S-adenosyl-L-methionine Binding Research Articles

In silico Design of Novel SAM Analogs as Potential Inhibitors Against N2G966 16s rRNA Methyltransferase (RsmD)

Introduction: Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), has its natural history tracing back to 70,000 years. Latent M.tb infection is the reservoir of the TB epidemic. M.tb is becoming more prevalent and acquiring multidrug resistance among the first-line antibiotics. Methods: Methylation is one of the main mechanisms through which bacteria develop resistance, hence targeting methyltransferases provides the opportunity to achieve two-birds-with-one-stone: a) antibiotic: inhibiting the translation activity and b) anti-resistance: eliminating methylation as a mode of resistance. Currently, no known drugs or lead molecules are targeting the methyltransferases, in general, and rRNA Small Subunit Methyltransferase D (RsmD) family, in particular, in M.tb species. Results and Discussion: S-Adenosyl-L-methionine(SAM) is known as the universal donor of a methyl group which is an indispensable cofactor for the proper functioning of SAM-dependent methyltransferases. This in silico study attempts to design and develop novel SAM-analog inhibitors against RsmD, which in turn affects the growth and survival of M.tb in TB patients. The SAM-analogs were designed, after careful study and analysis of RsmD pharmacophore and SAM binding properties. The functional groups such as amide, amine, acetamide, formamide, hydroxyl, fluorine, iodine, and bromine were used to design novel analogs with the aim to improve the binding of analog with RsmD. The analogs that gave better docking scores followed by favourable binding affinities and ADMET properties than native SAM were ranked. Conclusion: Among the library of SAM analogs, the top two analogs with IDs: SAM_172 and SAM_153 need testing and validation for their efficacy through in vitro and in vivo studies.

S-adenosyl-l-methionine interaction signatures in methyltransferases

The switching on or off of methylation, a change from a normal methylation to hyper or hypo methylation is implicated in many diseases that include cancers, infectious, neurodegenerative diseases and others. Methyltransferases are one of the most sought targets that have diversified for the methylation of a variety of substrates. However, without S-adenosyl-l-methionine (SAM), the universal methyl donor, the majority of the methyltransferases remain functionally inactive. In this article, we did a comprehensive analysis of all available SAM-receptor crystal structures at atom, moiety and structure levels to gain deeper insights into the structure and function of SAM. SAM demonstrated flexibility in binding to a variety of receptors irrespective of the size of the binding pockets. Further analysis of the binding pockets resulted in all SAM conformations clustering into four natural shapes. The conserved interaction analysis provides an unambiguous orientation of SAM binding to receptors which has been elusive till now. SAM peptide moiety (SPM) and SAM nucleobase moiety (SNM) show up to 89% interactions with receptors whereas only 11% interactions with SAM ribose moiety (SRM). It is found that SPM and SNM terminal atoms anchor to the highly conserved receptor subsites creating a workbench for catalysis. It is seen that every interacting atom and its position is crucial in the methyl transfer phenomenon. A very unique observation is that the methyl group of SAM does not have even one interaction with the receptor. The deep insights gained help in the design and development of novel drugs against the methyltransferases. Communicated by Ramaswamy H. Sarma

Computational engineering of previously crystallized pyruvate formate-lyase activating enzyme reveals insights into SAM binding and reductive cleavage

