Observation of conformational changes that underlie the catalytic cycle of Xrn2.

Nuclear magnetic resonance (NMR) methods that quantitatively probe motions on molecular and atomic levels have propelled the understanding of biomolecular processes for which static structures cannot provide a satisfactory description. In this work, we studied the structure and dynamics of the essential 100-kDa eukaryotic 5'→3' exoribonuclease Xrn2. A combination of complementary fluorine and methyl-TROSY NMR spectroscopy reveals that the apo enzyme is highly dynamic around the catalytic center. These observed dynamics are in agreement with a transition of the enzyme from the ground state into a catalytically competent state. We show that the conformational equilibrium in Xrn2 shifts substantially toward the active state in the presence of substrate and magnesium. Finally, our data reveal that the dynamics in Xrn2 correlate with the RNA degradation rate, as a mutation that attenuates motions also affects catalytic activity. In that light, our results stress the importance of studies that go beyond static structural information.

Modulations of DNA Contacts by Linker Histones and Post-translational Modifications Determine the Mobility and Modifiability of Nucleosomal H3 Tails.

Post-translational histone modifications and linker histone incorporation regulate chromatin structure and genome activity. How these systems interface on a molecular level is unclear. Using biochemistry and NMR spectroscopy, we deduced mechanistic insights into the modification behavior of N-terminal histone H3 tails in different nucleosomal contexts. We find that linker histones generally inhibit modifications of different H3 sites and reduce H3 tail dynamics in nucleosomes. These effects are caused by modulations of electrostatic interactions of H3 tails with linker DNA and largely depend on the C-terminal domains of linker histones. In agreement, linker histone occupancy and H3 tail modifications segregate on a genome-wide level. Charge-modulating modifications such as phosphorylation and acetylation weaken transient H3 tail-linker DNA interactions, increase H3 tail dynamics, and, concomitantly, enhance general modifiability. We propose that alterations of H3 tail-linker DNA interactions by linker histones and charge-modulating modifications execute basal control mechanisms of chromatin function.

Structural basis for the activation of the DEAD-box RNA helicase DbpA by the nascent ribosome.

The adenosine triphosphate (ATP)-dependent DEAD-box RNA helicase DbpA from Escherichia coli functions in ribosome biogenesis. DbpA is targeted to the nascent 50S subunit by an ancillary, carboxyl-terminal RNA recognition motif (RRM) that specifically binds to hairpin 92 (HP92) of the 23S ribosomal RNA (rRNA). The interaction between HP92 and the RRM is required for the helicase activity of the RecA-like core domains of DbpA. Here, we elucidate the structural basis by which DbpA activity is endorsed when the enzyme interacts with the maturing ribosome. We used nuclear magnetic resonance (NMR) spectroscopy to show that the RRM and the carboxyl-terminal RecA-like domain tightly interact. This orients HP92 such that this RNA hairpin can form electrostatic interactions with a positively charged patch in the N-terminal RecA-like domain. Consequently, the enzyme can stably adopt the catalytically important, closed conformation. The substrate binding mode in this complex reveals that a region 5' to helix 90 in the maturing ribosome is specifically targeted by DbpA. Finally, our results indicate that the ribosome maturation defects induced by a dominant negative DbpA mutation are caused by a delayed dissociation of DbpA from the nascent ribosome. Taken together, our findings provide unique insights into the important regulatory mechanism that modulates the activity of DbpA.

Assessing the applicability of 19F labeled tryptophan residues to quantify protein dynamics.

