UFMylation: a ubiquitin-like modification.

Post-translational modifications (PTMs) add a major degree of complexity to the proteome and are essential controllers of protein homeostasis. Amongst the hundreds of PTMs identified, ubiquitin and ubiquitin-like (UBL) modifications are recognized as key regulators of cellular processes through their ability to affect protein-protein interactions, protein stability, and thus the functions of their protein targets. Here, we focus on the most recently identified UBL, ubiquitin-fold modifier 1 (UFM1), and the machinery responsible for its transfer to substrates (UFMylation) or its removal (deUFMylation). We first highlight the biochemical peculiarities of these processes, then we develop on how UFMylation and its machinery control various intertwined cellular processes and we highlight some of the outstanding research questions in this emerging field.

Phosphorylation of GCN2 by mTOR confers adaptation to conditions of hyper-mTOR activation under stress.

Adaptation to shortage in free amino acids (AA) is mediated by two pathways, the integrated stress response (ISR) and the mechanistic target of rapamycin (mTOR). In response to reduced levels, primarily of leucine or arginine, mTOR in its complex 1 configuration (mTORC1) is suppressed leading to a decrease in translation initiation and elongation. The eIF2α kinase general control nonderepressible 2 (GCN2) is activated by uncharged tRNAs, leading to induction of the ISR in response to a broader range of AA shortage. ISR confers a reduced translation initiation, while promoting the selective synthesis of stress proteins, such as ATF4. To efficiently adapt to AA starvation, the two pathways are cross-regulated at multiple levels. Here we identified a new mechanism of ISR/mTORC1 crosstalk that optimizes survival under AA starvation, when mTORC1 is forced to remain active. mTORC1 activation during acute AA shortage, augmented ATF4 expression in a GCN2-dependent manner. Under these conditions, enhanced GCN2 activity was not dependent on tRNA sensing, inferring a different activation mechanism. We identified a labile physical interaction between GCN2 and mTOR that results in a phosphorylation of GCN2 on serine 230 by mTOR, which promotes GCN2 activity. When examined under prolonged AA starvation, GCN2 phosphorylation by mTOR promoted survival. Our data unveils an adaptive mechanism to AA starvation, when mTORC1 evades inhibition.

Dess-Martin periodinane-mediated oxidation of the primary alcohol of cytidine into a carboxylic acid.

Herein the first example of conversion of alcohols into carboxylic acids by use of the Dess-Martin Periodinane (DMP), which is otherwise routinely employed for the conversion to aldehydes, is reported. This methodology will have significant potential utility in the synthesis of cytidine analogues and other related biologically important molecules.

Multiscale In Silico Study of the Mechanism of Activation of the RtcB Ligase by the PTP1B Phosphatase.

Inositol-requiring enzyme 1 (IRE1) is a transmembrane sensor that is part of a trio of sensors responsible for controlling the unfolded protein response within the endoplasmic reticulum (ER). Upon the accumulation of unfolded or misfolded proteins in the ER, IRE1 becomes activated and initiates the cleavage of a 26-nucleotide intron from human X-box-containing protein 1 (XBP1). The cleavage is mediated by the RtcB ligase enzyme, which splices together two exons, resulting in the formation of the spliced isoform XBP1s. The XBP1s isoform activates the transcription of genes involved in ER-associated degradation to maintain cellular homeostasis. The catalytic activity of RtcB is controlled by the phosphorylation and dephosphorylation of three tyrosine residues (Y306, Y316, and Y475), which are regulated by the ABL1 tyrosine kinase and PTP1B phosphatase, respectively. This study focuses on investigating the mechanism by which the PTP1B phosphatase activates the RtcB ligase using a range of advanced in silico methods. Protein-protein docking identified key interacting residues between RtcB and PTP1B. Notably, the phosphorylated Tyr306 formed hydrogen bonds and salt bridge interactions with the "gatekeeper" residues Arg47 and Lys120 of the inactive PTP1B. Classical molecular dynamics simulation emphasized the crucial role of Asp181 in the activation of PTP1B, driving the conformational change from an open to a closed state of the WPD-loop. Furthermore, QM/MM-MD simulations provided insights into the free energy landscape of the dephosphorylation reaction mechanism of RtcB, which is mediated by the PTP1B phosphatase.

Insights into the structure and function of the RNA ligase RtcB.

