Knot or not? Identifying unknotted proteins in knotted families with sequence-based Machine Learning model.

Knotted proteins, although scarce, are crucial structural components of certain protein families, and their roles continue to be a topic of intense research. Capitalizing on the vast collection of protein structure predictions offered by AlphaFold (AF), this study computationally examines the entire UniProt database to create a robust dataset of knotted and unknotted proteins. Utilizing this dataset, we develop a machine learning (ML) model capable of accurately predicting the presence of knots in protein structures solely from their amino acid sequences. We tested the model's capabilities on 100 proteins whose structures had not yet been predicted by AF and found agreement with our local prediction in 92% cases. From the point of view of structural biology, we found that all potentially knotted proteins predicted by AF can be classified only into 17 families. This allows us to discover the presence of unknotted proteins in families with a highly conserved knot. We found only three new protein families: UCH, DUF4253, and DUF2254, that contain both knotted and unknotted proteins, and demonstrate that deletions within the knot core could potentially account for the observed unknotted (trivial) topology. Finally, we have shown that in the majority of knotted families (11 out of 15), the knotted topology is strictly conserved in functional proteins with very low sequence similarity. We have conclusively demonstrated that proteins AF predicts as unknotted are structurally accurate in their unknotted configurations. However, these proteins often represent nonfunctional fragments, lacking significant portions of the knot core (amino acid sequence).

tRNA m1G9 modification depends on substrate-specific RNA conformational changes induced by the methyltransferase Trm10.

The methyltransferase Trm10 modifies a subset of tRNAs on the base N1 position of the ninth nucleotide in the tRNA core. Trm10 is conserved throughout Eukarya and Archaea, and mutations in the human gene (TRMT10A) have been linked to neurological disorders such as microcephaly and intellectual disability, as well as defects in glucose metabolism. Of the 26 tRNAs in yeast with guanosine at position 9, only 13 are substrates for Trm10. However, no common sequence or other posttranscriptional modifications have been identified among these substrates, suggesting the presence of some other tRNA feature(s) that allow Trm10 to distinguish substrate from nonsubstrate tRNAs. Here, we show that substrate recognition by Saccharomyces cerevisiae Trm10 is dependent on both intrinsic tRNA flexibility and the ability of the enzyme to induce specific tRNA conformational changes upon binding. Using the sensitive RNA structure-probing method SHAPE, conformational changes upon binding to Trm10 in tRNA substrates, but not nonsubstrates, were identified and mapped onto a model of Trm10-bound tRNA. These changes may play an important role in substrate recognition by allowing Trm10 to gain access to the target nucleotide. Our results highlight a novel mechanism of substrate recognition by a conserved tRNA modifying enzyme. Further, these studies reveal a strategy for substrate recognition that may be broadly employed by tRNA-modifying enzymes which must distinguish between structurally similar tRNA species.

Escherichia coli tRNA (Gm18) methyltransferase (TrmH) requires the correct localization of its methylation site (G18) in the D-loop for efficient methylation.

TrmH is a eubacterial tRNA methyltransferase responsible for formation of 2'-O-methylguaosine at position 18 (Gm18) in tRNA. In Escherichia coli cells, only 14 tRNA species possess the Gm18 modification. To investigate the substrate tRNA selection mechanism of E. coli TrmH, we performed biochemical and structural studies. Escherichia coli TrmH requires a high concentration of substrate tRNA for efficient methylation. Experiments using native tRNA SerCGA purified from a trmH gene disruptant strain showed that modified nucleosides do not affect the methylation. A gel mobility-shift assay reveals that TrmH captures tRNAs without distinguishing between relatively good and very poor substrates. Methylation assays using wild-type and mutant tRNA transcripts revealed that the location of G18 in the D-loop is very important for efficient methylation by E. coli TrmH. In the case of tRNASer, tRNATyrand tRNALeu, the D-loop structure formed by interaction with the long variable region is important. For tRNAGln, the short distance between G18 and A14 is important. Thus, our biochemical study explains all Gm18 modification patterns in E. coli tRNAs. The crystal structure of E. coli TrmH has also been solved, and the tRNA binding mode of E. coli TrmH is discussed based on the structure.

tRNA m1G9 modification depends on substrate-specific RNA conformational changes induced by the methyltransferase Trm10.