Nuclear magnetic resonance (NMR) spectroscopy is uniquely suited to study the dynamics of biomolecules in solution. Most NMR studies exploit the spins of proton, carbon and nitrogen isotopes, as these atoms are highly abundant in proteins and nucleic acids. As an alternative and complementary approach, fluorine atoms can be introduced into biomolecules at specific sites of interest. These labels can then be used as sensitive probes for biomolecular structure, dynamics or interactions. Here, we address if the replacement of tryptophan with 5-fluorotryptophan residues has an effect on the overall dynamics of proteins and if the introduced fluorine probe is able to accurately report on global exchange processes. For the four different model proteins (KIX, Dcp1, Dcp2 and DcpS) that we examined, we established that 15N CPMG relaxation dispersion or EXSY profiles are not affected by the 5-fluorotryptophan, indicating that this replacement of a proton with a fluorine has no effect on the protein motions. However, we found that the motions that the 5-fluorotryptophan reports on can be significantly faster than the backbone motions. This implies that care needs to be taken when interpreting fluorine relaxation data in terms of global protein motions. In summary, our results underscore the great potential of fluorine NMR methods, but also highlight potential pitfalls that need to be considered.

A structural biology view on the enzymes involved in eukaryotic mRNA turnover.

The cellular environment contains numerous ribonucleases that are dedicated to process mRNA transcripts that have been targeted for degradation. Here, we review the three dimensional structures of the ribonuclease complexes (Pan2-Pan3, Ccr4-Not, Xrn1, exosome) and the mRNA decapping enzymes (Dcp2, DcpS) that are involved in mRNA turnover. Structures of major parts of these proteins have been experimentally determined. These enzymes and factors do not act in isolation, but are embedded in interaction networks which regulate enzyme activity and ensure that the appropriate substrates are recruited. The structural details of the higher order complexes that form can, in part, be accurately deduced from known structural data of sub-complexes. Interestingly, many of the ribonuclease and decapping enzymes have been observed in structurally different conformations. Together with experimental data, this highlights that structural changes are often important for enzyme function. We conclude that the known structural data of mRNA decay factors provide important functional insights, but that static structural data needs to be complemented with information regarding protein motions to complete the picture of how transcripts are turned over. In addition, we highlight multiple aspects that influence mRNA turnover rates, but that have not been structurally characterized so far.

Molecular basis of the selective processing of short mRNA substrates by the DcpS mRNA decapping enzyme.

The 5' messenger RNA (mRNA) cap structure enhances translation and protects the transcript against exonucleolytic degradation. During mRNA turnover, this cap is removed from the mRNA. This decapping step is catalyzed by the Scavenger Decapping Enzyme (DcpS), in case the mRNA has been exonucleolyticly shortened from the 3' end by the exosome complex. Here, we show that DcpS only processes mRNA fragments that are shorter than three nucleotides in length. Based on a combination of methyl transverse relaxation optimized (TROSY) NMR spectroscopy and X-ray crystallography, we established that the DcpS substrate length-sensing mechanism is based on steric clashes between the enzyme and the third nucleotide of a capped mRNA. For longer mRNA substrates, these clashes prevent conformational changes in DcpS that are required for the formation of a catalytically competent active site. Point mutations that enlarge the space for the third nucleotide in the mRNA body enhance the activity of DcpS on longer mRNA species. We find that this mechanism to ensure that the enzyme is not active on translating long mRNAs is conserved from yeast to humans. Finally, we show that the products that the exosome releases after 3' to 5' degradation of the mRNA body are indeed short enough to be decapped by DcpS. Our data thus directly confirms the notion that mRNA products of the exosome are direct substrates for DcpS. In summary, we demonstrate a direct relationship between conformational changes and enzyme activity that is exploited to achieve substrate selectivity.

Multi-Site Conformational Exchange in the Synthetic Neomycin-Sensing Riboswitch Studied by 19 F NMR.

The synthetic neomycin-sensing riboswitch interacts with its cognate ligand neomycin as well as with the related antibiotics ribostamycin and paromomycin. Binding of these aminoglycosides induces a very similar ground state structure in the RNA, however, only neomycin can efficiently repress translation initiation. The molecular origin of these differences has been traced back to differences in the dynamics of the ligand:riboswitch complexes. Here, we combine five complementary fluorine based NMR methods to accurately quantify seconds to microseconds dynamics in the three riboswitch complexes. Our data reveal complex exchange processes with up to four structurally different states. We interpret our findings in a model that shows an interplay between different chemical groups in the antibiotics and specific bases in the riboswitch. More generally, our data underscore the potential of 19 F NMR methods to characterize complex exchange processes with multiple excited states.

