Modeling Flexible Protein Structure With AlphaFold2 and Crosslinking Mass Spectrometry.

We propose a pipeline that combines AlphaFold2 (AF2) and crosslinking mass spectrometry (XL-MS) to model the structure of proteins with multiple conformations. The pipeline consists of two main steps: ensemble generation using AF2 and conformer selection using XL-MS data. For conformer selection, we developed two scores-the monolink probability score (MP) and the crosslink probability score (XLP)-both of which are based on residue depth from the protein surface. We benchmarked MP and XLP on a large dataset of decoy protein structures and showed that our scores outperform previously developed scores. We then tested our methodology on three proteins having an open and closed conformation in the Protein Data Bank: Complement component 3 (C3), luciferase, and glutamine-binding periplasmic protein, first generating ensembles using AF2, which were then screened for the open and closed conformations using experimental XL-MS data. In five out of six cases, the most accurate model within the AF2 ensembles-or a conformation within 1 Å of this model-was identified using crosslinks, as assessed through the XLP score. In the remaining case, only the monolinks (assessed through the MP score) successfully identified the open conformation of glutamine-binding periplasmic protein, and these results were further improved by including the "occupancy" of the monolinks. This serves as a compelling proof-of-concept for the effectiveness of monolinks. In contrast, the AF2 assessment score was only able to identify the most accurate conformation in two out of six cases. Our results highlight the complementarity of AF2 with experimental methods like XL-MS, with the MP and XLP scores providing reliable metrics to assess the quality of the predicted models. The MP and XLP scoring functions mentioned above are available at https://gitlab.com/topf-lab/xlms-tools.

The SmAP2 RNA binding motif in the 3'UTR affects mRNA stability in the crenarchaeum Sulfolobus solfataricus.

Sm and Sm-like proteins represent an evolutionarily conserved family with key roles in RNA metabolism in Pro- and Eukaryotes. In this study, a collection of 53 mRNAs that co-purified with Sulfolobus solfataricus (Sso) SmAP2 were surveyed for a specific RNA binding motif (RBM). SmAP2 was shown to bind with high affinity to the deduced consensus RNA binding motif (SmAP2-cRBM) in vitro. Residues in SmAP2 interacting with the SmAP2-cRBM were mapped by UV-induced crosslinking in combination with mass-spectrometry, and verified by mutational analyses. The RNA-binding site on SmAP2 includes a modified uracil binding pocket containing a unique threonine (T40) located on the L3 face and a second residue, K25, located in the pore. To study the function of the SmAP2-RBM in vivo, three authentic RBMs were inserted in the 3'UTR of a lacS reporter gene. The presence of the SmAP2-RBM in the reporter-constructs resulted in decreased LacS activity and reduced steady state levels of lacS mRNA. Moreover, the presence of the SmAP2-cRBM in and the replacement of the lacS 3'UTR with that of Sso2194 encompassing a SmAP2-RBM apparently impacted on the stability of the chimeric transcripts. These results are discussed in light of the function(s) of eukaryotic Lsm proteins in RNA turnover.

The SmAP1/2 proteins of the crenarchaeon Sulfolobus solfataricus interact with the exosome and stimulate A-rich tailing of transcripts.

The conserved Sm and Sm-like proteins are involved in different aspects of RNA metabolism. Here, we explored the interactome of SmAP1 and SmAP2 of the crenarchaeon Sulfolobus solfataricus (Sso) to shed light on their physiological function(s). Both, SmAP1 and SmAP2 co-purified with several proteins involved in RNA-processing/modification, translation and protein turnover as well as with components of the exosome involved in 3΄ to 5΄ degradation of RNA. In follow-up studies a direct interaction with the poly(A) binding and accessory exosomal subunit DnaG was demonstrated. Moreover, elevated levels of both SmAPs resulted in increased abundance of the soluble exosome fraction, suggesting that they affect the subcellular localization of the exosome in the cell. The increased solubility of the exosome was accompanied by augmented levels of RNAs with A-rich tails that were further characterized using RNASeq. Hence, the observation that the Sso SmAPs impact on the activity of the exosome revealed a hitherto unrecognized function of SmAPs in archaea.

Back to translation: removal of aIF2 from the 5'-end of mRNAs by translation recovery factor in the crenarchaeon Sulfolobus solfataricus.