To be functional, some RNAs require a processing step involving splicing events. Each splicing event necessitates an RNA ligation step. RNA ligation is a process that can be achieved with various intermediaries such as self-catalysing RNAs, 5'-3' and 3'-5' RNA ligases. While several types of RNA ligation mechanisms occur in human, RtcB is the only 3'-5' RNA ligase identified in human cells to date. RtcB RNA ligation activity is well known to be essential for the splicing of XBP1, an essential transcription factor of the unfolded protein response; as well as for the maturation of specific intron-containing tRNAs. As such, RtcB is a core factor in protein synthesis and homeostasis. Taking advantage of the high homology between RtcB orthologues in archaea, bacteria and eukaryotes, this review will provide an introduction to the structure of RtcB and the mechanism of 3'-5' RNA ligation. This analysis is followed by a description of the mechanisms regulating RtcB activity and localisation, its known partners and its various functions from bacteria to human with a specific focus on human cancer.

Markov state models elucidate the stability of DNA influenced by the chiral 5S-Tg base.

The static and dynamic structures of DNA duplexes affected by 5S-Tg (Tg, Thymine glycol) epimers were studied using MD simulations and Markov State Models (MSMs) analysis. The results show that the 5S,6S-Tg base caused little perturbation to the helix, and the base-flipping barrier was determined to be 4.4 kcal mol-1 through the use of enhanced sampling meta-eABF calculations, comparable to 5.4 kcal mol-1 of the corresponding thymine flipping. Two conformations with the different hydrogen bond structures between 5S,6R-Tg and A19 were identified in several independent MD trajectories. The 5S,6R-Tg:O6HO6•••N1:A19 hydrogen bond is present in the high-energy conformation displaying a clear helical distortion, and near barrier-free Tg base flipping. The low-energy conformation always maintains Watson-Crick base pairing between 5S,6R-Tg and A19, and 5S-Tg base flipping is accompanied by a small barrier of ca. 2.0 KBT (T = 298 K). The same conformations are observed in the MSMs analysis. Moreover, the transition path and metastable structures of the damaged base flipping are for the first time verified through MSMs analysis. The data clearly show that the epimers have completely different influence on the stability of the DNA duplex, thus implying different enzymatic mechanisms for DNA repair.

Structural Insights into Pseudomonas aeruginosa Exotoxin A-Elongation Factor 2 Interactions: A Molecular Dynamics Study.

Exotoxin A (ETA) is an extracellular secreted toxin and a single-chain polypeptide with A and B fragments that is produced by Pseudomonas aeruginosa. It catalyzes the ADP-ribosylation of a post-translationally modified histidine (diphthamide) on eukaryotic elongation factor 2 (eEF2), which results in the inactivation of the latter and the inhibition of protein biosynthesis. Studies show that the imidazole ring of diphthamide plays an important role in the ADP-ribosylation catalyzed by the toxin. In this work, we employ different in silico molecular dynamics (MD) simulation approaches to understand the role of diphthamide versus unmodified histidine in eEF2 on the interaction with ETA. Crystal structures of the eEF2-ETA complexes with three different ligands NAD+, ADP-ribose, and ßTAD were selected and compared in the diphthamide and histidine containing systems. The study shows that NAD+ bound to ETA remains very stable in comparison with other ligands, enabling the transfer of ADP-ribose to the N3 atom of the diphthamide imidazole ring in eEF2 during ribosylation. We also show that unmodified histidine in eEF2 has a negative impact on ETA binding and is not a suitable target for the attachment of ADP-ribose. Analyzing of radius of gyration and COM distances for NAD+, ßTAD, and ADP-ribose complexes revealed that unmodified His affects the structure and destabilizes the complex with all different ligands throughout the MD simulations.

Footprints of natural selection at the mannose-6-phosphate isomerase locus in barnacles.