Radical S-adenosyl-l-methionine (SAM) enzymes are ubiquitous in nature and carry out a broad variety of difficult chemical transformations initiated by hydrogen atom abstraction. Although numerous radical SAM (RS) enzymes have been structurally characterized, many prove recalcitrant to crystallization needed for atomic-level structure determination using X-ray crystallography, and even those that have been crystallized for an initial study can be difficult to recrystallize for further structural work. We present here a method for computationally engineering previously observed crystallographic contacts and employ it to obtain more reproducible crystallization of the RS enzyme pyruvate formate-lyase activating enzyme (PFL-AE). We show that the computationally engineered variant binds a typical RS [4Fe-4S]2+/+ cluster that binds SAM, with electron paramagnetic resonance properties indistinguishable from the native PFL-AE. The variant also retains the typical PFL-AE catalytic activity, as evidenced by the characteristic glycyl radical electron paramagnetic resonance signal observed upon incubation of the PFL-AE variant with reducing agent, SAM, and PFL. The PFL-AE variant was also crystallized in the [4Fe-4S]2+ state with SAM bound, providing a new high-resolution structure of the SAM complex in the absence of substrate. Finally, by incubating such a crystal in a solution of sodium dithionite, the reductive cleavage of SAM is triggered, providing us with a structure in which the SAM cleavage products 5'-deoxyadenosine and methionine are bound in the active site. We propose that the methods described herein may be useful in the structural characterization of other difficult-to-resolve proteins.

Incoherent dual regulation by a SAM-II riboswitch controlling translation at a distance

ABSTRACT In Sinorhizobium meliloti, the methionine biosynthesis genes metA and metZ are preceded by S-adenosyl-L-methionine (SAM) riboswitches of the SAM-II class. Upon SAM binding, structural changes in the metZ riboswitch were predicted to cause transcriptional termination, generating the sRNA RZ. By contrast, the metA riboswitch was predicted to regulate translation from an AUG1 codon. However, downstream of the metA riboswitch, we found a putative Rho-independent terminator and an in-frame AUG2 codon, which may contribute to metA regulation. We validated the terminator between AUG1 and AUG2, which generates the sRNA RA1 that is processed to RA2. Under high SAM conditions, the activities of the metA and metZ promoters and the steady-state levels of the read-through metA and metZ mRNAs were decreased, while the levels of the RZ and RA2 sRNAs were increased. Under these conditions, the sRNAs and the mRNAs were stabilized. Reporter fusion experiments revealed that the Shine–Dalgarno (SD) sequence in the metA riboswitch is required for translation, which, however, starts 74 nucleotides downstream at AUG2, suggesting a novel translation initiation mechanism. Further, the reporter fusion data supported the following model of RNA-based regulation: Upon SAM binding by the riboswitch, the SD sequence is sequestered to downregulate metA translation, while the mRNA is stabilized. Thus, the SAM-II riboswitches fulfil incoherent, dual regulation, which probably serves to ensure basal metA and metZ mRNA levels under high SAM conditions. This probably helps to adapt to changing conditions and maintain SAM homoeostasis.

Detection of cofactor conformer exchange in the active sites of methyltransferases with vibrational spectroscopy

SAM (S‐adenosyl‐L‐Methionine) is the universal methyl donor used by cells, and is also the second most used cofactor after ATP. Methyltransferases catalyze the methylation of numerous biomolecules including DNA, protein, and RNA; and have a broad array of functions ranging from cell signaling to gene transcription. Abnormal levels of methyltransferases have been linked to malignant cellular states, cardiovascular diseases, and neurological diseases. Therefore, methyltransferases are an attractive drug target. Several inhibitors of histone PMTs (protein methyltransferases) have been used in the clinic, signifying PMTs as a target class. The SAM binding pocket in protein methyltransferases has been utilized by in drug design strategies. Yet, there is not high specificity when it comes to these drugs, making the drugs imperfect. Recent NMR work reveals that unbound SAM has high populations of different conformations in dynamic equilibrium, which means there may be multiple conformations when SAM is bound to methyltransferases. Having multiple conformations of SAM may allow for specific drugs to be designed to fit the unique pockets, allowing for higher drug specificity. Here we use vibrational spectroscopy, which is sensitive to molecular conformation and immune to timescale, to determine if these conformational dynamics persist when SAM is bound to PMTs. Molecular dynamic studies and small molecules studies support the hypothesis that various SAM conformations exist both when free and when bound to the proteins.

