How Natural Enzymes and Synthetic Ribozymes Generate Methylated Nucleotides in RNA.

Methylation of RNA nucleotides represents an important layer of gene expression regulation, and perturbation of the RNA methylome is associated with pathophysiology. In cells, RNA methylations are installed by RNA methyltransferases (RNMTs) that are specialized to catalyze particular types of methylation (ribose or different base positions). Furthermore, RNMTs must specifically recognize their appropriate target RNAs within the RNA-dense cellular environment. Some RNMTs are catalytically active alone and achieve target specificity via recognition of sequence motifs and/or RNA structures. Others function together with protein cofactors that can influence stability, S-adenosyl-L-methionine binding, and RNA affinity as well as aiding specific recruitment and catalytic activity. Association of RNMTs with guide RNAs represents an alternative mechanism to direct site-specific methylation by an RNMT that lacks intrinsic specificity. Recently, ribozyme-catalyzed methylation of RNA has been achieved in vitro, and here, we compare these different strategies for RNA methylation from structural and mechanistic perspectives.

Zona Pellucida Proteins, Fibrils, and Matrix.

The zona pellucida (ZP) is an extracellular matrix that surrounds all mammalian oocytes, eggs, and early embryos and plays vital roles during oogenesis, fertilization, and preimplantation development. The ZP is composed of three or four glycosylated proteins, ZP1-4, that are synthesized, processed, secreted, and assembled into long, cross-linked fibrils by growing oocytes. ZP proteins have an immunoglobulin-like three-dimensional structure and a ZP domain that consists of two subdomains, ZP-N and ZP-C, with ZP-N of ZP2 and ZP3 required for fibril assembly. A ZP2-ZP3 dimer is located periodically along ZP fibrils that are cross-linked by ZP1, a protein with a proline-rich N terminus. Fibrils in the inner and outer regions of the ZP are oriented perpendicular and parallel to the oolemma, respectively, giving the ZP a multilayered appearance. Upon fertilization of eggs, modification of ZP2 and ZP3 results in changes in the ZP's physical and biological properties that have important consequences. Certain structural features of ZP proteins suggest that they may be amyloid-like proteins.

How α-Helical Motifs Form Functionally Diverse Lipid-Binding Compartments.

Lipids are produced site-specifically in cells and then distributed nonrandomly among membranes via vesicular and nonvesicular trafficking mechanisms. The latter involves soluble amphitropic proteins extracting specific lipids from source membranes to function as molecular solubilizers that envelope their insoluble cargo before transporting it to destination sites. Lipid-binding and lipid transfer structural motifs range from multi-ß-strand barrels, to ß-sheet cups and baskets covered by α-helical lids, to multi-α-helical bundles and layers. Here, we focus on how α-helical proteins use amphipathic helical layering and bundling to form modular lipid-binding compartments and discuss the functional consequences. Preformed compartments generally rely on intramolecular disulfide bridging to maintain conformation (e.g., albumins, nonspecific lipid transfer proteins, saposins, nematode polyprotein allergens/antigens). Insights into nonpreformed hydrophobic compartments that expand and adapt to accommodate a lipid occupant are few and provided mostly by the three-layer, α-helical ligand-binding domain of nuclear receptors. The simple but elegant and nearly ubiquitous two-layer, α-helical glycolipid transfer protein (GLTP)-fold now further advances understanding.

The inner workings of an ancient biological clock.

Circadian clocks evolved in diverse organisms as an adaptation to the daily swings in ambient light and temperature that derive from Earth's rotation. These timing systems, based on intracellular molecular oscillations, synchronize organisms' behavior and physiology with the 24-h environmental rhythm. The cyanobacterial clock serves as a special model for understanding circadian rhythms because it can be fully reconstituted in vitro. This review summarizes recent advances that leverage new biochemical, biophysical, and mathematical approaches to shed light on the molecular mechanisms of cyanobacterial Kai proteins that support the clock, and their homologues in other bacteria. Many questions remain in circadian biology, and the tools developed for the Kai system will bring us closer to the answers.