The mannose-6-phosphate isomerase (Mpi) locus in Semibalanus balanoides has been studied as a candidate gene for balancing selection for more than two decades. Previous work has shown that Mpi allozyme genotypes (fast and slow) have different frequencies across Atlantic intertidal zones due to selection on postsettlement survival (i.e., allele zonation). We present the complete gene sequence of the Mpi locus and quantify nucleotide polymorphism in S. balanoides, as well as divergence to its sister taxon Semibalanus cariosus We show that the slow allozyme contains a derived charge-altering amino acid polymorphism, and both allozyme classes correspond to two haplogroups with multiple internal haplotypes. The locus shows several footprints of balancing selection around the fast/slow site: an enrichment of positive Tajima's D for nonsynonymous mutations, an excess of polymorphism, and a spike in the levels of silent polymorphism relative to silent divergence, as well as a site frequency spectrum enriched for midfrequency mutations. We observe other departures from neutrality across the locus in both coding and noncoding regions. These include a nonsynonymous trans-species polymorphism and a recent mutation under selection within the fast haplogroup. The latter suggests ongoing allelic replacement of functionally relevant amino acid variants. Moreover, predicted models of Mpi protein structure provide insight into the functional significance of the putatively selected amino acid polymorphisms. While footprints of selection are widespread across the range of S. balanoides, our data show that intertidal zonation patterns are variable across both spatial and temporal scales. These data provide further evidence for heterogeneous selection on Mpi.

A novel DPH5-related diphthamide-deficiency syndrome causing embryonic lethality or profound neurodevelopmental disorder.

PURPOSE: Diphthamide is a post-translationally modified histidine essential for messenger RNA translation and ribosomal protein synthesis. We present evidence for DPH5 as a novel cause of embryonic lethality and profound neurodevelopmental delays (NDDs). METHODS: Molecular testing was performed using exome or genome sequencing. A targeted Dph5 knockin mouse (C57BL/6Ncrl-Dph5em1Mbp/Mmucd) was created for a DPH5 p.His260Arg homozygous variant identified in 1 family. Adenosine diphosphate-ribosylation assays in DPH5-knockout human and yeast cells and in silico modeling were performed for the identified DPH5 potential pathogenic variants. RESULTS: DPH5 variants p.His260Arg (homozygous), p.Asn110Ser and p.Arg207Ter (heterozygous), and p.Asn174LysfsTer10 (homozygous) were identified in 3 unrelated families with distinct overlapping craniofacial features, profound NDDs, multisystem abnormalities, and miscarriages. Dph5 p.His260Arg homozygous knockin was embryonically lethal with only 1 subviable mouse exhibiting impaired growth, craniofacial dysmorphology, and multisystem dysfunction recapitulating the human phenotype. Adenosine diphosphate-ribosylation assays showed absent to decreased function in DPH5-knockout human and yeast cells. In silico modeling of the variants showed altered DPH5 structure and disruption of its interaction with eEF2. CONCLUSION: We provide strong clinical, biochemical, and functional evidence for DPH5 as a novel cause of embryonic lethality or profound NDDs with multisystem involvement and expand diphthamide-deficiency syndromes and ribosomopathies.

Dynamics of 5R-Tg Base Flipping in DNA Duplexes Based on Simulations─Agreement with Experiments and Beyond.

Damaged or mismatched DNA bases are normally thought to be able to flip out of the helical stack, providing enzymes with access to the faulty genetic information otherwise hidden inside the helix. Thymine glycol (Tg) is one of the most common products of nucleic acid damage. However, the static and dynamic structures of DNA duplexes affected by 5R-Tg epimers are still not clearly understood, including the ability of these to undergo spontaneous base flipping. Structural effects of the 5R-Tg epimers on the duplex DNA are herein studied using molecular dynamics together with reliable DFT based calculations. In comparison with the corresponding intact DNA, the cis-5R,6S-Tg epimer base causes little perturbation to the duplex DNA, and a barrier of 4.9 kcal mol-1 is obtained by meta-eABF for cis-5R,6S-Tg base flipping out of the duplex DNA, comparable to the 5.4 kcal mol-1 obtained for the corresponding thymine flipping in intact DNA. For the trans-5R,6R-Tg epimer, three stable local structures were identified, of which the most stable disrupts the Watson-Crick hydrogen-bonded G5/C20 base pair, leading to conformational distortion of the duplex. Interestingly, the relative barrier height of the 5R-Tg flipping is only 1.0 kcal mol-1 for one of these trans-5R,6R-Tg epimers. Water bridge interactions were identified to be essential for 5R-Tg flipping. The study clearly demonstrates the occurrence of partial trans-5R,6R-Tg epimer flipping in solution.

QM/MM Well-Tempered Metadynamics Study of the Mechanism of XBP1 mRNA Cleavage by Inositol Requiring Enzyme 1α RNase.