The translation initiation factor aIF2 of the crenarchaeon Sulfolobus solfataricus (Sso) recruits initiator tRNA to the ribosome and stabilizes mRNAs by binding via the γ-subunit to their 5'-triphosphate end. It has been hypothesized that the latter occurs predominantly during unfavorable growth conditions, and that aIF2 or aIF2-γ is released on relief of nutrient stress to enable in particular anew translation of leaderless mRNAs. As leaderless mRNAs are prevalent in Sso and aIF2-γ bound to the 5'-end of a leaderless RNA inhibited ribosome binding in vitro, we aimed at elucidating the mechanism underlying aIF2/aIF2-γ recycling from mRNAs. We have identified a protein termed Trf (translation recovery factor) that co-purified with trimeric aIF2 during outgrowth of cells from prolonged stationary phase. Subsequent in vitro studies revealed that Trf triggers the release of trimeric aIF2 from RNA, and that Trf directly interacts with the aIF2-γ subunit. The importance of Trf is further underscored by an impaired protein synthesis during outgrowth from stationary phase in a Sso trf deletion mutant.

Antisense regulation by transposon-derived RNAs in the hyperthermophilic archaeon Sulfolobus solfataricus.

We report the first example of antisense RNA regulation in a hyperthermophilic archaeon. In Sulfolobus solfataricus, the transposon-derived paralogous RNAs, RNA-257(1-4), show extended complementarity to the 3' UTR of the 1183 mRNA, encoding a putative phosphate transporter. Phosphate limitation results in decreased RNA-257(1) and increased 1183 mRNA levels. Correspondingly, the 1183 mRNA is faster degraded in vitro upon duplex formation with RNA-257(1). Insertion of the 1183 3' UTR downstream of the lacS gene results in strongly reduced lacS mRNA levels in transformed cells, indicating that antisense regulation can function in trans.

Crystal structure of the translation recovery factor Trf from Sulfolobus solfataricus.

During translation initiation, the heterotrimeric archaeal translation initiation factor 2 (aIF2) recruits the initiator tRNAi to the small ribosomal subunit. In the stationary growth phase and/or during nutrient stress, Sulfolobus solfataricus aIF2 has a second function: It protects leaderless mRNAs against degradation by binding to their 5'-ends. The S. solfataricus protein Sso2509 is a translation recovery factor (Trf) that interacts with aIF2 and is responsible for the release of aIF2 from bound mRNAs, thereby enabling translation re-initiation. It is a member of the domain of unknown function 35 (DUF35) protein family and is conserved in Sulfolobales as well as in other archaea. Here, we present the X-ray structure of S. solfataricus Trf solved to a resolution of 1.65 Å. Trf is composed of an N-terminal rubredoxin-like domain containing a bound zinc ion and a C-terminal oligosaccharide/oligonucleotide binding fold domain. The Trf structure reveals putative mRNA binding sites in both domains. Surprisingly, the Trf protein is structurally but not sequentially very similar to proteins linked to acyl-CoA utilization-for example, the Sso2064 protein from S. solfataricus-as well as to scaffold proteins found in the acetoacetyl-CoA thiolase/high-mobility group-CoA synthase complex of the archaeon Methanothermococcus thermolithotrophicus and in a steroid side-chain-cleaving aldolase complex from the bacterium Thermomonospora curvata. This suggests that members of the DUF35 protein family are able to act as scaffolding and binding proteins in a wide variety of biological processes.

X-ray structure of RLI, an essential twin cassette ABC ATPase involved in ribosome biogenesis and HIV capsid assembly.

The ABC ATPase RNase-L inhibitor (RLI) emerges as a key enzyme in ribosome biogenesis, formation of translation preinitiation complexes, and assembly of HIV capsids. To help reveal the structural mechanism of RLI, we determined the Mg2+-ADP bound crystal structure of the twin cassette ATPase of P. furiosus RLI at 1.9 A resolution and analyzed functional motifs in yeast in vivo. RLI shows similarities but also differences to known ABC enzyme structures. Twin nucleotide binding domains (NBD1 and NBD2) are arranged to form two composite active sites in their interface cleft, indicating they undergo the ATP-driven clamp-like motion of the NBDs of ABC transporters. An unusual "hinge" domain along the NBD1:NBD2 interface provides a frame for association and possibly ATP-driven conformational changes of the NBDs. Our results establish a first structural basis for ABC domain heterodimers and suggest that RLI may act as mechanochemical enzyme in ribosome and HIV capsid biogenesis.