The methyltransferase Trm10 modifies a subset of tRNAs on the base N1 position of the 9th nucleotide in the tRNA core. Trm10 is conserved throughout Eukarya and Archaea, and mutations in the human gene (TRMT10A) have been linked to neurological disorders such as microcephaly and intellectual disability, as well as defects in glucose metabolism. Of the 26 tRNAs in yeast with guanosine at position 9, only 14 are substrates for Trm10. However, no common sequence or other posttranscriptional modifications have been identified among these substrates, suggesting the presence of some other tRNA feature(s) which allow Trm10 to distinguish substrate from nonsubstrate tRNAs. Here, we show that substrate recognition by Saccharomyces cerevisiae Trm10 is dependent on both intrinsic tRNA flexibility and the ability of the enzyme to induce specific tRNA conformational changes upon binding. Using the sensitive RNA structure-probing method SHAPE, conformational changes upon binding to Trm10 in tRNA substrates, but not nonsubstrates, were identified and mapped onto a model of Trm10-bound tRNA. These changes may play an important role in substrate recognition by allowing Trm10 to gain access to the target nucleotide. Our results highlight a novel mechanism of substrate recognition by a conserved tRNA modifying enzyme. Further, these studies reveal a strategy for substrate recognition that may be broadly employed by tRNA-modifying enzymes which must distinguish between structurally similar tRNA species.

Are there double knots in proteins? Prediction and in vitro verification based on TrmD-Tm1570 fusion from C. nitroreducens.

We have been aware of the existence of knotted proteins for over 30 years-but it is hard to predict what is the most complicated knot that can be formed in proteins. Here, we show new and the most complex knotted topologies recorded to date-double trefoil knots (31 #31). We found five domain arrangements (architectures) that result in a doubly knotted structure in almost a thousand proteins. The double knot topology is found in knotted membrane proteins from the CaCA family, that function as ion transporters, in the group of carbonic anhydrases that catalyze the hydration of carbon dioxide, and in the proteins from the SPOUT superfamily that gathers 31 knotted methyltransferases with the active site-forming knot. For each family, we predict the presence of a double knot using AlphaFold and RoseTTaFold structure prediction. In the case of the TrmD-Tm1570 protein, which is a member of SPOUT superfamily, we show that it folds in vitro and is biologically active. Our results show that this protein forms a homodimeric structure and retains the ability to modify tRNA, which is the function of the single-domain TrmD protein. However, how the protein folds and is degraded remains unknown.

Tied up in knots: Untangling substrate recognition by the SPOUT methyltransferases.

The SpoU-TrmD (SPOUT) methyltransferase superfamily was designated when structural similarity was identified between the transfer RNA-modifying enzymes TrmH (SpoU) and TrmD. SPOUT methyltransferases are found in all domains of life and predominantly modify transfer RNA or ribosomal RNA substrates, though one instance of an enzyme with a protein substrate has been reported. Modifications placed by SPOUT methyltransferases play diverse roles in regulating cellular processes such as ensuring translational fidelity, altering RNA stability, and conferring bacterial resistance to antibiotics. This large collection of S-adenosyl-L-methionine-dependent methyltransferases is defined by a unique α/ß fold with a deep trefoil knot in their catalytic (SPOUT) domain. Herein, we describe current knowledge of SPOUT enzyme structure, domain architecture, and key elements of catalytic function, including S-adenosyl-L-methionine co-substrate binding, beginning with a new sequence alignment that divides the SPOUT methyltransferase superfamily into four major clades. Finally, a major focus of this review will be on our growing understanding of how these diverse enzymes accomplish the molecular feat of specific substrate recognition and modification, as highlighted by recent advances in our knowledge of protein-RNA complex structures and the discovery of the dependence of one SPOUT methyltransferase on metal ion binding for catalysis. Considering the broad biological roles of RNA modifications, developing a deeper understanding of the process of substrate recognition by the SPOUT enzymes will be critical for defining many facets of fundamental RNA biology with implications for human disease.