Metamorphic protein folding as evolutionary adaptation.

Metamorphic proteins switch reversibly between multiple distinct, stable structures, often with different functions. It was previously hypothesized that metamorphic proteins arose as intermediates in the evolution of a new fold - rare and transient exceptions to the 'one sequence, one fold' paradigm. However, as described herein, mounting evidence suggests that metamorphic folding is an adaptive feature, preserved and optimized over evolutionary time as exemplified by the NusG family and the chemokine XCL1. Analysis of extant protein families and resurrected protein ancestors demonstrates that large regions of sequence space are compatible with metamorphic folding. As a category that enhances biological fitness, metamorphic proteins are likely to employ fold switching to perform important biological functions and may be more common than previously thought.

Critical Role of the UBL Domain in Stimulating the E3 Ubiquitin Ligase Activity of UHRF1 toward Chromatin.

The RING E3 ubiquitin ligase UHRF1 controls DNA methylation through its ability to target the maintenance DNA methyltransferase DNMT1 to newly replicated chromatin. DNMT1 recruitment relies on ubiquitylation of histone H3 by UHRF1; however, how UHRF1 deposits ubiquitin onto the histone is unknown. Here, we demonstrate that the ubiquitin-like domain (UBL) of UHRF1 is essential for RING-mediated H3 ubiquitylation. Using chemical crosslinking and mass spectrometry, biochemical assays, and recombinant chromatin substrates, we show that the UBL participates in structural rearrangements of UHRF1 upon binding to chromatin and the E2 ubiquitin conjugating enzyme UbcH5a/UBE2D1. Similar to ubiquitin, the UBL exerts its effects through a hydrophobic patch that contacts a regulatory surface on the "backside" of the E2 to stabilize the E2-E3-chromatin complex. Our analysis of the enzymatic mechanism of UHRF1 uncovers an unexpected function of the UBL domain and defines a new role for this domain in DNMT1-dependent inheritance of DNA methylation.

Reversible switching between two common protein folds in a designed system using only temperature.

Naturally occurring metamorphic proteins have the ability to interconvert from one folded state to another through either a limited set of mutations or by way of a change in the local environment. Here, we show in a designed system that it is possible to switch reversibly between two of the most common monomeric folds employing only temperature changes. We demonstrate that a latent 3α state can be unmasked from an α/ß-plait topology with a single V90T amino acid substitution, populating both forms simultaneously. The equilibrium between these two states exhibits temperature dependence, such that the 3α state is predominant (>90%) at 5 °C, while the α/ß-plait fold is the major species (>90%) at 30 °C. We describe the structure and dynamics of these topologies, how mutational changes affect the temperature dependence, and the energetics and kinetics of interconversion. Additionally, we demonstrate how ligand-binding function can be tightly regulated by large amplitude changes in protein structure over a relatively narrow temperature range that is relevant to biology. The 3α/αß switch thus represents a potentially useful approach for designing proteins that alter their fold topologies in response to environmental triggers. It may also serve as a model for computational studies of temperature-dependent protein stability and fold switching.

Cross-sections of doubly curved sheets as confined elastica.

Although thin films are typically manufactured in planar sheets or rolls, they are often forced into three-dimensional (3D) shapes, producing a plethora of structures across multiple length scales. To understand this complex response, previous studies have either focused on the overall gross shape or the small-scale buckling that decorates it. A geometric model, which considers the sheet as inextensible yet free to compress, has been shown to capture the gross shape of the sheet. However, the precise meaning of such predictions, and how the gross shape constrains the fine features, remains unclear. Here, we study a thin-membraned balloon as a prototypical system that involves a doubly curved gross shape with large amplitude undulations. By probing its side profiles and horizontal cross-sections, we discover that the mean behavior of the film is the physical observable that is predicted by the geometric model, even when the buckled structures atop it are large. We then propose a minimal model for the horizontal cross-sections of the balloon, as independent elastic filaments subjected to an effective pinning potential around the mean shape. Despite the simplicity of our model, it reproduces a broad range of phenomena seen in the experiments, from how the morphology changes with pressure to the detailed shape of the wrinkles and folds. Our results establish a route to combine global and local features consistently over an enclosed surface, which could aid the design of inflatable structures, or provide insight into biological patterns.