Exploring the energy landscape of a SAM-I riboswitch

SAM-I riboswitches regulate gene expression through transcription termination upon binding a S-adenosyl-L-methionine (SAM) ligand. In previous work, we characterized the conformational energy landscape of the full-length Bacillus subtilis yitJ SAM-I riboswitch as a function of Mg2+ and SAM ligand concentrations. Here, we have extended this work with measurements on a structurally similar ligand, S-adenosyl-l-homocysteine (SAH), which has, however, a much lower binding affinity. Using single-molecule Förster resonance energy transfer (smFRET) microscopy and hidden Markov modeling (HMM) analysis, we identified major conformations and determined their fractional populations and dynamics. At high Mg2+ concentration, FRET analysis yielded four distinct conformations, which we assigned to two terminator and two antiterminator states. In the same solvent, but with SAM added at saturating concentrations, four states persisted, although their populations, lifetimes and interconversion dynamics changed. In the presence of SAH instead of SAM, HMM revealed again four well-populated states and, in addition, a weakly populated ‘hub’ state that appears to mediate conformational transitions between three of the other states. Our data show pronounced and specific effects of the SAM and SAH ligands on the RNA conformational energy landscape. Interestingly, both SAM and SAH shifted the fractional populations toward terminator folds, but only gradually, so the effect cannot explain the switching action. Instead, we propose that the noticeably accelerated dynamics of interconversion between terminator and antiterminator states upon SAM binding may be essential for control of transcription.

Crystal Structure and Regiospecificity of Catechol O-Methyltransferase from Niastella koreensis.

Catechol O-methyltransferase (COMT) is an enzyme that transfers a methyl group to the catechol-derivative substrates using S-adenosyl-l-methionine (SAM) and Mg2+. We report the biochemical and structural analysis of COMT from Niastella koreensis (NkCOMT). NkCOMT showed the highest activity with Mg2+, although the enzyme also showed a significant level of activity with Cu2+ and Zn2+. NkCOMT structures complexed with SAH and Mg2+ elucidated how the enzyme stabilized the cosubstrate and the metal ion and revealed that the region near the SAM binding site undergoes conformational changes upon the binding of the cosubstrate and the metal ion. We also identified the catechol binding pocket of the enzyme and explained a broad substrate specificity of the bacterial enzyme and its ability to accommodate the catechol derivatives. In addition, we developed the NkCOMTE211R and NkCOMTE211K variants that showed both enhanced activities and regiospecificity for the production of the para-forms. Our study provides a structural basis for regiospecificity of NkCOMT, which is related with the conformational change upon binding of SAM and Mg2+.

Computational Investigation of Structural Dynamics of SARS-CoV-2 Methyltransferase-Stimulatory Factor Heterodimer nsp16/nsp10 Bound to the Cofactor SAM.

Recently, a highly contagious novel coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, has emerged, posing a global threat to public health. Identifying a potential target and developing vaccines or antiviral drugs is an urgent demand in the absence of approved therapeutic agents. The 5′-capping mechanism of eukaryotic mRNA and some viruses such as coronaviruses (CoVs) are essential for maintaining the RNA stability and protein translation in the virus. SARS-CoV-2 encodes S-adenosyl-L-methionine (SAM) dependent methyltransferase (MTase) enzyme characterized by nsp16 (2′-O-MTase) for generating the capped structure. The present study highlights the binding mechanism of nsp16 and nsp10 to identify the role of nsp10 in MTase activity. Furthermore, we investigated the conformational dynamics and energetics behind the binding of SAM to nsp16 and nsp16/nsp10 heterodimer by employing molecular dynamics simulations in conjunction with the Molecular Mechanics Poisson-Boltzmann Surface Area (MM/PBSA) method. We observed from our simulations that the presence of nsp10 increases the favorable van der Waals and electrostatic interactions between SAM and nsp16. Thus, nsp10 acts as a stimulator for the strong binding of SAM to nsp16. The hydrophobic interactions were predominately identified for the nsp16-nsp10 interactions. Also, the stable hydrogen bonds between Ala83 (nsp16) and Tyr96 (nsp10), and between Gln87 (nsp16) and Leu45 (nsp10) play a vital role in the dimerization of nsp16 and nsp10. Besides, Computational Alanine Scanning (CAS) mutagenesis was performed, which revealed hotspot mutants, namely I40A, V104A, and R86A for the dimer association. Hence, the dimer interface of nsp16/nsp10 could also be a potential target in retarding the 2′-O-MTase activity in SARS-CoV-2. Overall, our study provides a comprehensive understanding of the dynamic and thermodynamic process of binding nsp16 and nsp10 that will contribute to the novel design of peptide inhibitors based on nsp16.

