Drug resistance through ribosome splitting and rRNA disordering in mycobacteria.

HflX is known to rescue stalled ribosomes and is implicated in antibiotic resistance in several bacteria. Here we present several high-resolution cryo-EM structures of mycobacterial HflX in complex with the ribosome and its 50S subunit, with and without antibiotics. These structures reveal a distinct mechanism for HflX- mediated ribosome splitting and antibiotic resistance in mycobacteria. In addition to dissociating ribosome into two subunits, mycobacterial HflX mediates persistent disordering of multiple 23S rRNA helices to generate an inactive pool of 50S subunits. Mycobacterial HflX also acts as an anti-association factor by binding to pre-dissociated 50S subunits. A mycobacteria-specific insertion in HflX reaches further into the peptidyl transferase center. The position of this insertion overlaps with ribosome-bound macrolides or lincosamide class of antibiotics. The extended conformation of insertion seen in the absence of these antibiotics retracts and adjusts around the bound antibiotics instead of physically displacing them. It therefore likely imparts antibiotic resistance by sequestration of the antibiotic- bound inactive 50S subunits.

Clinical Course, Antifungal Susceptibility, and Genomic Sequencing of Trichophyton indotineae.

Importance: Trichophyton indotineae is an emerging dermatophyte causing outbreaks of extensive tinea infections often unresponsive to terbinafine. This species has been detected worldwide and in multiple US states, yet detailed US data on infections with T indotineae are sparse and could improve treatment practices and medical understanding of transmission. Objective: To correlate clinical features of T indotineae infections with in vitro antifungal susceptibility testing results, squalene epoxidase gene sequence variations, and isolate relatedness using whole-genome sequencing. Design, Setting, and Participants: This retrospective cohort study of patients with T indotineae infections in New York City spanned May 2022 to May 2023. Patients with confirmed T indotineae infections were recruited from 6 New York City medical centers. Main Outcome and Measure: Improvement or resolution at the last follow-up assessment. Results: Among 11 patients with T indotineae (6 male and 5 female patients; median [range] age, 39 [10-65] years), 2 were pregnant; 1 had lymphoma; and the remainder were immunocompetent. Nine patients reported previous travel to Bangladesh. All had widespread lesions with variable scale and inflammation, topical antifungal monotherapy failure, and diagnostic delays (range, 3-42 months). Terbinafine treatment failed in 7 patients at standard doses (250 mg daily) for prolonged duration; these patients also had isolates with amino acid substitutions at positions 393 (L393S) or 397 (F397L) in squalene epoxidase that correlated with elevated terbinafine minimum inhibitory concentrations of 0.5 µg/mL or higher. Patients who were treated with fluconazole and griseofulvin improved in 2 of 4 and 2 of 5 instances, respectively, without correlation between outcomes and antifungal minimum inhibitory concentrations. Furthermore, 5 of 7 patients treated with itraconazole cleared or had improvement at the last follow-up, and 2 of 7 were lost to follow-up or stopped treatment. Based on whole-genome sequencing analysis, US isolates formed a cluster distinct from Indian isolates. Conclusion and Relevance: The results of this case series suggest that disease severity, diagnostic delays, and lack of response to typically used doses and durations of antifungals for tinea were common in this primarily immunocompetent patient cohort with T indotineae, consistent with published data. Itraconazole was generally effective, and the acquisition of infection was likely in Bangladesh.

The structure of a hibernating ribosome in a Lyme disease pathogen.

