Current Research in Structural Biology

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, can use host derived lipids as carbon and energy source for survival. Mammalian cell entry (Mce) associated membrane (Mam) proteins are important for the stability of lipid importing Mce complexes. Mtb has five homologs of Mam proteins referred as orphaned Mam (OmamA-E) proteins. A recent study suggested that OmamC (Rv1363c) is essential for the storage and utilization of lipids under starvation in Mtb. To understand the structure and interactions of OmamC, we generated a truncated soluble variant of OmamC (OmamC129-261). Here, we report on the challenges encountered during the crystallization and structure determination of OmamC129-261 and the strategies applied to overcome them. Despite the AlphaFold2 predicted model proving an initial molecular replacement solution, experimental phasing was necessary to determine the structure of OmamC129-261. Heat treatment of protein prior to crystallization setup removed partially unfolded protein present and played a critical role in enhancing the reproducibility and diffraction quality of OmamC129-261 crystals. Although reported earlier, it is not a widely used method. It is worth to try this method, especially, when faced with poor reproducibility and diffraction of crystals.

The three conventional isoforms of the Ras G-protein (H-, K-, N-Ras) function as molecular on-off switches that regulate a wide array of signaling pathways, including the Ras-PI3K-PIP3-PDK1-AKT pathway that is central to innate immunity and normal cell growth, and is dysregulated in many disease states. Activation of the pathway by Ras requires adequate Ras-PI3K binding affinity. Here we focus on the interface of known structure in the H-Ras:PI3K{gamma} co-complex essential to multiple pathways including directed pseudopod growth in leukocyte chemotaxis. At this interface 10 H-Ras residues, all 100% conserved between the H-, K- and N-Ras isomers, contact the Ras binding domain of PI3K{gamma} (PI3K{gamma}RBD). To investigate the degree to which the native H-Ras:PI3K{gamma}RBD interface is optimized by evolution for maximal binding affinity, 8 interfacial Ras mutations selected from the COSMIC database and the literature were introduced at the contact positions. All 8 Ras mutations were observed to alter the H-Ras:PI3K{gamma}RBD binding affinity, with 4 mutations yielding significant affinity increases and 4 yielding significant affinity decreases. These findings indicate that the native H-Ras:PI3K{gamma}RBD interface provides intermediate, rather than maximal, binding affinity. Such intermediate affinity is consistent with the substantial binding plasticity of the conserved H-, N-, K-Ras effector docking surface, which has evolved to bind a diverse array of effectors. Furthermore, the findings provide evidence that COSMIC-linked mutations at the H-Ras:PI3K{gamma}RBD interface frequently generate affinity increases as well as decreases, with potential implications for molecular mechanisms of disease and for tool development in cell biology.

We present an ultrahigh-field magic-angle spinning (MAS) solid-state NMR (ssNMR) study to characterize intact nontuberculous mycobacteria (NTM) at the molecular level. Hydrated and dried whole-cell Mycobacterium abscessus samples were investigated by combining conventional high-field ssNMR at 750 MHz with ultrahigh-field ssNMR at 1.2 GHz and ultrafast MAS at 100 kHz. To improve sensitivity and enable multidimensional experiments, 13C/15N isotope labeling was performed after growth in synthetic cystic fibrosis medium (SCFM). We utilized 1D 13C and multidimensional 1H-13C and 13C-13C ssNMR experiments to characterize the chemical composition, dynamics, and structural organization of the M. abscessus cell envelope. The isotope-labeling efficiency was found to be non-uniform across different molecular classes, with high incorporation into polysaccharides and lower incorporation into lipid and peptide-associated signals. INEPT- and CP-based experiments selectively probed flexible and rigid fractions of the samples, revealing substantial differences in linewidth, dynamics, and sensitivity between hydrated and dried preparations. Conventional 750 MHz experiments provided high-resolution multidimensional spectra and enabled identification of distinct chemical environments associated with peptidoglycan, arabinogalactan, mycolic acids, lipids, and peptide-associated components. Ultrahigh-field ssNMR at 1.2 GHz combined with ultrafast MAS and 1H detection substantially improved spectral resolution and sensitivity in particular per mg of sample amount, allowing detection of weak and previously unresolved resonances, including polysaccharide and possible nucleic-acid-associated signals. Together, these results demonstrate that ultra-high-field and ultrafast-MAS ssNMR enables detailed characterization of intact NTM cell envelopes under near-native conditions and provides a framework for future molecular investigations of antimicrobial interactions.