Identification of a Novel N-Acyl Homoserine Lactone Synthase, AhyI, in Aeromonas hydrophila and Structural Basis for Its Substrate Specificity.

In the Gram-negative bacterium Aeromonas hydrophila, N-acyl homoserine lactone (AHL)-mediated quorum sensing (QS) influences pathogenicity, protein secretion, and motility. However, the catalytic mechanism of AHL biosynthesis and the structural basis and substrate specificity for AhyI members remain unclear. In this study, we cloned the ahyI gene from the isolate A. hydrophila HX-3, and the overexpressed AhyI protein was confirmed to produce six types of AHLs by ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis, contrasting with previous reports that AhyI only produces N-butanoyl-l-homoserine lactone (C4-HSL) and N-hexanoyl-l-homoserine lactone (C6-HSL). The results of an in vitro biosynthetic assay showed that purified AhyI can catalyze the formation of C4-HSL using S-adenosyl-l-methionine (SAM) and butyryl-acyl carrier protein (ACP) as substrates and indicated that the fatty acyl substrate used in AhyI-mediated AHL synthesis is derived from acyl-ACP rather than acyl-CoA. The kinetic data of AhyI using butyryl-ACP as an acyl substrate indicated that the catalytic efficiency of the A. hydrophila HX-3 AhyI enzyme is within an order of magnitude compared to other LuxI homologues. In this study, for the first time, the tertiary structural modeling results of AhyI and those of molecular docking and structural and functional analyses showed the importance of several crucial residues, as well as the secondary structure with respect to acylation. A Phe125-Phe152 clamp grasps the terminal methyl group to assist in stabilizing the long acyl chains in a putative binding pocket. The stacking interactions within a strong hydrophobic environment, a hydrogen-bonding network, and a β bulge presumably stabilize the ACP acyl chain for the attack of the SAM α-amine toward the thioester carbon, offering a relatively reasonable explanation for how AhyI can synthesize AHLs with diverse acyl-chain lengths. Moreover, Trp34 participates in forming the binding pocket for C4-ACP and becomes ordered upon SAM binding, providing a good basis for catalysis. The novel finding that AhyI can produce both short- and long-chain AHLs enhances current knowledge regarding the variety of AHLs produced by this enzyme. These structural data are expected to serve as a molecular rationale for AHL synthesis by AhyI.

SAMbinder: A Web Server for Predicting S-Adenosyl-L-Methionine Binding Residues of a Protein From Its Amino Acid Sequence.

MotivationS-adenosyl-L-methionine (SAM) is an essential cofactor present in the biological system and plays a key role in many diseases. There is a need to develop a method for predicting SAM binding sites in a protein for designing drugs against SAM associated disease. To the best of our knowledge, there is no method that can predict the binding site of SAM in a given protein sequence.ResultThis manuscript describes a method SAMbinder, developed for predicting SAM interacting residue in a protein from its primary sequence. All models were trained, tested, and evaluated on 145 SAM binding protein chains where no two chains have more than 40% sequence similarity. Firstly, models were developed using different machine learning techniques on a balanced data set containing 2,188 SAM interacting and an equal number of non-interacting residues. Our random forest based model developed using binary profile feature got maximum Matthews Correlation Coefficient (MCC) 0.42 with area under receiver operating characteristics (AUROC) 0.79 on the validation data set. The performance of our models improved significantly from MCC 0.42 to 0.61, when evolutionary information in the form of the position-specific scoring matrix (PSSM) profile is used as a feature. We also developed models on a realistic data set containing 2,188 SAM interacting and 40,029 non-interacting residues and got maximum MCC 0.61 with AUROC of 0.89. In order to evaluate the performance of our models, we used internal as well as external cross-validation technique.Availability and Implementationhttps://webs.iiitd.edu.in/raghava/sambinder/.