The spirochete bacterial pathogen Borrelia (Borreliella) burgdorferi (Bbu) affects more than 10% of the world population and causes Lyme disease in about half a million people in the US annually. Therapy for Lyme disease includes antibiotics that target the Bbu ribosome. Here we present the structure of the Bbu 70S ribosome obtained by single particle cryo-electron microscopy at 2.9 Å resolution, revealing a bound hibernation promotion factor protein and two genetically non-annotated ribosomal proteins bS22 and bL38. The ribosomal protein uL30 in Bbu has an N-terminal α-helical extension, partly resembling the mycobacterial bL37 protein, suggesting evolution of bL37 and a shorter uL30 from a longer uL30 protein. Its analogy to proteins uL30m and mL63 in mammalian mitochondrial ribosomes also suggests a plausible evolutionary pathway for expansion of protein content in mammalian mitochondrial ribosomes. Computational binding free energy predictions for antibiotics reflect subtle distinctions in antibiotic-binding sites in the Bbu ribosome. Discovery of these features in the Bbu ribosome may enable better ribosome-targeted antibiotic design for Lyme disease treatment.

The structure of a hibernating ribosome in a Lyme disease pathogen.

The spirochete bacterial pathogen Borrelia ( Borreliella) burgdorferi ( Bbu ) affects more than 10% of the world population and causes Lyme disease in about half a million people in the US annually. Therapy for Lyme disease includes antibiotics that target the Bbu ribosome. We determined the structure of the Bbu 70S ribosome by single particle cryo-electron microscopy (cryo-EM) at a resolution of 2.9 Å, revealing its distinctive features. In contrast to a previous study suggesting that the single hibernation promoting factor protein present in Bbu (bbHPF) may not bind to its ribosome, our structure reveals a clear density for bbHPF bound to the decoding center of the small ribosomal 30S subunit. The 30S subunit has a non-annotated ribosomal protein, bS22, that has been found only in mycobacteria and Bacteroidetes so far. The protein bL38, recently discovered in Bacteroidetes, is also present in the Bbu large 50S ribosomal subunit. The protein bL37, previously seen only in mycobacterial ribosomes, is replaced by an N-terminal α-helical extension of uL30, suggesting that the two bacterial ribosomal proteins uL30 and bL37 may have evolved from one longer uL30 protein. The longer uL30 protein interacts with both the 23S rRNA and the 5S rRNA, is near the peptidyl transferase center (PTC), and could impart greater stability to this region. Its analogy to proteins uL30m and mL63 in mammalian mitochondrial ribosomes also suggests a plausible evolutionary pathway for expansion of protein content in mammalian mitochondrial ribosomes. Computational binding free energies are predicted for antibiotics, bound to the decoding center or PTC and are in clinical use for Lyme disease, that account for subtle distinctions in antibiotic-binding regions in the Bbu ribosome structure. Besides revealing unanticipated structural and compositional features for the Bbu ribosome, our study thus provides groundwork to enable ribosome-targeted antibiotic design for more effective treatment of Lyme disease.

Starvation sensing by mycobacterial RelA/SpoT homologue through constitutive surveillance of translation.

The stringent response, which leads to persistence of nutrient-starved mycobacteria, is induced by activation of the RelA/SpoT homolog (Rsh) upon entry of a deacylated-tRNA in a translating ribosome. However, the mechanism by which Rsh identifies such ribosomes in vivo remains unclear. Here, we show that conditions inducing ribosome hibernation result in loss of intracellular Rsh in a Clp protease-dependent manner. This loss is also observed in nonstarved cells using mutations in Rsh that block its interaction with the ribosome, indicating that Rsh association with the ribosome is important for Rsh stability. The cryo-EM structure of the Rsh-bound 70S ribosome in a translation initiation complex reveals unknown interactions between the ACT domain of Rsh and components of the ribosomal L7/L12 stalk base, suggesting that the aminoacylation status of A-site tRNA is surveilled during the first cycle of elongation. Altogether, we propose a surveillance model of Rsh activation that originates from its constitutive interaction with the ribosomes entering the translation cycle.

The small mycobacterial ribosomal protein, bS22, modulates aminoglycoside accessibility to its 16S rRNA helix-44 binding site.