A suite of 19F based relaxation dispersion experiments to assess biomolecular motions.

Proteins and nucleic acids are highly dynamic bio-molecules that can populate a variety of conformational states. NMR relaxation dispersion (RD) methods are uniquely suited to quantify the associated kinetic and thermodynamic parameters. Here, we present a consistent suite of 19F-based CPMG, on-resonance R1ρ and off-resonance R1ρ RD experiments. We validate these experiments by studying the unfolding transition of a 7.5 kDa cold shock protein. Furthermore we show that the 19F RD experiments are applicable to very large molecular machines by quantifying dynamics in the 360 kDa half-proteasome. Our approach significantly extends the timescale of chemical exchange that can be studied with 19F RD, adds robustness to the extraction of exchange parameters and can determine the absolute chemical shifts of excited states. Importantly, due to the simplicity of 19F NMR spectra, it is possible to record complete datasets within hours on samples that are of very low costs. This makes the presented experiments ideally suited to complement static structural information from cryo-EM and X-ray crystallography with insights into functionally relevant motions.

Correction to: A suite of 19F based relaxation dispersion experiments to assess biomolecular motions.

In the original publication, Figures 3 and 6 were displayed incorrectly due to a mistake made by the publisher. The correct version of Figs. 3 and 6 are given below.

Changes in conformational equilibria regulate the activity of the Dcp2 decapping enzyme.

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.

Myosin-1E interacts with FAK proline-rich region 1 to induce fibronectin-type matrix.

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase involved in development and human disease, including cancer. It is currently thought that the four-point one, ezrin, radixin, moesin (FERM)-kinase domain linker, which contains autophosphorylation site tyrosine (Y) 397, is not required for in vivo FAK function until late midgestation. Here, we directly tested this hypothesis by generating mice with FAK Y397-to-phenylalanine (F) mutations in the germline. We found that Y397F embryos exhibited reduced mesodermal fibronectin (FN) and osteopontin expression and died during mesoderm development akin to FAK kinase-dead mice. We identified myosin-1E (MYO1E), an actin-dependent molecular motor, to interact directly with the FAK FERM-kinase linker and induce FAK kinase activity and Y397 phosphorylation. Active FAK in turn accumulated in the nucleus where it led to the expression of osteopontin and other FN-type matrix in both mouse embryonic fibroblasts and human melanoma. Our data support a model in which FAK Y397 autophosphorylation is required for FAK function in vivo and is positively regulated by MYO1E.

The Rrp4-exosome complex recruits and channels substrate RNA by a unique mechanism.

The exosome is a large molecular machine involved in RNA degradation and processing. Here we address how the trimeric Rrp4 cap enhances the activity of the archaeal enzyme complex. Using methyl-TROSY NMR methods we identified a 50-Å long RNA binding path on each Rrp4 protomer. We show that the Rrp4 cap can thus simultaneously recruit three substrates, one of which is degraded in the core while the others are positioned for subsequent degradation rounds. The local interaction energy between the substrate and the Rrp4-exosome increases from the periphery of the complex toward the active sites. Notably, the intrinsic interaction strength between the cap and the substrate is weakened as soon as substrates enter the catalytic barrel, which provides a means to reduce friction during substrate movements toward the active sites. Our data thus reveal a sophisticated exosome-substrate interaction mechanism that enables efficient RNA degradation.

A synergistic network of interactions promotes the formation of in vitro processing bodies and protects mRNA against decapping.