Mechanism of Bacterial Ribosome 30S and 50S Subunit Recognition and Modification by M. tuberculosis TlyA

RNA (rRNA) modification is important for correct ribosome assembly, can alter ribosome function, and can confer resistance to clinically important ribosome‐targeting antibiotics. While the activity of most ribosome‐targeting antibiotics is blocked by rRNA methylation, the tuberactinomycin antibiotic capreomycin requires ribose 2′‐OH methylation at C1409 of the 16S rRNA within the small ribosome subunit (30S) and C1920 of the 23S rRNA within the large ribosome subunit (50S). TlyA is the 2′‐O‐methyltransferase that incorporates both modifications using S‐adenosyl‐L‐methionine (SAM) as a methyl group donor. The X‐ray crystal structure of the C‐terminal domain (CTD) of TlyA showed that the domain adopts a Class I methyltransferase fold, while homology modeling suggests the N‐terminal domain (NTD) adopts an S4 ribosomal protein fold. Additionally, the TlyA CTD structural studies revealed that the short interdomain linker was able to adopt two different conformations and was unexpectedly critical for SAM binding within the CTD. This project will test the resulting hypothesis that the interdomain linker acts a “molecular switch,” altering the interaction between the NTD and the CTD to control TlyA activity upon correct recognition of its two structurally distinct substrates. Using a sequence alignment and structure‐guided mutagenesis approach coupled with fluorescence polarization (FP) TlyA‐30S binding and methyltransferase activity assays, the role of the NTD and individual residues in this domain in 30S and 50S binding will be defined. Using complementary studies of TlyA protein dynamics (using HDX‐MS) and structure (using X‐ray crystallography and cryo‐EM), the long‐term goal of this project is to determine TlyA's molecular mechanism of site‐specific ribosome subunit recognition and modification. Defining these molecular details will help complete the picture of TlyA activity and its role in bacterial drug sensitivity or resistance, as well as furthering our general understanding of rRNA modification processes that are prevalent in all domains of life.Support or Funding InformationThis work was supported in part by the Graduate Division of Biological and Biomedical Sciences (GDBBS) of the Laney Graduate School (LGS) of Emory University, NIH grant R01‐AI088025, and the Antibiotic Resistance and Therapeutic Discovery Training Program (ARTDTP) T32‐AI 106699.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Kinetic Analysis of PRMT1 Reveals Multifactorial Processivity and a Sequential Ordered Mechanism