Treatment of tuberculosis continues to be challenging due to the widespread latent form of the disease and the emergence of antibiotic-resistant strains of the pathogen, Mycobacterium tuberculosis. Bacterial ribosomes are a common and effective target for antibiotics. Several second line anti-tuberculosis drugs, e.g. kanamycin, amikacin, and capreomycin, target ribosomal RNA to inhibit protein synthesis. However, M. tuberculosis can acquire resistance to these drugs, emphasizing the need to identify new drug targets. Previous cryo-EM structures of the M. tuberculosis and M. smegmatis ribosomes identified two novel ribosomal proteins, bS22 and bL37, in the vicinity of two crucial drug-binding sites: the mRNA-decoding center on the small (30S), and the peptidyl-transferase center on the large (50S) ribosomal subunits, respectively. The functional significance of these two small proteins is unknown. In this study, we observe that an M. smegmatis strain lacking the bs22 gene shows enhanced susceptibility to kanamycin compared to the wild-type strain. Cryo-EM structures of the ribosomes lacking bS22 in the presence and absence of kanamycin suggest a direct role of bS22 in modulating the 16S rRNA kanamycin-binding site. Our structures suggest that amino-acid residue Lys-16 of bS22 interacts directly with the phosphate backbone of helix 44 of 16S rRNA to influence the micro-configuration of the kanamycin-binding pocket. Our analysis shows that similar interactions occur between eukaryotic homologues of bS22, and their corresponding rRNAs, pointing to a common mechanism of aminoglycoside resistance in higher organisms.

Development and Validation of TaqMan Chemistry Probe-Based Rapid Assay for the Detection of Echinocandin-Resistance in Candida auris.

Candida auris is a multidrug-resistant yeast pathogen causing outbreaks in health care facilities worldwide, and the emergence of echinocandin-resistant C. auris is a concern. Currently used Clinical and Laboratory Standards Institute (CLSI) and commercial antifungal susceptibility tests (AFST) are phenotype-based, slow, and not scalable, limiting their effectiveness in the surveillance of echinocandin-resistant C. auris. The urgent need for accurate and rapid methods of assessment of echinocandin resistance cannot be overstated, as this class of antifungal drugs is preferred for patient management. We report the development and validation of a TaqMan chemistry probe-based fluorescence melt curve analysis (FMCA) following asymmetric polymerase chain reaction (PCR) to assess mutations within the hot spot one (HS1) region of FKS1, the gene responsible for encoding 1,3-ß-d-glucan synthase that is a target for echinocandins. The assay correctly identified F635C, F635Y, F635del, F635S, S639F or S639Y, S639P, and D642H/R645T mutations. Of these mutations, F635S and D642H/R645T were not involved in echinocandin resistance, while the rest were, as confirmed by AFST. Of 31 clinical cases, the predominant mutation conferring echinocandin resistance was S639F/Y (20 cases) followed by S639P (4 cases), F635del (4 cases), F635Y (2 cases), and F635C (1 case). The FMCA assay was highly specific and did not cross-react with closely and distantly related Candida and other yeast and mold species. Structural modeling of the Fks1 protein, its mutants, and docked conformations of three echinocandin drugs suggest a plausible Fks1 binding orientation for echinocandins. These findings lay the groundwork for future evaluations of additional FKS1 mutations and their impact on the development of drug resistance. The TaqMan chemistry probe-based FMCA would allow rapid, high throughput, and accurate detection of FKS1 mutations conferring echinocandin resistance in C. auris.

Distinct mechanisms of the human mitoribosome recycling and antibiotic resistance.

Ribosomes are recycled for a new round of translation initiation by dissociation of ribosomal subunits, messenger RNA and transfer RNA from their translational post-termination complex. Here we present cryo-EM structures of the human 55S mitochondrial ribosome (mitoribosome) and the mitoribosomal large 39S subunit in complex with mitoribosome recycling factor (RRFmt) and a recycling-specific homolog of elongation factor G (EF-G2mt). These structures clarify an unusual role of a mitochondria-specific segment of RRFmt, identify the structural distinctions that confer functional specificity to EF-G2mt, and show that the deacylated tRNA remains with the dissociated 39S subunit, suggesting a distinct sequence of events in mitoribosome recycling. Furthermore, biochemical and structural analyses reveal that the molecular mechanism of antibiotic fusidic acid resistance for EF-G2mt is markedly different from that of mitochondrial elongation factor EF-G1mt, suggesting that the two human EF-Gmts have evolved diversely to negate the effect of a bacterial antibiotic.