Dopaminergic neurons in the substantia nigra depend on iron for dopamine synthesis but are vulnerable to iron-induced oxidative stress. Many of these neurons synthesize neuromelanin, an iron-chelating pigment that accumulates across the lifespan and makes them vulnerable in Parkinsons disease. It remains unclear whether their selective vulnerability arises from neuromelanin overload or from the release of toxic labile iron from the oversaturated pigment. We quantified iron and neuromelanin at the single-cell level across the lifespan of chimpanzees, a species closely resembling humans in pigment and iron accumulation. Combining quantitative MRI, X-ray fluorescence imaging, and microscopic colorimetry, we found that the iron-to-neuromelanin ratio remains stable with age across large neuronal populations. Chemical equilibrium modeling of the iron binding in neuromelanin indicated that cytosolic labile iron concentrations remain low throughout adulthood. We have found no evidence for neuromelanin saturation or increased iron-mediated toxicity with age. This finding challenges the hypothesis that neuromelanin saturation drives age-related dopaminergic vulnerability. The presented method provides a quantitative framework for studying iron homeostasis in these neurons.

A proteomic analysis of Ixodes scapularis nymph saliva identified 252 proteins, including six tubular lipid-binding proteins (TULIPs). Comparing nymphs fed on mice that were uninfected or infected with Borrelia burgdorferi, twelve salivary proteins showed significant differences in the amounts detected, including XP_040079658.2, which we refer to as TULIP2. Considering the known immunity-related functions of some TULIPs, we expressed and purified TULIP2 from Escherichia coli and analyzed its interaction with B. burgdorferi lipids. The purification of TULIP2 from E. coli presented many obstacles, due to insolubility, which is consistent with previous reports from studies of other TULIP family members. The binding results showed specificity for B. burgdorferi lipids, with evidence for cholesteryl {beta}-galactoside as a major binding target. Molecular modeling of TULIP2 did not show any strong lipid binding sites. We used molecular dynamics simulation of TULIP2 to explore its conformational landscape by thermal unfolding. The earliest unfolding intermediate opened a hydrophobic pocket to which cholesteryl {beta}-galactoside was predicted to bind strongly. We propose that a specific lipid bilayer interaction with TULIP2 triggers the opening of the ligand-binding site.

Serine-aspartate repeat-containing protein D (SdrD) is a Staphylococcus aureus cell wall-anchored, calcium-binding adhesin member of the MSCRAMM Sdr subfamily that may contribute to bacterial adhesion and virulence. S. aureus is the most common cause of periprosthetic joint infection (PJI). Population-level distribution and sequence diversity of SdrD among clinical PJI isolates have not been systematically characterized, and the SdrD binding mechanism is still not well understood. To address these gaps, sdrD alleles were queried across 156 newly sequenced PJI isolates and compared to publicly available S. aureus genomes, and nucleotide- and protein-level phylogenies of the sdrCDE locus constructed. The SdrD crystal structure from S. aureus JH1 was determined, with solution small-angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations, and assessment of conformational changes with calcium depletion. Three dominant sdrD subtypes were defined, associating with USA300, JH1, and TCH60; the JH1 sdrD subtype was predominant among PJI isolates. Structural studies showed that the conformation of individual domains and interdomain organization of the multidomain SdrD have limited flexibility in solution, and that the calcium-binding B domain retains its core fold under conditions of calcium depletion. Together, the findings presented support functional diversification among Sdr family members in mediating host attachment and inform a re-evaluation of the ligand-binding mechanism previously proposed for SdrD. AUTHOR SUMMARYStaphylococcus aureus is the leading cause of infections that develop around joint implants (periprosthetic joint infection, PJI). This bacterium has a large arsenal of surface proteins that allow it to stick to human tissues and implanted devices. This work focused on one such protein, SdrD, which has been linked to implant-associated infections but the structure and diversity of which among patients with PJI had not been well characterized. The genetic sequences of SdrD were analyzed across thousands of bacterial genomes, including those from patients with PJI. Distinct genetic variants of the protein were found, one of which was particularly common with PJI. The three-dimensional structure of SdrD was determined at atomic resolution and solution small-angle X-ray scattering (SAXS) and molecular dynamics used to study how it moves and responds to changes in its environment. Contrary to what was previously described, SdrD was shown to be relatively rigid. These findings change how SdrDs mechanism of action should be considered, potentially informing design strategies to block bacterial attachment before infection takes hold.