A range of in silico methodologies were herein employed to study the unconventional XBP1 mRNA cleavage mechanism performed by the unfolded protein response (UPR) mediator Inositol Requiring Enzyme 1α (IRE1). Using Protein-RNA molecular docking along with a series of extensive restrained/unrestrained atomistic molecular dynamics (MD) simulations, the dynamical behavior of the system was evaluated and a reliable model of the IRE1/XBP1 mRNA complex was constructed. From a series of well-converged quantum mechanics molecular mechanics well-tempered metadynamics (QM/MM WT-MetaD) simulations using the Grimme dispersion interaction corrected semiempirical parametrization method 6 level of theory (PM6-D3) and the AMBER14SB-OL3 force field, the free energy profile of the cleavage mechanism was determined, along with intermediates and transition state structures. The results show two distinct reaction paths based on general acid-general base type mechanisms, with different activation energies that perfectly match observations from experimental mutagenesis data. The study brings unique atomistic insights into the cleavage mechanism of XBP1 mRNA by IRE1 and clarifies the roles of the catalytic residues H910 and Y892. Increased understanding of the details in UPR signaling can assist in the development of new therapeutic agents for its modulation.

Virtual Screening Expands the Non-Natural Amino Acid Palette for Peptide Optimization.

Peptides are an important modality in drug discovery. While current peptide optimization focuses predominantly on the small number of natural and commercially available non-natural amino acids, the chemical spaces available for small molecule drug discovery are in the billions of molecules. In the present study, we describe the development of a large virtual library of readily synthesizable non-natural amino acids that can power the virtual screening protocols and aid in peptide optimization. To that end, we enumerated nearly 380 thousand amino acids and demonstrated their vast chemical diversity compared to the 20 natural and commercial residues. Furthermore, we selected a diverse ten thousand amino acid subset to validate our virtual screening workflow on the Keap1-Neh2 complex model system. Through in silico mutations of Neh2 peptide residues to those from the virtual library, our docking-based protocol identified a number of possible solutions with a significantly higher predicted affinity toward the Keap1 protein. This protocol demonstrates that the non-natural amino acid chemical space can be massively extended and virtually screened with a reasonable computational cost.

Structural and molecular bases to IRE1 activity modulation.

The Unfolded Protein response is an adaptive pathway triggered upon alteration of endoplasmic reticulum (ER) homeostasis. It is transduced by three major ER stress sensors, among which the Inositol Requiring Enzyme 1 (IRE1) is the most evolutionarily conserved. IRE1 is an ER-resident type I transmembrane protein exhibiting an ER luminal domain that senses the protein folding status and a catalytic kinase and RNase cytosolic domain. In recent years, IRE1 has emerged as a relevant therapeutic target in various diseases including degenerative, inflammatory and metabolic pathologies and cancer. As such several drugs altering IRE1 activity were developed that target either catalytic activity and showed some efficacy in preclinical pathological mouse models. In this review, we describe the different drugs identified to target IRE1 activity as well as their mode of action from a structural perspective, thereby identifying common and different modes of action. Based on this information we discuss on how new IRE1-targeting drugs could be developed that outperform the currently available molecules.

Molecular dynamics study of the recognition of ATP by nucleic acid aptamers.

Despite their great success in recognizing small molecules in vitro, nucleic acid aptamers are rarely used in clinical settings. This is partially due to the lack of structure-based mechanistic information. In this work, atomistic molecular dynamics simulations are used to study the static and dynamic supramolecular structures relevant to the process of the wild-type (wt) nucleic acid aptamer recognition and binding of ATP. The effects brought about by mutation of key residues in the recognition site are also explored. The simulations reveal that the aptamer displays a high degree of rigidity and is structurally very little affected by the binding of ATP. Interaction energy decomposition shows that dispersion forces from π-stacking between ATP and the G6 and A23 nucleobases in the aptamer binding site plays a more important role in stabilizing the supramolecular complex, compared to hydrogen-bond interaction between ATP and G22. Moreover, metadynamics simulations show that during the association process, water molecules act as essential bridges connecting ATP with G22, which favors the dynamic stability of the complex. The calculations carried out on three mutated aptamer structures confirm the crucial role of the hydrogen bonds and π-stacking interactions for the binding affinity of the ATP nucleic acid aptamer.

The stressosome, a caspase-8-activating signalling complex assembled in response to cell stress in an ATG5-mediated manner.