Ribosome hibernation: a new molecular framework for targeting nonreplicating persisters of mycobacteria.

Treatment of tuberculosis requires a multi-drug regimen administered for at least 6 months. The long-term chemotherapy is attributed in part to a minor subpopulation of nonreplicating Mycobacterium tuberculosis cells that exhibit phenotypic tolerance to antibiotics. The origins of these cells in infected hosts remain unclear. Here we discuss some recent evidence supporting the hypothesis that hibernation of ribosomes in M. tuberculosis, induced by zinc starvation, could be one of the primary mechanisms driving the development of nonreplicating persisters in hosts. We further analyse inconsistencies in previously reported studies to clarify the molecular principles underlying mycobacterial ribosome hibernation.

Structures of the human mitochondrial ribosome bound to EF-G1 reveal distinct features of mitochondrial translation elongation.

The mammalian mitochondrial ribosome (mitoribosome) and its associated translational factors have evolved to accommodate greater participation of proteins in mitochondrial translation. Here we present the 2.68-3.96 Å cryo-EM structures of the human 55S mitoribosome in complex with the human mitochondrial elongation factor G1 (EF-G1mt) in three distinct conformational states, including an intermediate state and a post-translocational state. These structures reveal the role of several mitochondria-specific (mito-specific) mitoribosomal proteins (MRPs) and a mito-specific segment of EF-G1mt in mitochondrial tRNA (tRNAmt) translocation. In particular, the mito-specific C-terminal extension in EF-G1mt is directly involved in translocation of the acceptor arm of the A-site tRNAmt. In addition to the ratchet-like and independent head-swiveling motions exhibited by the small mitoribosomal subunit, we discover significant conformational changes in MRP mL45 at the nascent polypeptide-exit site within the large mitoribosomal subunit that could be critical for tethering of the elongating mitoribosome onto the inner-mitochondrial membrane.

The Mechanism of Cholesterol Modification of Hedgehog Ligand.

Hedgehog (Hh) proteins are important components of signal transduction pathways involved in animal development, and their defects are implicated in carcinogenesis. Their N-terminal domain (HhN) acts as a signaling ligand, and their C-terminal domain (HhC) performs an autocatalytic function of cleaving itself away, while adding a cholesterol moiety to HhN. HhC has two sub-domains: a hedgehog/intein (hint) domain that primarily performs the autocatalytic activity, and a sterol-recognition region (SRR) that binds to cholesterol and properly positions it with respect to HhN. The three-dimensional details of this autocatalytic mechanism remain unknown, as does the structure of the precursor Hh protein. In this study, a complete cholesterol-bound precursor form of the drosophila Hh precursor is modeled using known crystal structures of HhN and the hint domain, and a hypothesized similarity of SRR to an unrelated but similar-sized cholesterol binding protein. The restrained geometries and topology switching (RGATS) strategy is then used to predict atomic-detail pathways for the full autocatalytic reaction starting from the precursor and ending in a cholesterol-linked HhN domain and a cleaved HhC domain. The RGATS explicit solvent simulations indicate the roles of individual HhC residues in facilitating the reaction, which can be confirmed through mutational experiments. These simulations also provide plausible structural models for the N/S acyl transfer intermediate and the product states of this reaction. This study thus provides a good framework for future computational and experimental studies to develop a full structural and dynamic understanding of Hh autoprocessing. © 2019 Wiley Periodicals, Inc.

Spliceosomal Prp8 intein at the crossroads of protein and RNA splicing.