Polypropylene Composites Reinforced by Nonmetallic from Waste Printed Circuit Boards Using Spout-Fluid Bed Coating with PP Particles Enhance Fluidization.

Nonmetallic materials recycled from waste printed circuit boards (N-WPCBs) were modified by coating KH-550 in a spout-fluid bed. To improve the effect of the modification, PP particles were used to enhance the fluidization quality of the N-WPCB particles in the coating modification. Then, the modified N-WPCBs were used as fillers to fabricate PP/N-WPCB composites. The method of coating in a spout-fluid bed with PP particles enhanced fluidization and showed the best modification effect compared to other coating methods. The FT-IR and SEM results demonstrated that interfacial bonding between N-WPCBs and PP could be enhanced by modified N-WPCBs, which improved the mechanical properties of the composites. When the mass ratio of PP to N-WPCBs is 100:75 and the dose of KH-550 is 4 phr, the flexural strength, tensile strength, and impact strength of the composites increase by 16.60%, 23.22%, and 23.64%, respectively. This would realize the high-value utilization of N-WPCBs with coating modification in the spout-fluid bed.

Distinct substrate specificities of the human tRNA methyltransferases TRMT10A and TRMT10B.

The tRNA m1R9 methyltransferase (Trm10) family is conserved throughout Eukarya and Archaea. Despite the presence of a single Trm10 gene in Archaea and most single-celled eukaryotes, metazoans encode up to three homologs of Trm10. Several disease states correlate with a deficiency in the human homolog TRMT10A, despite the presence of another cytoplasmic enzyme, TRMT10B. Here we investigate these phenomena and demonstrate that human TRMT10A (hTRMT10A) and human TRMT10B (hTRMT10B) are not biochemically redundant. In vitro activity assays with purified hTRMT10A and hTRMT10B reveal a robust activity for hTRMT10B as a tRNAAsp-specific m1A9 methyltransferase and suggest that it is the relevant enzyme responsible for this newly discovered m1A9 modification in humans. Moreover, a comparison of the two cytosolic enzymes with multiple tRNA substrates exposes the enzymes' distinct substrate specificities, and suggests that hTRMT10B exhibits a restricted selectivity hitherto unseen in the Trm10 enzyme family. Single-turnover kinetics and tRNA binding assays highlight further differences between the two enzymes and eliminate overall tRNA affinity as a primary determinant of substrate specificity for either enzyme. These results increase our understanding of the important biology of human tRNA modification systems, which can aid in understanding the molecular basis for diseases in which their aberrant function is increasingly implicated.

Untying a Protein Knot by Circular Permutation.

Topologically knotted proteins are tantalizing examples of how polypeptide chains can explore complex free energy landscapes to efficiently attain defined knotted conformations. The evolution trails of protein knots, however, remain elusive. We used circular permutation to change an evolutionally conserved topologically knotted SPOUT RNA methyltransferase into an unknotted form. The unknotted variant adopted the same three-dimensional structure and oligomeric state as its knotted parent, but its folding stability was markedly reduced with accelerated folding kinetics and its ligand binding was abrogated. Our findings support the hypothesis that the universally conserved knotted topology of the SPOUT superfamily evolved from unknotted forms through circular permutation under selection pressure for folding robustness and, more importantly, for functional requirements associated with the knotted structural element.

Structural and biochemical analysis of the dual-specificity Trm10 enzyme from Thermococcus kodakaraensis prompts reconsideration of its catalytic mechanism.