EMBER multidimensional spectral microscopy enables quantitative determination of disease- and cell-specific amyloid strains.

In neurodegenerative diseases, proteins fold into amyloid structures with distinct conformations (strains) that are characteristic of different diseases. However, there is a need to rapidly identify amyloid conformations in situ. Here, we use machine learning on the full information available in fluorescent excitation/emission spectra of amyloid-binding dyes to identify six distinct different conformational strains in vitro, as well as amyloid-ß (Aß) deposits in different transgenic mouse models. Our EMBER (excitation multiplexed bright emission recording) imaging method rapidly identifies conformational differences in Aß and tau deposits from Down syndrome, sporadic and familial Alzheimer's disease human brain slices. EMBER has in situ identified distinct conformational strains of tau inclusions in astrocytes, oligodendrocytes, and neurons from Pick's disease. In future studies, EMBER should enable high-throughput measurements of the fidelity of strain transmission in cellular and animal neurodegenerative diseases models, time course of amyloid strain propagation, and identification of pathogenic versus benign strains.

De novo protein fold design through sequence-independent fragment assembly simulations.

De novo protein design generally consists of two steps, including structure and sequence design. Many protein design studies have focused on sequence design with scaffolds adapted from native structures in the PDB, which renders novel areas of protein structure and function space unexplored. We developed FoldDesign to create novel protein folds from specific secondary structure (SS) assignments through sequence-independent replica-exchange Monte Carlo (REMC) simulations. The method was tested on 354 non-redundant topologies, where FoldDesign consistently created stable structural folds, while recapitulating on average 87.7% of the SS elements. Meanwhile, the FoldDesign scaffolds had well-formed structures with buried residues and solvent-exposed areas closely matching their native counterparts. Despite the high fidelity to the input SS restraints and local structural characteristics of native proteins, a large portion of the designed scaffolds possessed global folds completely different from natural proteins in the PDB, highlighting the ability of FoldDesign to explore novel areas of protein fold space. Detailed data analyses revealed that the major contributions to the successful structure design lay in the optimal energy force field, which contains a balanced set of SS packing terms, and REMC simulations, which were coupled with multiple auxiliary movements to efficiently search the conformational space. Additionally, the ability to recognize and assemble uncommon super-SS geometries, rather than the unique arrangement of common SS motifs, was the key to generating novel folds. These results demonstrate a strong potential to explore both structural and functional spaces through computational design simulations that natural proteins have not reached through evolution.

Tau filaments from amyotrophic lateral sclerosis/parkinsonism-dementia complex adopt the CTE fold.

The amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC) of the island of Guam and the Kii peninsula of Japan is a fatal neurodegenerative disease of unknown cause that is characterized by the presence of abundant filamentous tau inclusions in brains and spinal cords. Here, we used electron cryo-microscopy to determine the structures of tau filaments from the cerebral cortex of three cases of ALS/PDC from Guam and eight cases from Kii, as well as from the spinal cord of two of the Guam cases. Tau filaments had the chronic traumatic encephalopathy (CTE) fold, with variable amounts of Type I and Type II filaments. Paired helical tau filaments were also found in three Kii cases and tau filaments with the corticobasal degeneration fold in one Kii case. We identified a new Type III CTE tau filament, where protofilaments pack against each other in an antiparallel fashion. ALS/PDC is the third known tauopathy with CTE-type filaments and abundant tau inclusions in cortical layers II/III, the others being CTE and subacute sclerosing panencephalitis. Because these tauopathies are believed to have environmental causes, our findings support the hypothesis that ALS/PDC is caused by exogenous factors.