The spliceosome is a large ribonucleoprotein complex that removes introns from pre-mRNAs. At its functional core lies the essential pre-mRNA processing factor 8 (Prp8) protein. Across diverse eukaryotes, this protein cofactor of RNA catalysis harbors a self-splicing element called an intein. Inteins in Prp8 are extremely pervasive and are found at 7 different sites in various species. Here, we focus on the Prp8 intein from Cryptococcus neoformans (Cne), a human fungal pathogen. We solved the crystal structure of this intein, revealing structural homology among protein splicing sequences in eukaryotes, including the Hedgehog C terminus. Working with the Cne Prp8 intein in a reporter assay, we find that the biologically relevant divalent metals copper and zinc inhibit intein splicing, albeit by 2 different mechanisms. Copper likely stimulates reversible modifications on a catalytically important cysteine, whereas zinc binds at the terminal asparagine and the same critical cysteine. Importantly, we also show that copper treatment inhibits Prp8 protein splicing in Cne. Lastly, an intein-containing Prp8 precursor model is presented, suggesting that metal-induced protein splicing inhibition would disturb function of both Prp8 and the spliceosome. These results indicate that Prp8 protein splicing can be modulated, with potential functional implications for the spliceosome.

Sterol A-ring plasticity in hedgehog protein cholesterolysis supports a primitive substrate selectivity mechanism.

Cholesterolysis of Hedgehog family proteins couples endoproteolysis to protein C-terminal sterylation. The transformation is self-catalyzed by HhC, a partially characterized enzymatic domain found in precursor forms of Hedgehog. Here we explore spatial ambiguity in sterol recognition by HhC, using a trio of derivatives where the sterol A-ring is contracted, fused, or distorted. Sterylation assays indicate that these geometric variants react as substrates with relative activity: cholesterol, 1.000 > A-ring contracted, 0.100 > A-ring fused, 0.020 > A-ring distorted, 0.005. Experimental results and computational sterol docking into the first HhC homology model suggest a partially unstructured binding site with substrate recognition governed in large part by hydrophobic interactions.

Mycobacterial DnaB helicase intein as oxidative stress sensor.

Inteins are widespread self-splicing protein elements emerging as potential post-translational environmental sensors. Here, we describe two inteins within one protein, the Mycobacterium smegmatis replicative helicase DnaB. These inteins, DnaBi1 and DnaBi2, have homology to inteins in pathogens, splice with vastly varied rates, and are differentially responsive to environmental stressors. Whereas DnaBi1 splicing is reversibly inhibited by oxidative and nitrosative insults, DnaBi2 is not. Using a reporter that measures splicing in a native intein-containing organism and western blotting, we show that H2O2 inhibits DnaBi1 splicing in M. smegmatis. Intriguingly, upon oxidation, the catalytic cysteine of DnaBi1 forms an intramolecular disulfide bond. We report a crystal structure of the class 3 DnaBi1 intein at 1.95 Å, supporting our findings and providing insight into this splicing mechanism. We propose that this cysteine toggle allows DnaBi1 to sense stress, pausing replication to maintain genome integrity, and then allowing splicing immediately when permissive conditions return.

Conditional Protein Splicing Switch in Hyperthermophiles through an Intein-Extein Partnership.