tRNA molecules get heavily modified post-transcriptionally. The N-1 methylation of purines at position 9 of eukaryal and archaeal tRNA is catalyzed by the SPOUT methyltranferase Trm10. Remarkably, while certain Trm10 orthologs are specific for either guanosine or adenosine, others show a dual specificity. Structural and functional studies have been performed on guanosine- and adenosine-specific enzymes. Here we report the structure and biochemical analysis of the dual-specificity enzyme from Thermococcus kodakaraensis (TkTrm10). We report the first crystal structure of a construct of this enzyme, consisting of the N-terminal domain and the catalytic SPOUT domain. Moreover, crystal structures of the SPOUT domain, either in the apo form or bound to S-adenosyl-l-methionine or S-adenosyl-l-homocysteine reveal the conformational plasticity of two active site loops upon substrate binding. Kinetic analysis shows that TkTrm10 has a high affinity for its tRNA substrates, while the enzyme on its own has a very low methyltransferase activity. Mutation of either of two active site aspartate residues (Asp206 and Asp245) to Asn or Ala results in only modest effects on the N-1 methylation reaction, with a small shift toward a preference for m1G formation over m1A formation. Only a double D206A/D245A mutation severely impairs activity. These results are in line with the recent finding that the single active-site aspartate was dispensable for activity in the guanosine-specific Trm10 from yeast, and suggest that also dual-specificity Trm10 orthologs use a noncanonical tRNA methyltransferase mechanism without residues acting as general base catalysts.

How Much Can We do by Reverse Superficial Sural Artery Flap to Potentiate Its Effects: Introducing Spout Sural Flap as a New Modification.

BACKGROUND: Due to shortage of local donor tissue and unreliable blood supply, free flaps were the mainstay of treatment for tissue defects in the lower leg and foot region, but it requires a qualified microvascular surgeon. Recently, attention has been paid to reverse superficial sural artery flap (RSSAF) and its modifications as a good alternative to pave the way to simple and friendly techniques. METHODS: Excluding each patient with septic and severely ischemic foot, every patient with tissue defect in distal leg and proximal foot region were studied. Various methods were applied including spout technique with sufficient follow up. No imaging was used to evaluate the blood supply. RESULTS: Five patients underwent spout technique with excellent results in four cases. Spout technique in one case failed due to narrow base. In five cases, RSSAF was performed with creating skin tunnel and very good results. CONCLUSION: RSSAF is a good alternative for free flap to cover the leg and foot tissue defects. We also advise wide base pedicle (>4 cm) in every patient.

Backbone assignments for the SPOUT methyltransferase MTT Tm , a knotted protein from Thermotoga maritima.

The SPOUT family of methyltransferase proteins is noted for containing a deep trefoil knot in their defining backbone fold. This unique fold is of high interest for furthering the understanding of knots in proteins. Here, we report the 1H, 13C, 15N assignments for MTT Tm , a canonical member of the SPOUT family. This protein is unique, as it is one of the smallest members of the family, making it an ideal system for probing the unique properties of the knot. Our present work represents the foundation for further studies into the topology of MTT Tm , and understanding how its structure affects both its folding and function.

Functional roles in S-adenosyl-L-methionine binding and catalysis for active site residues of the thiostrepton resistance methyltransferase.

Resistance to the antibiotic thiostrepton, in producing Streptomycetes, is conferred by the S-adenosyl-L-methionine (SAM)-dependent SPOUT methyltransferase Tsr. For this and related enzymes, the roles of active site amino acids have been inadequately described. Herein, we have probed SAM interactions in the Tsr active site by investigating the catalytic activity and the thermodynamics of SAM binding by site-directed Tsr mutants. Two arginine residues were demonstrated to be critical for binding, one of which appears to participate in the catalytic reaction. Additionally, evidence consistent with the involvement of an asparagine in the structural organization of the SAM binding site is presented.

Structural basis for methyl-donor-dependent and sequence-specific binding to tRNA substrates by knotted methyltransferase TrmD.

The deep trefoil knot architecture is unique to the SpoU and tRNA methyltransferase D (TrmD) (SPOUT) family of methyltransferases (MTases) in all three domains of life. In bacteria, TrmD catalyzes the N(1)-methylguanosine (m(1)G) modification at position 37 in transfer RNAs (tRNAs) with the (36)GG(37) sequence, using S-adenosyl-l-methionine (AdoMet) as the methyl donor. The m(1)G37-modified tRNA functions properly to prevent +1 frameshift errors on the ribosome. Here we report the crystal structure of the TrmD homodimer in complex with a substrate tRNA and an AdoMet analog. Our structural analysis revealed the mechanism by which TrmD binds the substrate tRNA in an AdoMet-dependent manner. The trefoil-knot center, which is structurally conserved among SPOUT MTases, accommodates the adenosine moiety of AdoMet by loosening/retightening of the knot. The TrmD-specific regions surrounding the trefoil knot recognize the methionine moiety of AdoMet, and thereby establish the entire TrmD structure for global interactions with tRNA and sequential and specific accommodations of G37 and G36, resulting in the synthesis of m(1)G37-tRNA.