Bovine tuberculosis (TB), caused by Mycobacterium bovis, has become a global concern over the last two decades. Bovine TB primarily affects cattle, but other domestic livestock are also affected and it is more common in less developed and developing countries. The significant loss of livestock leads to trade restrictions and economic crises. Zoonotic potential of bovine TB raises health concerns for the public. Currently, no effective treatment is available and animal slaughtering is usually undertaken to reduce the burden of it in the environment. Antibiotic therapy can be used on animals living in captivity, but it is not reliable for herd or free-grazing animals. The BCG vaccine is another option available for treating the disease, but it shows limited efficacy in cattle. The prevention of bovine TB is a long-term goal that can only be accomplished by developing a more effective vaccine than BCG and designing new drugs. In this research, we propose therapeutic drug targets and vaccine for treating bovine TB. The conceptual framework for vaccine developed in this study uses a number of bioinformatics approaches to identify potential vaccine candidates and construct an in-silico epitope-based vaccine. Our holistic framework identified potential therapeutic candidates by directly analysing the proteome of TB bacterial strains. Specifically, we performed a comparative proteomic analysis of 11 Mycobacterium bovis strains to cover the diversity and identify conserved proteins among those strains for developing the bovine TB vaccine. An extensive reverse vaccinology and immunoinformatics analysis provided 26 highly immunogenic, non-toxic and non-allergenic epitopes (CTL epitopes-8, HTL epitopes-2 and B-cell epitopes-16) for Mycobacterium bovis required for three-dimensional structure construction of TB vaccine. The constructed epitope-based vaccine showed a potent interaction inside the host, thus generating efficient cell-mediated and humoral immune responses. Next, a framework based on a novel subtractive proteomic approach was developed for identifying bovine TB drug targets. We performed this approach on the 11 Mycobacterium bovis strains and identified nine drug targets that are conserved, essential, antigenic and have unique metabolic pathways in Mycobacterium bovis. These drug targets could further help investigate therapeutic drugs for the treatment of bovine TB. Several bioinformatics prediction tools were used together to ensure checks and balances, aiming to reduce the chance of errors and provide accurate results. The vaccine and drug targets developed in this study can be tested experimentally with confidence for further validation as therapeutics with the potential to eradicate bovine TB globally. The strategies implemented in the study are generic and can be used for other zoonotic infectious diseases. This study would be a game changer in the field of bovine tuberculosis treatment.

WD40 domains share a widespread {beta}-propeller fold, and often act as versatile scaffold proteins. Despite their central role in organizing dynamic cellular complexes, the molecular and structural mechanisms of many WD40 proteins remain poorly understood. Among them, DCAF7, an ubiquitously expressed and essential gene in human, also encodes a highly conserved WD40 protein in eukaryotic organisms. It is known to interact with multiple and functionnally diverse partners to coordinates cellular activity of several protein kinases as well as transcriptional regulators, thereby modulating key cellular processes such as cell growth, differentiation, and transcriptional regulation. However, the precise mode of action of DCAF7 is unknown and its important divergence in sequence from better characterize WD40 prevent information transfer by similarity. Structural interactomic can reveal how protein-protein interactions (PPIs) occur within an organism and are essential for understanding biological functions and developing new therapeutic strategies. Using SLiMAn2, AlphaFold2/3 and PSSMsearch, we identified a conserved -helical short linear motif (SLiM) in several well known DCAF7 partners that binds to the top surface of its {beta}-propeller. This motif was subsequently used to generate a regular expression, to identify potential new direct binders across the DCAF7 meta-interactome and the human proteome. Domain-domain interactions were also predicted for some other partners. Finally, modeling of oligomeric complexes with such new hits reveals the structural basis of DCAF7 scaffolding, with links to neurodevelopmental disorders such as autism.