Identification of a conserved α-helical domain at the N terminus of human DNA methyltransferase 1.

In vertebrates, DNA methyltransferase 1 (DNMT1) contributes to preserving DNA methylation patterns, ensuring the stability and heritability of epigenetic marks important for gene expression regulation and the maintenance of cellular identity. Previous structural studies have elucidated the catalytic mechanism of DNMT1 and its specific recognition of hemimethylated DNA. Here, using solution nuclear magnetic resonance spectroscopy and small-angle X-ray scattering, we demonstrate that the N-terminal region of human DNMT1, while flexible, encompasses a conserved globular domain with a novel α-helical bundle-like fold. This work expands our understanding of the structure and dynamics of DNMT1 and provides a structural framework for future functional studies in relation with this new domain.

Characterization of alternate encounter assemblies of SARS-CoV-2 main protease.

The assembly of two monomeric constructs spanning segments 1-199 (MPro1-199) and 10-306 (MPro10-306) of SARS-CoV-2 main protease (MPro) was examined to assess the existence of a transient heterodimer intermediate in the N-terminal autoprocessing pathway of MPro model precursor. Together, they form a heterodimer population accompanied by a 13-fold increase in catalytic activity. Addition of inhibitor GC373 to the proteins increases the activity further by ∼7-fold with a 1:1 complex and higher order assemblies approaching 1:2 and 2:2 molecules of MPro1-199 and MPro10-306 detectable by analytical ultracentrifugation and native mass estimation by light scattering. Assemblies larger than a heterodimer (1:1) are discussed in terms of alternate pathways of domain III association, either through switching the location of helix 201 to 214 onto a second helical domain of MPro10-306 and vice versa or direct interdomain III contacts like that of the native dimer, based on known structures and AlphaFold 3 prediction, respectively. At a constant concentration of MPro1-199 with molar excess of GC373, the rate of substrate hydrolysis displays first order dependency on the MPro10-306 concentration and vice versa. An equimolar composition of the two proteins with excess GC373 exhibits half-maximal activity at ∼6 µM MPro1-199. Catalytic activity arises primarily from MPro1-199 and is dependent on the interface interactions involving the N-finger residues 1 to 9 of MPro1-199 and E290 of MPro10-306. Importantly, our results confirm that a single N-finger region with its associated intersubunit contacts is sufficient to form a heterodimeric MPro intermediate with enhanced catalytic activity.

A Conserved Ribosomal Protein Has Entirely Dissimilar Structures in Different Organisms.

Ribosomes from different species can markedly differ in their composition by including dozens of ribosomal proteins that are unique to specific lineages but absent in others. However, it remains unknown how ribosomes acquire new proteins throughout evolution. Here, to help answer this question, we describe the evolution of the ribosomal protein msL1/msL2 that was recently found in ribosomes from the parasitic microorganism clade, microsporidia. We show that this protein has a conserved location in the ribosome but entirely dissimilar structures in different organisms: in each of the analyzed species, msL1/msL2 exhibits an altered secondary structure, an inverted orientation of the N-termini and C-termini on the ribosomal binding surface, and a completely transformed 3D fold. We then show that this fold switching is likely caused by changes in the ribosomal msL1/msL2-binding site, specifically, by variations in rRNA. These observations allow us to infer an evolutionary scenario in which a small, positively charged, de novo-born unfolded protein was first captured by rRNA to become part of the ribosome and subsequently underwent complete fold switching to optimize its binding to its evolving ribosomal binding site. Overall, our work provides a striking example of how a protein can switch its fold in the context of a complex biological assembly, while retaining its specificity for its molecular partner. This finding will help us better understand the origin and evolution of new protein components of complex molecular assemblies-thereby enhancing our ability to engineer biological molecules, identify protein homologs, and peer into the history of life on Earth.