Structural basis for Sfm1 functioning as a protein arginine methyltransferase.

SPOUT proteins constitute one class of methyltransferases, which so far are found to exert activity mainly towards RNAs. Previously, yeast Sfm1 was predicted to contain a SPOUT domain but can methylate ribosomal protein S3. Here we report the crystal structure of Sfm1, which comprises of a typical SPOUT domain and a small C-terminal domain. The active site is similar to that of protein arginine methyltransferases but different from that of RNA methyltransferases. In addition, Sfm1 exhibits a negatively charged surface surrounding the active site unsuitable for RNA binding. Our biochemical data show that Sfm1 exists as a monomer and has high activity towards ribosomal protein S3 but no activity towards RNA. It can specifically catalyze the methylation of Arg146 of S3 and the C-terminal domain is critical for substrate binding and activity. These results together provide the structural basis for Sfm1 functioning as a PRMT for ribosomal protein S3.

Characterization of two homologous 2'-O-methyltransferases showing different specificities for their tRNA substrates.

The 2'-O-methylation of the nucleoside at position 32 of tRNA is found in organisms belonging to the three domains of life. Unrelated enzymes catalyzing this modification in Bacteria (TrmJ) and Eukarya (Trm7) have already been identified, but until now, no information is available for the archaeal enzyme. In this work we have identified the methyltransferase of the archaeon Sulfolobus acidocaldarius responsible for the 2'-O-methylation at position 32. This enzyme is a homolog of the bacterial TrmJ. Remarkably, both enzymes have different specificities for the nature of the nucleoside at position 32. While the four canonical nucleosides are substrates of the Escherichia coli enzyme, the archaeal TrmJ can only methylate the ribose of a cytidine. Moreover, the two enzymes recognize their tRNA substrates in a different way. We have solved the crystal structure of the catalytic domain of both enzymes to gain better understanding of these differences at a molecular level.

The structure of Rv2372c identifies an RsmE-like methyltransferase from Mycobacterium tuberculosis.

U1498 of 16S rRNA plays an important role in translation fidelity as well as in antibiotic response. U1498 is present in a methylated form in the decoding centre of the ribosome. In this study, Rv2372c from Mycobacterium tuberculosis has been identified as an RsmE-like methyltransferase which specifically methylates U1498 of 16S rRNA at the N3 position and can complement RsmE-deleted Escherichia coli. The crystal structure of Rv2372c has been determined, and reveals that the protein belongs to a distinct class in the SPOUT superfamily and exists as a dimer. The deletion of critical residues at the C-terminus of Rv2372c leads to an inability of the protein to form stable dimers and to abolition of the methyltransferase activity. A ternary model of Rv2372c with its cofactor S-adenosylmethionine (SAM) and the 16S rRNA fragment (1487)16S rRNA(1510) helps to identify binding pockets for SAM (in the deep trefoil knot) and substrate RNA (at the dimer interface) and suggests an S(N)2 mechanism for the methylation of N3 of U1498 in 16S rRNA.

Cholinesterase inhibitor, donepezil, improves visual contrast detectability in freely behaving rats.