Inteins are intervening proteins that undergo an autocatalytic splicing reaction that ligates flanking host protein sequences termed exteins. Some intein-containing proteins have evolved to couple splicing to environmental signals; this represents a new form of posttranslational regulation. Of particular interest is RadA from the archaeon Pyrococcus horikoshii, for which long-range intein-extein interactions block splicing, requiring temperature and single-stranded DNA (ssDNA) substrate to splice rapidly and accurately. Here, we report that splicing of the intein-containing RadA from another archaeon, Thermococcus sibericus, is activated by significantly lower temperatures than is P. horikoshii RadA, consistent with differences in their growth environments. Investigation into variations between T. sibericus and P. horikoshii RadA inteins led to the discovery that a nonconserved region (NCR) of the intein, a flexible loop where a homing endonuclease previously resided, is critical to splicing. Deletion of the NCR leads to a substantial loss in the rate and accuracy of P. horikoshii RadA splicing only within native exteins. The influence of the NCR deletion can be largely overcome by ssDNA, demonstrating that the splicing-competent conformation can be achieved. We present a model whereby the NCR is a flexible hinge which acts as a switch by controlling distant intein-extein interactions that inhibit active site assembly. These results speak to the repurposing of the vestigial endonuclease loop to control an intein-extein partnership, which ultimately allows exquisite adaptation of protein splicing upon changes in the environment.IMPORTANCE Inteins are mobile genetic elements that interrupt coding sequences (exteins) and are removed by protein splicing. They are abundant elements in microbes, and recent work has demonstrated that protein splicing can be controlled by environmental cues, including the substrate of the intein-containing protein. Here, we describe an intein-extein collaboration that controls temperature-induced splicing of RadA from two archaea and how variation in this intein-extein partnership results in fine-tuning of splicing to closely match the environment. Specifically, we found that a small sequence difference between the two inteins, a flexible loop that likely once housed a homing endonuclease used for intein mobility, acts as a switch to control intein-extein interactions that block splicing. Our results argue strongly that some inteins have evolved away from a purely parasitic lifestyle to control the activity of host proteins, representing a new form of posttranslational regulation that is potentially widespread in the microbial world.

Structural Articulation of Biochemical Reactions Using Restrained Geometries and Topology Switching.

A strategy named "restrained geometries and topology switching" (RGATS) is presented to obtain detailed trajectories for complex biochemical reactions using molecular mechanics (MM) methods. It enables prediction of realistic dynamical pathways for chemical reactions, especially for accurately characterizing the structural adjustments of highly complex environments to any proximal biochemical reaction. It can be used to generate reactive conformations, model stepwise or concerted reactions in complex environments, and probe the influence of changes in the environment. Its ability to take reactively nonoptimal conformations and generate favorable starting conformations for a biochemical reaction is illustrated for a proton transfer between two model compounds. Its ability to study concerted reactions in explicit solvent is illustrated using proton transfers between an ammonium ion and two conserved histidines in an ammonia transporter channel embedded in a lipid membrane. Its ability to characterize the changes induced by subtle differences in the active site environment is illustrated using nucleotide addition by a DNA polymerase in the presence of two versus three Mg2+ ions. RGATS can be employed within any MM program and requires no additional software implementation. This allows the full assortment of computational methods implemented in all available MM programs to be used to tackle virtually any question about biochemical reactions that is answerable without using a quantum mechanical (QM) model. It can also be applied to generate reasonable starting structures for more detailed and expensive QM or QM/MM methods. In particular, this strategy enables rapid prediction of reactant, intermediary, or product state structures in any macromolecular context, with the only requirement being that the structure in any one of these states is either known or can be accurately modeled.

Chemical activation of adenylyl cyclase Rv1625c inhibits growth of Mycobacterium tuberculosis on cholesterol and modulates intramacrophage signaling.

Mycobacterium tuberculosis (Mtb) uses a complex 3', 5'-cyclic AMP (cAMP) signaling network to sense and respond to changing environments encountered during infection, so perturbation of cAMP signaling might be leveraged to disrupt Mtb pathogenesis. However, understanding of cAMP signaling pathways is hindered by the presence of at least 15 distinct adenylyl cyclases (ACs). Recently, the small molecule V-58 was shown to inhibit Mtb replication within macrophages and stimulate cAMP production in Mtb. Here we determined that V-58 rapidly and directly activates Mtb AC Rv1625c to produce high levels of cAMP regardless of the bacterial environment or growth medium. Metabolic inhibition by V-58 was carbon source dependent in Mtb and did not occur in Mycobacterium smegmatis, suggesting that V-58-mediated growth inhibition is due to interference with specific Mtb metabolic pathways rather than a generalized cAMP toxicity. Chemical stimulation of cAMP production by Mtb within macrophages also caused down regulation of TNF-α production by the macrophages, indicating a complex role for cAMP in Mtb pathogenesis. Together these studies describe a novel approach for targeted stimulation of cAMP production in Mtb, and provide new insights into the myriad roles of cAMP signaling in Mtb, particularly during Mtb's interactions with macrophages.

