyrococcus horikoshii, which efficiently cleaves RNA-substrates containing the D-site of the preribosomal RNA in a manganese-dependent manner. The structure of PhNob1 solved by nuclear magnetic resonance spectroscopy revealed a PIN domain common with many nucleases and a zinc ribbon domain, which are structurally connected by a flexible linker. We show that amino acid residues required for substrate binding reside in the PIN domain whereas the zinc ribbon domain alone is sufficient to bind helix 40 of the small subunit rRNA. This suggests that the zinc ribbon domain acts as an anchor point for the protein on the nascent subunit positioning it in the proximity of the cleavage site.

Imidazole glycerol phosphate synthase (HisFH) is a heterodimeric bienzyme complex operating at a central branch point of metabolism. HisFH is responsible for the HisH-catalyzed hydrolysis of glutamine to glutamate and ammonia, which is then used for a cyclase reaction by HisF. The HisFH complex is allosterically regulated but the underlying mechanism is not well understood. Here, we elucidate the molecular basis of the long range, allosteric activation of HisFH. We establish that the catalytically active HisFH conformation is only formed when the substrates of both HisH and HisF are bound. We show that in this conformation an oxyanion hole in the HisH active site is established, which rationalizes the observed 4500-fold allosteric activation compared to the inactive conformation. In solution, the inactive and active conformations are in a dynamic equilibrium and the HisFH turnover rates correlate with the population of the active conformation, which is in accordance with the ensemble model of allostery.

Significance Ribosomes are complex macromolecular machines universal to all domains of life that synthesize all cellular proteins. They consist of many different protein and RNA building blocks. Their biogenesis—a paradigm for the assembly of macromolecular machines in general—is a highly complex and tightly regulated process in eukaryotes requiring the concerted action of many ribosome assembly factors. In this study we analyze the cross-talk between two of these assembly factors and ribosomal building blocks with biophysical methods and show how it might contribute to the fidelity and efficiency of ribosome assembly.