BACKGROUNDProtein arginine N‐methyltransferases (PRMTs) are responsible for the transfer of one or two methyl groups from the methyl donor S‐adenosyl‐L‐methionine (SAM) to produce mono‐ or dimethylated arginine residues and the spent cofactor S‐adenosyl‐L‐homocysteine (SAH). Methylated arginines in eukaryotic cells are involved in several crucial biological processes, including transcription control, DNA damager repair, and RNA processing. However, contrasting findings for two major questions in the field have generated debate regarding PRMT mechanisms. The purpose of this research was to elucidate the PRMT1 mechanism and answer these two highly debated questions: do PRMTs dimethylate their substrates processively or distributively, and do PRMTs bind their substrates using a random or sequential method of substrate binding?METHODSTo explore these mechanisms, we assayed PRMT1 with well‐characterized peptide substrates. We used tandem mass spectrometry to quantitate the ratios of mono‐ and dimethylarginine species produced to assess PRMT1 processivity. We determined the mechanism of bisubstrate binding using radioactive SAM in enzyme assays to measure methyl transfer to a target peptide in the presence of various concentrations of product inhibitors.RESULTSWe demonstrate that PRMT1 processivity differs depending on the substrate sequence, which is congruent with previous reports. However, we show for the first time that as cofactor and/or enzyme concentration increases, the ratio of dimethylarginine to monomethylarginine substantially increases, suggesting the enzyme becomes more processive. Further, the steady‐state inhibition patterns favour a sequential ordered mechanism over a random mechanism, indicating that for catalysis to occur, SAM binding must precede peptide binding.CONCLUSIONSThe degree of PRMT1 processivity is a multifactorial effect that is heavily influenced by experimental design considerations, so previous conclusions about processivity may require reassessment. We also find that PRMT1 uses a sequential ordered substrate binding mechanism, in contrast with recent findings. Use of nonlinear regression and presentation of our data using three linear plots strengthens our conclusions. Considering the conserved active sites within the human PRMT family, we anticipate that our results regarding PRMT1 kinetic and processive mechanisms may extend to other human PRMTs.Support or Funding InformationThis work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) RGPIN‐2015–04450 Discovery Grant (A.F.), NSERC CGS‐M (J.I.B.), the Shaughnessy Hospital Volunteer Society Fellowship in Health Care (J.I.B.), and the Walter C. Koerner Fellowship (J.I.B.). Additional support provided by Utrecht University and the Netherlands Organization for Scientific Research is acknowledged (T.K., J.v.S., and N.I.M.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Energy Landscape Analysis of the Full-Length SAM-I Riboswitch using Single-Molecule FRET Spectroscopy

Binding of the ligand S-adenosyl-L-methionine (SAM) produces major structural changes in the SAM-I riboswitch (RS) and thereby regulates gene expression via transcription termination. As yet, the conformations and motions governing the function of the full-length Bacillus subtilis yitJ SAM-I RS have not been deeply investigated. We have studied its conformational energy landscape as a function of Mg2+ and SAM ligand concentrations using single-molecule Förster resonance energy transfer (smFRET) microscopy. smFRET histograms of differently FRET-labeled constructs were so complicated that they could only be resolved with the help of kinetic experiments on immobilized riboswitches and hidden Markov modeling (HMM) analysis. At least four conformational states were identified, both in the presence and the absence of SAM. We determined their Mg2+-dependent fractional populations and conformational dynamics, including state lifetimes, interconversion rate coefficients and equilibration timescales. Riboswitches with terminator and antiterminator folds were found to coexist under all conditions; SAM binding induced only a gradual increase in the population of terminator states. Conformational transitions were much faster with bound SAM, which may be crucial for off-switching during the brief decision window before expression of the downstream gene.

Single-molecule FRET reveals the energy landscape of the full-length SAM-I riboswitch.

S-adenosyl-L-methionine (SAM) ligand binding induces major structural changes in SAM-I riboswitches, through which gene expression is regulated via transcription termination. Little is known about the conformations and motions governing the function of the full-length Bacillus subtilis yitJ SAM-I riboswitch. Therefore, we have explored its conformational energy landscape as a function of Mg2+ and SAM ligand concentrations using single-molecule Förster resonance energy transfer (smFRET) microscopy and hidden Markov modeling analysis. We resolved four conformational states both in the presence and the absence of SAM and determined their Mg2+-dependent fractional populations and conformational dynamics, including state lifetimes, interconversion rate coefficients and equilibration timescales. Riboswitches with terminator and antiterminator folds coexist, and SAM binding only gradually shifts the populations toward terminator states. We observed a pronounced acceleration of conformational transitions upon SAM binding, which may be crucial for off-switching during the brief decision window before expression of the downstream gene.

Binding of the Methyl Donor S-Adenosyl-l-Methionine to Middle East Respiratory Syndrome Coronavirus 2'-O-Methyltransferase nsp16 Promotes Recruitment of the Allosteric Activator nsp10.