The Heptameric SmAP1 and SmAP2 Proteins of the Crenarchaeon Sulfolobus Solfataricus Bind to Common and Distinct RNA Targets.

Sm and Sm-like proteins represent an evolutionarily conserved family with key roles in RNA metabolism. Sm-based regulation is diverse and can range in scope from eukaryotic mRNA splicing to bacterial quorum sensing, with at least one step in these processes being mediated by an RNA-associated molecular assembly built on Sm proteins. Despite the availability of several 3D-structures of Sm-like archaeal proteins (SmAPs), their function has remained elusive. The aim of this study was to shed light on the function of SmAP1 and SmAP2 of the crenarchaeon Sulfolobus solfataricus (Sso). Using co-purification followed by RNASeq different classes of non-coding RNAs and mRNAs were identified that co-purified either with both paralogues or solely with Sso-SmAP1 or Sso-SmAP2. The large number of associated intron-containing tRNAs and tRNA/rRNA modifying RNAs may suggest a role of the two Sso-SmAPs in tRNA/rRNA processing. Moreover, the 3D structure of Sso-SmAP2 was elucidated. Like Sso-SmAP1, Sso-SmAP2 forms homoheptamers. The binding of both proteins to distinct RNA substrates is discussed in terms of surface conservation, structural differences in the RNA binding sites and differences in the electrostatic surface potential of the two Sso-SmAP proteins. Taken together, this study may hint to common and different functions of both Sso-SmAPs in Sso RNA metabolism.

Binding of the 5'-Triphosphate End of mRNA to the γ-Subunit of Translation Initiation Factor 2 of the Crenarchaeon Sulfolobus solfataricus.

The heterotrimeric archaeal IF2 orthologue of eukaryotic translation initiation factor 2 consists of the α-subunit, ß-subunit and γ-subunit. Previous studies showed that the γ-subunit of aIF2, besides its central role in Met-tRNAi binding, has an additional function: it binds to the 5'-triphosphorylated end of mRNA and protects its 5'-part from degradation. Competition studies with nucleotides and mRNA, as well as structural and kinetic analyses of aIF2γ mutants, strongly implicate the canonical GTP/GDP-binding pocket in binding to the 5'-triphosphate end of mRNAs. The biological implication of these findings is being discussed.

Alterations of the transcriptome of Sulfolobus acidocaldarius by exoribonuclease aCPSF2.

Recent studies identified a 5´ to 3´ exoribonuclease termed Sso-RNase J in the crenarchaeon Sulfolobus solfataricus (Sso), which has been reclassified to the aCPSF2 (archaeal cleavage and polyadenylation specificity factor 2) group of ß-CASP proteins. In this study, the Sso-aCPSF2 orthologue of Sulfolobus acidocaldarius (Saci-aCPSF2) was functionally characterized. Like Sso-aCPSF2, Saci-aCPSF2 degrades RNA with 5´ to 3´ directionality in vitro. To address the biological significance of Saci-aCPSF2, a deletion mutant was constructed, and the influence of Saci-aCPSF2 on the transcriptome profile was assessed employing high throughput RNA sequencing. This analysis revealed 560 genes with differential transcript abundance, suggesting a considerable role of this enzyme in RNA metabolism. In addition, bioinformatic analyses revealed several transcripts that are preferentially degraded at the 5´ end. This was exemplarily verified for two transcripts by Northern-blot analyses, showing for the first time that aCPSF2 proteins play a role in 5' to 3' directional mRNA decay in the crenarchaeal clade of Archaea.

Structural biology of RNA polymerase III: subcomplex C17/25 X-ray structure and 11 subunit enzyme model.

We obtained an 11 subunit model of RNA polymerase (Pol) III by combining a homology model of the nine subunit core enzyme with a new X-ray structure of the subcomplex C17/25. Compared to Pol II, Pol III shows a conserved active center for RNA synthesis but a structurally different upstream face for specific initiation complex assembly during promoter selection. The Pol III upstream face includes a HRDC domain in subunit C17 that is translated by 35 A and rotated by 150 degrees compared to its Pol II counterpart. The HRDC domain is essential in vivo, folds independently in vitro, and, unlike other HRDC domains, shows no indication of nucleic acid binding. Thus, the HRDC domain is a functional module that could account for the role of C17 in Pol III promoter-specific initiation. During elongation, C17/25 may bind Pol III transcripts emerging from the adjacent exit pore, because the subcomplex binds to tRNA in vitro.