Acetylcholine (ACh) modulates neuronal activities in extensive brain regions to play an essential role in various brain functions including attention, learning and memory, and cognition. Although ACh is known to modulate information processing in the primary visual cortex (V1) in many species including rodent, its functional role in visual ability has remained unknown. We examined whether and how ACh influences behavioral contrast detectability in rat. The detectability was assessed as the contrast sensitivity (CS) to a grating stimulus. Measurements were performed in a two-alternative forced-choice task combined with a staircase method in freely behaving rats. The contrast sensitivity function of rats under the no drug condition showed a low-pass spatial frequency (SF) tuning peaking at 0.1 cycles/degree (cpd) of SF (SF(peak)) that bottomed at 0.5 cpd (SF(bottom)), which was sensitive to the stimulus size, but to neither the temporal frequency nor orientation of the stimulus. The stimulus size was correlated with the CS only at the low SF range. The effect of donepezil on the size- and SF-dependency of the CS was examined using three stimulus conditions: an easy detectability condition with large grating at SF(peak), a difficult detectability condition with small grating at SF(peak), and an upper limit SF condition with large grating at SF(bottom). Donepezil improved the CS at SF(peak), especially in the difficult detectability condition. Therefore, we conclude that ACh plays an important role in enhancing behavioral CS at sensitive SF ranges, but not in improving the upper limit of SF.

The catalytic domain of topological knot tRNA methyltransferase (TrmH) discriminates between substrate tRNA and nonsubstrate tRNA via an induced-fit process.

A conserved guanosine at position 18 (G18) in the D-loop of tRNAs is often modified to 2'-O-methylguanosine (Gm). Formation of Gm18 in eubacterial tRNA is catalyzed by tRNA (Gm18) methyltransferase (TrmH). TrmH enzymes can be divided into two types based on their substrate tRNA specificity. Type I TrmH, including Thermus thermophilus TrmH, can modify all tRNA species, whereas type II TrmH, for example Escherichia coli TrmH, modifies only a subset of tRNA species. Our previous crystal study showed that T. thermophilus TrmH is a class IV S-adenosyl-l-methionine-dependent methyltransferase, which maintains a topological knot structure in the catalytic domain. Because TrmH enzymes have short stretches at the N and C termini instead of a clear RNA binding domain, these stretches are believed to be involved in tRNA recognition. In this study, we demonstrate by site-directed mutagenesis that both N- and C-terminal regions function in tRNA binding. However, in vitro and in vivo chimera protein studies, in which four chimeric proteins of type I and II TrmHs were used, demonstrated that the catalytic domain discriminates substrate tRNAs from nonsubstrate tRNAs. Thus, the N- and C-terminal regions do not function in the substrate tRNA discrimination process. Pre-steady state analysis of complex formation between mutant TrmH proteins and tRNA by stopped-flow fluorescence measurement revealed that the C-terminal region works in the initial binding process, in which nonsubstrate tRNA is not excluded, and that structural movement of the motif 2 region of the catalytic domain in an induced-fit process is involved in substrate tRNA discrimination.

Qualidade da água de bicas localizadas nos municípios de Santos e São Vicente, Estado de São Paulo, Brasil / Quality of water from spouts located in Santos and São Vicente municipalities, São Paulo state, Brazil

A água oriunda das bicas geralmente não é tratada e requer atenção especial das autoridades sanitárias. Neste estudo foi avaliada a qualidade da água das bicas localizadas nos municípios de Santos e São Vicente, Estado de São Paulo. Em 2008, foram coletadas 31 amostras e foram analisadas quanto à presença de coliformes totais e termo tolerantes, além das seguintes características físico-químicas cloreto, cloro residual livre (nas águas tratadas), cor aparente, dureza, ferro, nitrato, nitrito, odor, pH, sólidos totais dissolvidos, sulfato e turbidez. Durante a coleta das amostras, foi aplicado um questionário ao usuário ou morador das proximidades da bica e esclarecido que não se tratava de fiscalização. Das 31 amostras analisadas, apenas seis foram aprovadas, de acordo com a Portaria 518/2004 do Ministério da Saúde. Foi detectada a presença de coliformes fecais em 13 amostras. O nitrato foi insatisfatório em 14 amostras, o pH em 12 e a cor aparente em uma amostra. Houve resistência de algumas pessoas em responder ao questionário, temerosos pelo fechamento das bicas e alegando que esse tipo de água ser melhor do que a tratada. O monitoramento constante da água de bicas pelas vigilâncias sanitárias municipais será de grande valia, bem como o estabelecimento de campanha entre os usuários para desmistificar essa questão cultural sobre a qualidade de água proveniente de bica.