Stress-induced apoptosis is mediated primarily through the intrinsic pathway that involves caspase-9. We previously reported that in caspase-9-deficient cells, a protein complex containing ATG5 and Fas-associated death domain (FADD) facilitated caspase-8 activation and cell death in response to endoplasmic reticulum (ER) stress. Here, we investigated whether this complex could be activated by other forms of cell stress. We show that diverse stress stimuli, including etoposide, brefeldin A and paclitaxel, as well as heat stress and gamma-irradiation, caused formation of a complex containing ATG5-ATG12, FADD and caspase-8 leading to activation of downstream caspases in caspase-9-deficient cells. We termed this complex the 'stressosome'. However, in these cells, only ER stress and heat shock led to stressosome-dependent cell death. Using in silico molecular modelling, we propose the structure of the stressosome complex, with FADD acting as an adaptor protein, interacting with pro-caspase-8 through their respective death effector domains (DEDs) and interacting with ATG5-ATG12 through its death domain (DD). This suggests that the complex could be regulated by cellular FADD-like interleukin-1ß-converting enzyme-inhibitory protein (cFLIPL ), which was confirmed experimentally. This study provides strong evidence for an alternative mechanism of caspase-8 activation involving the stressosome complex.

Reconstruction of the Fas-Based Death-Inducing Signaling Complex (DISC) Using a Protein-Protein Docking Meta-Approach.

The death-inducing signaling complex (DISC) is a fundamental multiprotein complex, which triggers the extrinsic apoptosis pathway through stimulation by death ligands. DISC consists of different death domain (DD) and death effector domain (DED) containing proteins such as the death receptor Fas (CD95) in complex with FADD, procaspase-8, and cFLIP. Despite many experimental and theoretical studies in this area, there is no global agreement neither on the DISC architecture nor on the mechanism of action of the involved species. In the current work, we have tried to reconstruct the DISC structure by identifying key protein interactions using a new protein-protein docking meta-approach. We combined the benefits of five of the most employed protein-protein docking engines, HADDOCK, ClusPro, HDOCK, GRAMM-X, and ZDOCK, in order to improve the accuracy of the predicted docking complexes. Free energy of binding and hot spot interacting residues were calculated and determined for each protein-protein interaction using molecular mechanics generalized Born surface area and alanine scanning techniques, respectively. In addition, a series of in-cellulo protein-fragment complementation assays were conducted to validate the protein-protein docking procedure. The results show that the DISC formation initiates by dimerization of adjacent FasDD trimers followed by recruitment of FADD through homotypic DD interactions with the oligomerized death receptor. Furthermore, the in-silico outcomes indicate that cFLIP cannot bind directly to FADD; instead, cFLIP recruitment to the DISC is a hierarchical and cooperative process where FADD initially recruits procaspase-8, which in turn recruits and heterodimerizes with cFLIP. Finally, a possible structure of the entire DISC is proposed based on the docking results.

Protein kinase A controls yeast growth in visible light.

BACKGROUND: A wide variety of photosynthetic and non-photosynthetic species sense and respond to light, having developed protective mechanisms to adapt to damaging effects on DNA and proteins. While the biology of UV light-induced damage has been well studied, cellular responses to stress from visible light (400-700 nm) remain poorly understood despite being a regular part of the life cycle of many organisms. Here, we developed a high-throughput method for measuring growth under visible light stress and used it to screen for light sensitivity in the yeast gene deletion collection. RESULTS: We found genes involved in HOG pathway signaling, RNA polymerase II transcription, translation, diphthamide modifications of the translational elongation factor eEF2, and the oxidative stress response to be required for light resistance. Reduced nuclear localization of the transcription factor Msn2 and lower glycogen accumulation indicated higher protein kinase A (cAMP-dependent protein kinase, PKA) activity in many light-sensitive gene deletion strains. We therefore used an ectopic fluorescent PKA reporter and mutants with constitutively altered PKA activity to show that repression of PKA is essential for resistance to visible light. CONCLUSION: We conclude that yeast photobiology is multifaceted and that protein kinase A plays a key role in the ability of cells to grow upon visible light exposure. We propose that visible light impacts on the biology and evolution of many non-photosynthetic organisms and have practical implications for how organisms are studied in the laboratory, with or without illumination.

Oxygen Dependent Purine Lesions in Double-Stranded Oligodeoxynucleotides: Kinetic and Computational Studies Highlight the Mechanism for 5',8-Cyclopurine Formation.