Genetic analysis of translation initiation in bacteria: An initiator tRNA-centric view.

Translation of messenger RNA (mRNA) in bacteria occurs in the steps of initiation, elongation, termination, and ribosome recycling. The initiation step comprises multiple stages and uses a special transfer RNA (tRNA) called initiator tRNA (i-tRNA), which is first aminoacylated and then formylated using methionine and N10 -formyl-tetrahydrofolate (N10 -fTHF), respectively. Both methionine and N10 -fTHF are produced via one-carbon metabolism, linking translation initiation with active cellular metabolism. The fidelity of i-tRNA binding to the ribosomal peptidyl-site (P-site) is attributed to the structural features in its acceptor stem, and the highly conserved three consecutive G-C base pairs (3GC pairs) in the anticodon stem. The acceptor stem region is important in formylation of the amino acid attached to i-tRNA and in its initial binding to the P-site. And, the 3GC pairs are crucial in transiting the i-tRNA through various stages of initiation. We utilized the feature of 3GC pairs to investigate the nuanced layers of scrutiny that ensure fidelity of translation initiation through i-tRNA abundance and its interactions with the components of the translation apparatus. We discuss the importance of i-tRNA in the final stages of ribosome maturation, as also the roles of the Shine-Dalgarno sequence, ribosome heterogeneity, initiation factors, ribosome recycling factor, and coevolution of the translation apparatus in orchestrating a delicate balance between the fidelity of initiation and/or its leakiness to generate proteome plasticity in cells to confer growth fitness advantages in response to the dynamic nutritional states.

DNMT1 inhibition by pUG-fold quadruplex RNA.

Aberrant DNA methylation is one of the earliest hallmarks of cancer. DNMT1 is responsible for methylating newly replicated DNA, but the precise regulation of DNMT1 to ensure faithful DNA methylation remains poorly understood. A link between RNA and chromatin-associated proteins has recently emerged, and several studies have shown that DNMT1 can be regulated by a variety of RNAs. In this study, we have confirmed that human DNMT1 indeed interacts with multiple RNAs, including its own nuclear mRNA. Unexpectedly, we found that DNMT1 exhibits a strong and specific affinity for GU-rich RNAs that form a pUG-fold, a noncanonical G-quadruplex. We find that pUG-fold-capable RNAs inhibit DNMT1 activity by inhibiting binding of hemimethylated DNA, and we additionally provide evidence for multiple RNA binding modes with DNMT1. Together, our data indicate that a human chromatin-associated protein binds to and is regulated by pUG-fold RNA.

Multiple domains of the integral KREPA3 protein are critical for the structure and precise functions of RNA editing catalytic complexes in Trypanosoma brucei.

The gRNA directed U-insertion and deletion editing of mitochondrial mRNAs that is essential in different life-cycle stages for the protozoan parasite Trypanosoma brucei is performed by three similar multiprotein catalytic complexes (CCs) that contain the requisite enzymes. These CCs also contain a common set of eight proteins that have no apparent direct catalytic function, including six that have an OB-fold domain. We show here that one of these OB-fold proteins, KREPA3 (A3), has structural homology to other editing proteins, is essential for editing, and is multifunctional. We investigated A3 function by analyzing the effects of single amino acid loss of function mutations, most of which were identified by screening bloodstream form (BF) parasites for loss of growth following random mutagenesis. Mutations in the zinc fingers (ZFs), an intrinsically disordered region (IDR), and several within or near the carboxy-terminal OB-fold domain variably impacted CC structural integrity and editing. Some mutations resulted in almost complete loss of CCs and its proteins and editing, whereas others retained CCs but had aberrant editing. All but a mutation which is near the OB-fold affected growth and editing in BF but not procyclic form (PF) parasites. These data indicate that multiple positions within A3 have essential functions that contribute to the structural integrity of CCs, the precision of editing and the developmental differences in editing between BF and PF stages.

