High-resolution structure of eukaryotic Fibrillarin interacting with Nop56 amino-terminal domain.

Ribosomal RNA (rRNA) carries extensive 2'-O-methyl marks at functionally important sites. This simple chemical modification is thought to confer stability, promote RNA folding, and contribute to generate a heterogenous ribosome population with a yet-uncharacterized function. 2'-O-methylation occurs both in archaea and eukaryotes and is accomplished by the Box C/D RNP enzyme in an RNA-guided manner. Extensive and partially conflicting structural information exists for the archaeal enzyme, while no structural data is available for the eukaryotic enzyme. The yeast Box C/D RNP consists of a guide RNA, the RNA-primary binding protein Snu13, the two scaffold proteins Nop56 and Nop58, and the enzymatic module Nop1. Here we present the high-resolution structure of the eukaryotic Box C/D methyltransferase Nop1 from Saccharomyces cerevisiae bound to the amino-terminal domain of Nop56. We discuss similarities and differences between the interaction modes of the two proteins in archaea and eukaryotes and demonstrate that eukaryotic Nop56 recruits the methyltransferase to the Box C/D RNP through a protein-protein interface that differs substantially from the archaeal orthologs. This study represents a first achievement in understanding the evolution of the structure and function of these proteins from archaea to eukaryotes.

Recruitment of phospholipase Cγ1 to the non-structural membrane protein pK15 of Kaposi Sarcoma-associated herpesvirus promotes its Src-dependent phosphorylation.

Kaposi Sarcoma-associated herpesvirus (KSHV) causes three human malignancies, Kaposi Sarcoma (KS), Primary Effusion Lymphoma (PEL) and the plasma cell variant of multicentric Castleman's Disease (MCD), as well as an inflammatory cytokine syndrome (KICS). Its non-structural membrane protein, pK15, is among a limited set of viral proteins expressed in KSHV-infected KS tumor cells. Following its phosphorylation by Src family tyrosine kinases, pK15 recruits phospholipase C gamma 1 (PLCγ1) to activate downstream signaling cascades such as the MEK/ERK, NFkB and PI3K pathway, and thereby contributes to the increased proliferation and migration as well as the spindle cell morphology of KSHV-infected endothelial cells. Here, we show that a phosphorylated Y481EEVL motif in pK15 preferentially binds into the PLCγ1 C-terminal SH2 domain (cSH2), which is involved in conformational changes occurring during the activation of PLCγ1 by receptor tyrosine kinases. We determined the crystal structure of a pK15 12mer peptide containing the phosphorylated pK15 Y481EEVL motif in complex with a shortened PLCγ1 tandem SH2 (tSH2) domain. This structure demonstrates that the pK15 peptide binds to the PLCγ1 cSH2 domain in a position that is normally occupied by the linker region connecting the PLCγ1 cSH2 and SH3 domains. We also show that longer pK15 peptides containing the phosphorylated pK15 Y481EEVL motif can increase the Src-mediated phosphorylation of the PLCγ1 tSH2 region in vitro. This pK15-induced increase in Src-mediated phosphorylation of PLCγ1 can be inhibited with the small pK15-derived peptide which occupies the PLCγ1 cSH2 domain. Our findings thus suggest that pK15 may act as a scaffold protein to promote PLCγ1 activation in a manner similar to the cellular scaffold protein SLP-76, which has been shown to promote PLCγ1 activation in the context of T-cell receptor signaling. Reminiscent of its positional homologue in Epstein-Barr Virus, LMP2A, pK15 may therefore mimic aspects of antigen-receptor signaling. Our findings also suggest that it may be possible to inhibit the recruitment and activation of PLCγ1 pharmacologically.

Identification of RNA Base Pairs and Complete Assignment of Nucleobase Resonances by Proton-Detected Solid-State NMR Spectroscopy at 100†kHz MAS.

Knowledge of RNA structure, either in isolation or in complex, is fundamental to understand the mechanism of cellular processes. Solid-state NMR (ssNMR) is applicable to high molecular-weight complexes and does not require crystallization; thus, it is well-suited to study RNA as part of large multicomponent assemblies. Recently, we solved the first structures of both RNA and an RNA-protein complex by ssNMR using conventional 13 C- and 15 N-detection. This approach is limited by the severe overlap of the RNA peaks together with the low sensitivity of multidimensional experiments. Here, we overcome the limitations in sensitivity and resolution by using 1 H-detection at fast MAS rates. We develop experiments that allow the identification of complete nucleobase spin-systems together with their site-specific base pair pattern using sub-milligram quantities of one uniformly labelled RNA sample. These experiments provide rapid access to RNA secondary structure by ssNMR in protein-RNA complexes of any size.

Observing Protein Degradation by the PAN-20S Proteasome by Time-Resolved Neutron Scattering.

The proteasome is a key player of regulated protein degradation in all kingdoms of life. Although recent atomic structures have provided snapshots on a number of conformations, data on substrate states and populations during the active degradation process in solution remain scarce. Here, we use time-resolved small-angle neutron scattering of a deuterium-labeled GFPssrA substrate and an unlabeled archaeal PAN-20S system to obtain direct structural information on substrate states during ATP-driven unfolding and subsequent proteolysis in solution. We find that native GFPssrA structures are degraded in a biexponential process, which correlates strongly with ATP hydrolysis, the loss of fluorescence, and the buildup of small oligopeptide products. Our solution structural data support a model in which the substrate is directly translocated from PAN into the 20S proteolytic chamber, after a first, to our knowledge, successful unfolding process that represents a point of no return and thus prevents dissociation of the complex and the release of harmful, aggregation-prone products.