Cellular liquid-liquid phase separation (LLPS) results in the formation of dynamic granules that play an important role in many biological processes. On a molecular level, the clustering of proteins into a confined space results from an indefinite network of intermolecular interactions. Here, we introduce and exploit a novel high-throughput bottom-up approach to study how the interactions between RNA, the Dcp1:Dcp2 mRNA decapping complex and the scaffolding proteins Edc3 and Pdc1 result in the formation of processing bodies. We find that the LLPS boundaries are close to physiological concentrations upon inclusion of multiple proteins and RNA. Within in vitro processing bodies the RNA is protected against endonucleolytic cleavage and the mRNA decapping activity is reduced, which argues for a role of processing bodies in temporary mRNA storage. Interestingly, the intrinsically disordered region (IDR) in the Edc3 protein emerges as a central hub for interactions with both RNA and mRNA decapping factors. In addition, the Edc3 IDR plays a role in the formation of irreversible protein aggregates that are potentially detrimental for cellular homeostasis. In summary, our data reveal insights into the mechanisms that lead to cellular LLPS and into the way this influences enzymatic activity.

The S. pombe mRNA decapping complex recruits cofactors and an Edc1-like activator through a single dynamic surface.

The removal of the 5' 7-methylguanosine mRNA cap structure (decapping) is a central step in the 5'-3' mRNA degradation pathway and is performed by the Dcp1:Dcp2 decapping complex. The activity of this complex is tightly regulated to prevent premature degradation of the transcript. Here, we establish that the aromatic groove of the EVH1 domain of Schizosaccharomyces pombe Dcp1 can interact with proline-rich sequences in the exonuclease Xrn1, the scaffolding protein Pat1, the helicase Dhh1, and the C-terminal disordered region of Dcp2. We show that this region of Dcp1 can also recruit a previously unidentified enhancer of decapping protein (Edc1) and solved the crystal structure of the complex. NMR relaxation dispersion experiments reveal that the Dcp1 binding site can adopt multiple conformations, thus providing the plasticity that is required to accommodate different ligands. We show that the activator Edc1 makes additional contacts with the regulatory domain of Dcp2 and that an activation motif in Edc1 increases the RNA affinity of Dcp1:Dcp2. Our data support a model where Edc1 stabilizes the RNA in the active site, which results in enhanced decapping rates. In summary, we show that multiple decapping factors, including the Dcp2 C-terminal region, compete with Edc1 for Dcp1 binding. Our data thus reveal a network of interactions that can fine-tune the catalytic activity of the decapping complex.

A general method for rapid and cost-efficient large-scale production of 5' capped RNA.

The eukaryotic mRNA 5' cap structure is indispensible for pre-mRNA processing, mRNA export, translation initiation, and mRNA stability. Despite this importance, structural and biophysical studies that involve capped RNA are challenging and rare due to the lack of a general method to prepare mRNA in sufficient quantities. Here, we show that the vaccinia capping enzyme can be used to produce capped RNA in the amounts that are required for large-scale structural studies. We have therefore designed an efficient expression and purification protocol for the vaccinia capping enzyme. Using this approach, the reaction scale can be increased in a cost-efficient manner, where the yields of the capped RNA solely depend on the amount of available uncapped RNA target. Using a large number of RNA substrates, we show that the efficiency of the capping reaction is largely independent of the sequence, length, and secondary structure of the RNA, which makes our approach generally applicable. We demonstrate that the capped RNA can be directly used for quantitative biophysical studies, including fluorescence anisotropy and high-resolution NMR spectroscopy. In combination with (13)C-methyl-labeled S-adenosyl methionine, the methyl groups in the RNA can be labeled for methyl TROSY NMR spectroscopy. Finally, we show that our approach can produce both cap-0 and cap-1 RNA in high amounts. In summary, we here introduce a general and straightforward method that opens new means for structural and functional studies of proteins and enzymes in complex with capped RNA.

The oligomeric architecture of the archaeal exosome is important for processive and efficient RNA degradation.

The exosome plays an important role in RNA degradation and processing. In archaea, three Rrp41:Rrp42 heterodimers assemble into a barrel like structure that contains a narrow RNA entrance pore and a lumen that contains three active sites. Here, we demonstrate that this quaternary structure of the exosome is important for efficient RNA degradation. We find that the entrance pore of the barrel is required for nM substrate affinity. This strong interaction is crucial for processive substrate degradation and prevents premature release of the RNA from the enzyme. Using methyl TROSY NMR techniques, we establish that the 3' end of the substrate remains highly flexible inside the lumen. As a result, the RNA jumps between the three active sites that all equally participate in substrate degradation. The RNA jumping rate is, however, much faster than the cleavage rate, indicating that not all active site:substrate encounters result in catalysis. Enzymatic turnover therefore benefits from the confinement of the active sites and substrate in the lumen, which ensures that the RNA is at all times bound to one of the active sites. The evolution of the exosome into a hexameric complex and the optimization of its catalytic efficiency were thus likely co-occurring events.