The Middle East respiratory syndrome coronavirus (MERS-CoV) nonstructural protein 16 (nsp16) is an S-adenosyl-l-methionine (SAM)-dependent 2'-O-methyltransferase (2'-O-MTase) that is thought to methylate the ribose 2'-OH of the first transcribed nucleotide (N1) of viral RNA cap structures. This 2'-O-MTase activity is regulated by nsp10. The 2'-O methylation prevents virus detection by cell innate immunity mechanisms and viral translation inhibition by the interferon-stimulated IFIT-1 protein. To unravel the regulation of nsp10/nsp16 2'-O-MTase activity, we used purified MERS-CoV nsp16 and nsp10. First, we showed that nsp16 recruited N7-methylated capped RNA and SAM. The SAM binding promotes the assembly of the enzymatically active nsp10/nsp16 complex that converted 7mGpppG (cap-0) into 7mGpppG2'Om (cap-1) RNA by 2'-OH methylation of N1 in a SAM-dependent manner. The subsequent release of SAH speeds up nsp10/nsp16 dissociation that stimulates the reaction turnover. Alanine mutagenesis and RNA binding assays allowed the identification of the nsp16 residues involved in RNA recognition forming the RNA binding groove (K46, K170, E203, D133, R38, Y47, and Y181) and the cap-0 binding site (Y30, Y132, and H174). Finally, we found that nsp10/nsp16 2'-O-MTase activity is sensitive to known MTase inhibitors, such as sinefungin and cap analogues. This characterization of the MERS-CoV 2'-O-MTase is a preliminary step toward the development of molecules to inhibit cap 2'-O methylation and to restore the host antiviral response. IMPORTANCE MERS-CoV codes for a cap 2'-O-methyltransferase that converts cap-0 into cap-1 structure in order to prevent virus detection by cell innate immunity mechanisms. We report the biochemical properties of MERS-CoV 2'O-methyltransferase, which is stimulated by nsp10 acting as an allosteric activator of the nsp16 2'-O-methyltransferase possibly through enhanced RNA binding affinity. In addition, we show that SAM promotes the formation of the active nsp10/nsp16 complex. Conversely, after cap methylation, the reaction turnover is speeded up by cap-1 RNA release and nsp10/nsp16 complex dissociation, at the low intracellular SAH concentration. These results suggest that SAM/SAH balance is a regulator of the 2'-O-methyltransferase activity and raises the possibility that SAH hydrolase inhibitors might interfere with CoV replication cycle. The enzymatic and RNA binding assays developed in this work were also used to identify nsp16 residues involved in cap-0 RNA recognition and to understand the action mode of known methyltransferase inhibitors.

A Novel Motif for S-Adenosyl-l-methionine Binding by the Ribosomal RNA Methyltransferase TlyA from Mycobacterium tuberculosis

Capreomycin is a potent ribosome-targeting antibiotic that is an essential component of current antituberculosis treatments, particularly in the case of multidrug-resistant Mycobacterium tuberculosis (Mtb). Optimal capreomycin binding and Mtb ribosome inhibition requires ribosomal RNA methylation in both ribosome subunits by TlyA (Rv1694), an enzyme with dual 2'-O-methytransferase and putative hemolytic activities. Despite the important role of TlyA in capreomycin sensitivity and identification of inactivating mutations in the corresponding Mtb gene tlyA, which cause resistance to capreomycin, our current structural and mechanistic understanding of TlyA action remains limited. Here, we present structural and functional analyses of Mtb TlyA interaction with its obligatory co-substrate for methyltransferase activity, S-adenosyl-l-methionine (SAM). Despite adopting a complete class I methyltransferase fold containing conserved SAM-binding and catalytic motifs, the isolated TlyA carboxyl-terminal domain exhibits no detectable affinity for SAM. Further analyses identify a tetrapeptide motif (RXWV) in the TlyA interdomain linker as indispensable for co-substrate binding. Our results also suggest that structural plasticity of the RXWV motif could contribute to TlyA domain interactions, as well as specific recognition of its two structurally distinct ribosomal RNA targets. Our findings thus reveal a novel motif requirement for SAM binding by TlyA and set the stage for future mechanistic studies of TlyA substrate recognition and modification that underpin Mtb sensitivity to capreomycin.