Geometric Patterns for Neighboring Bases Near the Stacked State in Nucleic Acid Strands.

Structural variation in base stacking has been analyzed frequently in isolated double helical contexts for nucleic acids, but not as often in nonhelical geometries or in complex biomolecular environments. In this study, conformations of two neighboring bases near their stacked state in any environment are comprehensively characterized for single-strand dinucleotide (SSD) nucleic acid crystal structure conformations. An ensemble clustering method is used to identify a reduced set of representative stacking geometries based on pairwise distances between select atoms in consecutive bases, with multiple separable conformational clusters obtained for categories divided by nucleic acid type (DNA/RNA), SSD sequence, stacking face orientation, and the presence or absence of a protein environment. For both DNA and RNA, SSD conformations are observed that are either close to the A-form, or close to the B-form, or intermediate between the two forms, or further away from either form, illustrating the local structural heterogeneity near the stacked state. Among this large variety of distinct conformations, several common stacking patterns are observed between DNA and RNA, and between nucleic acids in isolation or in complex with proteins, suggesting that these might be stable stacking orientations. Noncanonical face/face orientations of the two bases are also observed for neighboring bases in the same strand, but their frequency is much lower, with multiple SSD sequences across categories showing no occurrences of such unusual stacked conformations. The resulting reduced set of stacking geometries is directly useful for stacking-energy comparisons between empirical force fields, prediction of plausible localized variations in single-strand structures near their canonical states, and identification of analogous stacking patterns in newly solved nucleic acid containing structures.

Maintenance of electrostatic stabilization in altered tubulin lateral contacts may facilitate formation of helical filaments in foraminifera.

Microtubules in foraminiferan protists (forams) can convert into helical filament structures, in which longitudinal intraprotofilament interactions between tubulin heterodimers are thought to be lost, while lateral contacts across protofilaments are still maintained. The coarse geometric features of helical filaments are known through low-resolution negative stain electron microscopy (EM). In this study, geometric restraints derived from these experimental data were used to generate an average atomic-scale helical filament model, which anticipated a modest reorientation in the lateral tubulin heterodimer interface. Restrained molecular dynamics (MD) simulations of the nearest neighbor interactions combined with a Genalized Born implicit solvent model were used to assess the lateral, longitudinal, and seam contacts in 13-3 microtubules and the reoriented lateral contacts in the helical filament model. This electrostatic analysis suggests that the change in the lateral interface in the helical filament does not greatly diminish the lateral electrostatic interaction. After longitudinal dissociation, the 13-3 seam interaction is much weaker than the reoriented lateral interface in the helical filament model, providing a plausible atomic-detail explanation for seam-to-lateral contact transition that enables the transition to a helical filament structure.

RNA approaches the B-form in stacked single strand dinucleotide contexts.

Duplex RNA adopts an A-form structure, while duplex DNA interconverts between the A- and B-forms depending on the environment. The C2'-endo sugar pucker seen in B-form DNA can occur infrequently in ribose sugars as well, but RNA is not understood to assume B-form conformations. Through analysis of over 45,000 stacked single strand dinucleotide (SSD) crystal structure conformations, this study demonstrates that RNA is capable of adopting a wide conformational range between the canonical A- and B-forms at the localized SSD level, including many B-form-like conformations. It does so through C2'-endo ribose conformations in one or both nucleotides, and B-form-like neighboring base stacking patterns. As chemical reactions on nucleic acids involve localized changes in chemical bonds, the understanding of how enzymes distinguish between DNA and RNA nucleotides is altered by the energetic accessibility of these rare B-form-like RNA SSD conformations. The existence of these conformations also has direct implications in parametrization of molecular mechanics energy functions used extensively to model nucleic acid behavior., 2016. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 65-82, 2016.