M3: an integrative framework for structure determination of molecular machines.

We present a broadly applicable, user-friendly protocol that incorporates sparse and hybrid experimental data to calculate quasi-atomic-resolution structures of molecular machines. The protocol uses the HADDOCK framework, accounts for extensive structural rearrangements both at the domain and atomic levels and accepts input from all structural and biochemical experiments whose data can be translated into interatomic distances and/or molecular shapes.

Structural characterization of the Asf1-Rtt109 interaction and its role in histone acetylation.

Acetylation of histone H3 at lysine-56 by the histone acetyltransferase Rtt109 in lower eukaryotes is important for maintaining genomic integrity and is required for C. albicans pathogenicity. Rtt109 is activated by association with two different histone chaperones, Vps75 and Asf1, through an unknown mechanism. Here, we reveal that the Rtt109 C-terminus interacts directly with Asf1 and elucidate the structural basis of this interaction. In addition, we find that the H3 N-terminus can interact via the same interface on Asf1, leading to a competition between the two interaction partners. This, together with the recruitment and position of the substrate, provides an explanation of the role of the Rtt109 C-terminus in Asf1-dependent Rtt109 activation.

Structure of a Protein-RNA Complex by Solid-State NMR Spectroscopy.

Solid-state NMR (ssNMR) is applicable to high molecular-weight (MW) protein assemblies in a non-amorphous precipitate. The technique yields atomic resolution structural information on both soluble and insoluble particles without limitations of MW or requirement of crystals. Herein, we propose and demonstrate an approach that yields the structure of protein-RNA complexes (RNP) solely from ssNMR data. Instead of using low-sensitivity magnetization transfer steps between heteronuclei of the protein and the RNA, we measure paramagnetic relaxation enhancement effects elicited on the RNA by a paramagnetic tag coupled to the protein. We demonstrate that this data, together with chemical-shift-perturbation data, yields an accurate structure of an RNP complex, starting from the bound structures of its components. The possibility of characterizing protein-RNA interactions by ssNMR may enable applications to large RNP complexes, whose structures are not accessible by other methods.

The structure of the box C/D enzyme reveals regulation of RNA methylation.

Post-transcriptional modifications are essential to the cell life cycle, as they affect both pre-ribosomal RNA processing and ribosome assembly. The box C/D ribonucleoprotein enzyme that methylates ribosomal RNA at the 2'-O-ribose uses a multitude of guide RNAs as templates for the recognition of rRNA target sites. Two methylation guide sequences are combined on each guide RNA, the significance of which has remained unclear. Here we use a powerful combination of NMR spectroscopy and small-angle neutron scattering to solve the structure of the 390 kDa archaeal RNP enzyme bound to substrate RNA. We show that the two methylation guide sequences are located in different environments in the complex and that the methylation of physiological substrates targeted by the same guide RNA occurs sequentially. This structure provides a means for differential control of methylation levels at the two sites and at the same time offers an unexpected regulatory mechanism for rRNA folding.

Isotope labeling for studying RNA by solid-state NMR spectroscopy.

Nucleic acids play key roles in most biological processes, either in isolation or in complex with proteins. Often they are difficult targets for structural studies, due to their dynamic behavior and high molecular weight. Solid-state nuclear magnetic resonance spectroscopy (ssNMR) provides a unique opportunity to study large biomolecules in a non-crystalline state at atomic resolution. Application of ssNMR to RNA, however, is still at an early stage of development and presents considerable challenges due to broad resonances and poor dispersion. Isotope labeling, either as nucleotide-specific, atom-specific or segmental labeling, can resolve resonance overlaps and reduce the line width, thus allowing ssNMR studies of RNA domains as part of large biomolecules or complexes. In this review we discuss the methods for RNA production and purification as well as numerous approaches for isotope labeling of RNA. Furthermore, we give a few examples that emphasize the instrumental role of isotope labeling and ssNMR for studying RNA as part of large ribonucleoprotein complexes.

Archaea box C/D enzymes methylate two distinct substrate rRNA sequences with different efficiency.

RNA modifications confer complexity to the 4-nucleotide polymer; nevertheless, their exact function is mostly unknown. rRNA 2'-O-ribose methylation concentrates to ribosome functional sites and is important for ribosome biogenesis. The methyl group is transferred to rRNA by the box C/D RNPs: The rRNA sequence to be methylated is recognized by a complementary sequence on the guide RNA, which is part of the enzyme. In contrast to their eukaryotic homologs, archaeal box C/D enzymes can be assembled in vitro and are used to study the mechanism of 2'-O-ribose methylation. In Archaea, each guide RNA directs methylation to two distinct rRNA sequences, posing the question whether this dual architecture of the enzyme has a regulatory role. Here we use methylation assays and low-resolution structural analysis with small-angle X-ray scattering to study the methylation reaction guided by the sR26 guide RNA fromPyrococcus furiosus We find that the methylation efficacy at sites D and D' differ substantially, with substrate D' turning over more efficiently than substrate D. This observation correlates well with structural data: The scattering profile of the box C/D RNP half-loaded with substrate D' is similar to that of the holo complex, which has the highest activity. Unexpectedly, the guide RNA secondary structure is not responsible for the functional difference at the D and D' sites. Instead, this difference is recapitulated by the nature of the first base pair of the guide-substrate duplex. We suggest that substrate turnover may occur through a zip mechanism that initiates at the 5'-end of the product.