Pseudomonas aeruginosa EftM Is a Thermoregulated Methyltransferase

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that trimethylates elongation factor-thermo-unstable (EF-Tu) on lysine 5. Lysine 5 methylation occurs in a temperature-dependent manner and is generally only seen when P. aeruginosa is grown at temperatures close to ambient (25 °C) but not at higher temperatures (37 °C). We have previously identified the gene, eftM (for EF-Tu-modifying enzyme), responsible for this modification and shown its activity to be associated with increased bacterial adhesion to and invasion of respiratory epithelial cells. Bioinformatic analyses predicted EftM to be a Class I S-adenosyl-l-methionine (SAM)-dependent methyltransferase. An in vitro methyltransferase assay was employed to show that, in the presence of SAM, EftM directly trimethylates EF-Tu. A natural variant of EftM, with a glycine to arginine substitution at position 50 in the predicted SAM-binding domain, lacks both SAM binding and enzyme activity. Mass spectrometry analysis of the in vitro methyltransferase reaction products revealed that EftM exclusively methylates at lysine 5 of EF-Tu in a distributive manner. Consistent with the in vivo temperature dependence of methylation of EF-Tu, preincubation of EftM at 37 °C abolished methyltransferase activity, whereas this activity was retained when EftM was preincubated at 25 °C. Irreversible protein unfolding at 37 °C was observed, and we propose that this instability is the molecular basis for the temperature dependence of EftM activity. Collectively, our results show that EftM is a thermolabile, SAM-dependent methyltransferase that directly trimethylates lysine 5 of EF-Tu in P. aeruginosa.

Molecular Mechanism underlying PRMT1 Dimerization for SAM Binding and Methylase Activity.

Protein arginine methyltransferases (PRMTs) catalyze the posttranslational methylation of arginine, which is important in a range of biological processes, including epigenetic regulation, signal transduction, and cancer progression. Although previous studies of PRMT1 mutants suggest that the dimerization arm and the N-terminal region of PRMT1 are important for activity, the contributions of these regions to the structural architecture of the protein and its catalytic methylation activity remain elusive. Molecular dynamics (MD) simulations performed in this study showed that both the dimerization arm and the N-terminal region undergo conformational changes upon dimerization. Because a correlation was found between the two regions despite their physical distance, an allosteric pathway mechanism was proposed based on a network topological analysis. The mutation of residues along the allosteric pathways markedly reduced the methylation activity of PRMT1, which may be attributable to the destruction of dimer formation and accordingly reduced S-adenosyl-L-methionine (SAM) binding. This study provides the first demonstration of the use of a combination of MD simulations, network topological analysis, and biochemical assays for the exploration of allosteric regulation upon PRMT1 dimerization. These findings illuminate the results of mechanistic studies of PRMT1, which have revealed that dimer formation facilitates SAM binding and catalytic methylation, and provided direction for further allosteric studies of the PRMT family.

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.

A putative ATPase mediates RNA transcription and capping in a dsRNA virus.

mRNA transcription in dsRNA viruses is a highly regulated process but the mechanism of this regulation is not known. Here, by nucleoside triphosphatase (NTPase) assay and comparisons of six high-resolution (2.9-3.1 Å) cryo-electron microscopy structures of cytoplasmic polyhedrosis virus with bound ligands, we show that the large sub-domain of the guanylyltransferase (GTase) domain of the turret protein (TP) also has an ATP-binding site and is likely an ATPase. S-adenosyl-L-methionine (SAM) acts as a signal and binds the methylase-2 domain of TP to induce conformational change of the viral capsid, which in turn activates the putative ATPase. ATP binding/hydrolysis leads to an enlarged capsid for efficient mRNA synthesis, an open GTase domain for His217-mediated guanylyl transfer, and an open methylase-1 domain for SAM binding and methyl transfer. Taken together, our data support a role of the putative ATPase in mediating the activation of mRNA transcription and capping within the confines of the virus.