An excess of catalytically required motions inhibits the scavenger decapping enzyme.

The scavenger decapping enzyme hydrolyzes the protective 5' cap structure on short mRNA fragments that are generated from the exosomal degradation of mRNAs. From static crystal structures and NMR data, it is apparent that the dimeric enzyme has to undergo large structural changes to bind its substrate in a catalytically competent conformation. Here we studied the yeast enzyme and showed that the associated opening and closing motions can be orders of magnitude faster than the catalytic turnover rate. This excess of motion is induced by the binding of a second ligand to the enzyme, which occurs at high substrate concentrations. We designed a mutant that disrupted the allosteric pathway that links the second binding event to the dynamics and showed that this mutant enzyme is hyperactive. Our data reveal a unique mechanism of substrate inhibition in which motions that are required for catalytic activity also inhibit efficient turnover when they are present in excess.

Solid-State NMR H-N-(C)-H and H-N-C-C 3D/4D Correlation Experiments for Resonance Assignment of Large Proteins.

Solid-state NMR spectroscopy can provide insight into protein structure and dynamics at the atomic level without inherent protein size limitations. However, a major hurdle to studying large proteins by solid-state NMR spectroscopy is related to spectral complexity and resonance overlap, which increase with molecular weight and severely hamper the assignment process. Here the use of two sets of experiments is shown to expand the tool kit of 1 H-detected assignment approaches, which correlate a given amide pair either to the two adjacent CO-CA pairs (4D hCOCANH/hCOCAcoNH), or to the amide 1 H of the neighboring residue (3D HcocaNH/HcacoNH, which can be extended to 5D). The experiments are based on efficient coherence transfers between backbone atoms using INEPT transfers between carbons and cross-polarization for heteronuclear transfers. The utility of these experiments is exemplified with application to assemblies of deuterated, fully amide-protonated proteins from approximately 20 to 60 kDa monomer, at magic-angle spinning (MAS) frequencies from approximately 40 to 55 kHz. These experiments will also be applicable to protonated proteins at higher MAS frequencies. The resonance assignment of a domain within the 50.4 kDa bacteriophage T5 tube protein pb6 is reported, and this is compared to NMR assignments of the isolated domain in solution. This comparison reveals contacts of this domain to the core of the polymeric tail tube assembly.

The structural basis of Edc3- and Scd6-mediated activation of the Dcp1:Dcp2 mRNA decapping complex.

The Dcp1:Dcp2 decapping complex catalyses the removal of the mRNA 5' cap structure. Activator proteins, including Edc3 (enhancer of decapping 3), modulate its activity. Here, we solved the structure of the yeast Edc3 LSm domain in complex with a short helical leucine-rich motif (HLM) from Dcp2. The motif interacts with the monomeric Edc3 LSm domain in an unprecedented manner and recognizes a noncanonical binding surface. Based on the structure, we identified additional HLMs in the disordered C-terminal extension of Dcp2 that can interact with Edc3. Moreover, the LSm domain of the Edc3-related protein Scd6 competes with Edc3 for the interaction with these HLMs. We show that both Edc3 and Scd6 stimulate decapping in vitro, presumably by preventing the Dcp1:Dcp2 complex from adopting an inactive conformation. In addition, we show that the C-terminal HLMs in Dcp2 are necessary for the localization of the Dcp1:Dcp2 decapping complex to P-bodies in vivo. Unexpectedly, in contrast to yeast, in metazoans the HLM is found in Dcp1, suggesting that details underlying the regulation of mRNA decapping changed throughout evolution.