The reaction of HO• radical with DNA is intensively studied both mechanistically and analytically for lesions formation. Several aspects related to the reaction paths of purine moieties with the formation of 5',8-cyclopurines (cPu), 8-oxopurines (8-oxo-Pu), and their relationship are not well understood. In this study, we investigated the reaction of HO• radical with a 21-mer double-stranded oligodeoxynucleotide (ds-ODNs) in γ-irradiated aqueous solutions under various oxygen concentrations and accurately quantified the six purine lesions (i.e., four cPu and two 8-oxo-Pu) by LC-MS/MS analysis using isotopomeric internal standards. In the absence of oxygen, 8-oxo-Pu lesions are only ∼4 times more than cPu lesions. By increasing oxygen concentration, the 8-oxo-Pu and the cPu gradually increase and decrease, respectively, reaching a gap of ∼130 times at 2.01 × 10-4 M of O2. Kinetic treatment of the data allows to estimate the C5' radical competition between cyclization and oxygen trapping in ds-ODNs, and lastly the rate constants of the four cyclization steps. Tailored computational studies by means of dispersion-corrected DFT calculations were performed on the CGC and TAT in their double-strand models for each cPu diastereoisomer along with the complete reaction pathways of the cyclization steps. Our findings reveal unheralded reaction mechanisms that resolve the long-standing issues with C5' radical cyclization in purine moieties of DNA sequences.

Effect of Kinase Inhibiting RNase Attenuator (KIRA) Compounds on the Formation of Face-to-Face Dimers of Inositol-Requiring Enzyme 1: Insights from Computational Modeling.

Inositol-requiring enzyme 1α (IRE1α) is a transmembrane dual kinase/ribonuclease protein involved in propagation of the unfolded protein response (UPR). Inositol-requiring enzyme 1α is currently being explored as a potential drug target due to the growing evidence of its role in variety of disease conditions. Upon activation, IRE1 cleaves X-box binding protein 1 (XBP1) mRNA through its RNase domain. Small molecules targeting the kinase site are known to either increase or decrease RNase activity, but the allosteric relationship between the kinase and RNase domains of IRE1α is poorly understood. Subsets of IRE1 kinase inhibitors (known as "KIRA" compounds) bind to the ATP-binding site and allosterically impede the RNase activity. The KIRA compounds are able to regulate the RNase activity by stabilizing the monomeric form of IRE1α. In the present work, computational analysis, protein-protein and protein-ligand docking studies, and molecular dynamics simulations were applied to different IRE1 dimer systems to provide structural insights into the perturbation of IRE1 dimers by small molecules kinase inhibitors that regulate the RNase activity. By analyzing structural deviations, energetic components, and the number of hydrogen bonds in the interface region, we propose that the KIRA inhibitors act at an early stage of IRE1 activation by interfering with IRE1 face-to-face dimer formation thus disabling the activation of the RNase domain. This work sheds light on the mechanism of action of KIRA compounds and may assist in development of further compounds in, for example, cancer therapeutics. The work also provides information on the sequence of events and protein-protein interactions initiating the unfolded protein response.

QM/MM Studies of Dph5 - A Promiscuous Methyltransferase in the Eukaryotic Biosynthetic Pathway of Diphthamide.

Eukaryotic diphthine synthase, Dph5, is a promiscuous methyltransferase that catalyzes an extraordinary N, O-tetramethylation of 2-(3-carboxy-3-aminopropyl)-l-histidine (ACP) to yield diphthine methyl ester (DTM). These are intermediates in the biosynthesis of the post-translationally modified histidine residue diphthamide (DTA), a unique and essential residue part of the eukaryotic elongation factor 2 (eEF2). Herein, the promiscuity of Saccharomyces cerevisiae Dph5 has been studied with in silico approaches, including homology modeling to provide the structure of Dph5, protein-protein docking and molecular dynamics to construct the Dph5-eEF2 complex, and quantum mechanics/molecular mechanics (QM/MM) calculations to outline a plausible mechanism. The calculations show that the methylation of ACP follows a typical SN2 mechanism, initiating with a complete methylation (trimethylation) at the N-position, followed by the single O-methylation. For each of the three N-methylation reactions, our calculations support a stepwise mechanism, which first involve proton transfer through a bridging water to a conserved aspartate residue D165, followed by a methyl transfer. Once fully methylated, the trimethyl amino group forms a weak electrostatic interaction with D165, which allows the carboxylate group of diphthine to attain the right orientation for the final methylation step to be accomplished.