The evolutionary novelty of insect defensins: from bacterial killing to toxin neutralization.

Insect host defense comprises two complementary dimensions, microbial killing-mediated resistance and microbial toxin neutralization-mediated resilience, both jointly providing protection against pathogen infections. Insect defensins are a class of effectors of innate immunity primarily responsible for resistance to Gram-positive bacteria. Here, we report a newly originated gene from an ancestral defensin via genetic deletion following gene duplication in Drosophila virilis, which confers an enhanced resilience to Gram-positive bacterial infection. This gene encodes an 18-mer arginine-rich peptide (termed DvirARP) with differences from its parent gene in its pattern of expression, structure and function. DvirARP specifically expresses in D. virilis female adults with a constitutive manner. It adopts a novel fold with a 310 helix and a two CXC motif-containing loop stabilized by two disulfide bridges. DvirARP exhibits no activity on the majority of microorganisms tested and only a weak activity against two Gram-positive bacteria. DvirARP knockout flies are viable and have no obvious defect in reproductivity but they are more susceptible to the DvirARP-resistant Staphylococcus aureus infection than the wild type files, which can be attributable to its ability in neutralization of the S. aureus secreted toxins. Phylogenetic distribution analysis reveals that DvirARP is restrictedly present in the Drosophila subgenus, but independent deletion variations also occur in defensins from the Sophophora subgenus, in support of the evolvability of this class of immune effectors. Our work illustrates for the first time how a duplicate resistance-mediated gene evolves an ability to increase the resilience of a subset of Drosophila species against bacterial infection.

Fluid protein fold space and its implications.

Fold-switching proteins, which remodel their secondary and tertiary structures in response to cellular stimuli, suggest a new view of protein fold space. For decades, experimental evidence has indicated that protein fold space is discrete: dissimilar folds are encoded by dissimilar amino acid sequences. Challenging this assumption, fold-switching proteins interconnect discrete groups of dissimilar protein folds, making protein fold space fluid. Three recent observations support the concept of fluid fold space: (1) some amino acid sequences interconvert between folds with distinct secondary structures, (2) some naturally occurring sequences have switched folds by stepwise mutation, and (3) fold switching is evolutionarily selected and likely confers advantage. These observations indicate that minor amino acid sequence modifications can transform protein structure and function. Consequently, proteomic structural and functional diversity may be expanded by alternative splicing, small nucleotide polymorphisms, post-translational modifications, and modified translation rates.

Creative destruction: New protein folds from old.

Mechanisms of emergence and divergence of protein folds pose central questions in biological sciences. Incremental mutation and stepwise adaptation explain relationships between topologically similar protein folds. However, the universe of folds is diverse and riotous, suggesting more potent and creative forces are at play. Sequence and structure similarity are observed between distinct folds, indicating that proteins with distinct folds may share common ancestry. We found evidence of common ancestry between three distinct ß-barrel folds: Scr kinase family homology (SH3), oligonucleotide/oligosaccharide-binding (OB), and cradle loop barrel (CLB). The data suggest a mechanism of fold evolution that interconverts SH3, OB, and CLB. This mechanism, which we call creative destruction, can be generalized to explain many examples of fold evolution including circular permutation. In creative destruction, an open reading frame duplicates or otherwise merges with another to produce a fused polypeptide. A merger forces two ancestral domains into a new sequence and spatial context. The fused polypeptide can explore folding landscapes that are inaccessible to either of the independent ancestral domains. However, the folding landscapes of the fused polypeptide are not fully independent of those of the ancestral domains. Creative destruction is thus partially conservative; a daughter fold inherits some motifs from ancestral folds. After merger and refolding, adaptive processes such as mutation and loss of extraneous segments optimize the new daughter fold. This model has application in disease states characterized by genetic instability. Fused proteins observed in cancer cells are likely to experience remodeled folding landscapes and realize altered folds, conferring new or altered functions.