The INO80 chromatin remodeling complex plays a central role in DNA repair, transcription, and replication. Yet, a comprehensive understanding of its structural organization remains incomplete due to the dynamic nature of several of its subunits and the sharing of several subunits with related remodeling complexes. Here, we report a computational model of the three-dimensional structure of the S. cerevisiae INO80 complex using an integrative approach that combines experimental crosslinking mass spectrometry, molecular docking, and molecular dynamics simulations. Our results reveal the spatial and dynamical organization of key modules--ARP8, ARP5, NHP10, and RVB1/2--within the intact complex. The resulting structural model agrees with crosslinking constraints, highlighting the architecture of the previously uncharacterized NHP10 module. This module, including the C-terminal region of the Ino80 scaffolding protein, has remained elusive due to its intrinsic flexibility and lack of high-resolution structural data. To facilitate this integrative modeling workflow and make it broadly accessible, we presented INTEGRATOR: (INTEGRAtive TempOral and stRuctural Analysis of protein modules), a versatile workflow package designed as a tool to elucidate the structure and dynamics of large, flexible macromolecular assemblies using well-established softwares. Our findings demonstrate the power of integrative modeling in resolving the role of the highly disordered NPH10 module in recruiting other dynamic modules into INO80 large protein assemblies and offer a generalizable framework for determining the architecture of similarly complex and heterogeneous molecular machines. This work carries broad implications for understanding the structural basis of chromatin regulation in microbial organisms and the implications for the dysregulation in diseases such as cancer.

Species of Bacillus bacteria including Bacillus safensis and Bacillus subtilis are finding increasing uses in biotechnology and bioremediation, thanks in part to their metabolic robustness. Malate dehydrogenase (MDH) is at the heart of central metabolism and thus a better understanding of Bacillus MDH proteins could aid in the optimization of these applications. MDH of Bacillus spp. belong to the lactate dehydrogenase (LDH)-like class of MDHs, otherwise known as the MDH3 class. Despite wide prevalence in nature among prokaryotes and archaea, this typically homotetrameric class is understudied compared to the MDH1 and MDH2 classes found in eukaryotes. We therefore recombinantly expressed and purified MDH proteins from two societally relevant Bacillus spp.-B. safensis and B. subtilis-and characterized them biophysically (via Size Exclusion Chromatography-Small Angle X-ray Scattering (SEC-SAXS) and Differential Scanning Fluorimetry (DSF)) and enzymatically (via spectroscopic activity assays). As expected based on their high sequence identity, the two MDH orthologs had similar properties in most regards, including a tetrameric structure and high susceptibility to substrate inhibition. However, we uncovered differences in conditional thermal stability, in addition to subtle differences in enzymatic activity that offer insight into the workings of LDH-like MDH. Summary statementMalate dehydrogenase (MDH) is a fundamental metabolic enzyme, from microbes to mammals, yet comparably little is known about microbial MDH, especially MDH of the tetrameric MDH3 class. We compare the biophysical and enzymatic properties of two such enzymes from the societally relevant bacterial species Bacillus subtilis and Bacillus safensis, offering useful insight with potential biotechnological implications.

The Notch and Wnt/{beta}-catenin signaling pathways are essential regulators for cell-fate decisions, cellular patterning, and tissue homeostasis. Multiple studies point to their orchestrated role during development, but the molecular mechanism of the protein-protein crosstalk is largely unknown. Here, after screening effects of Wnt/{beta}-catenin component loss on NOTCH1 protein, we identify Casein Kinase 1 (CK1) as a positive regulator of NOTCH1 activity in vitro and in vivo. We demonstrate that CK1 associates with NOTCH1 and that its kinase activity is required to sustain Notch-driven transcription. Using UltraID proximity-assay, we revealed that CK1 is required for the NOTCH1 interactivity with transporter proteins, and MAML1 both prior and after ligand-induced activation. Combining structural modelling, NMR, and mass spectrometry, we identified Serine 1970 (S1970) as a previously unreported residue within the Notch1 Intracellular Domain (N1ICD) essential for its signaling competence. Our modeling predicts that the phosphorylation of S1970 facilitates an intra-domain conformational switch with R1937 and R1962 residues altering the assembly of the N1ICD-MAML1-RBPJk transcriptional complex. Finally, we demonstrate the biological significance of N1ICD S1970 in vivo using Xenopus laevis axis-duplication rescue assay. Our results establish CK1 as a key positive mediator of the Notch receptor transcriptional activity.

Conformational plasticity of RNAs plays important roles in recognizing RNA-binding proteins, and is often modulated by their binding partners. Here, we investigate RNA conformational preferences in a non-redundant dataset of 263 protein-RNA complexes to characterize the structural landscape associated with protein recognition. RNA dinucleotide segments are analyzed using seven backbone torsion angles ({delta}1, {varepsilon}1, {zeta}1, 2, {beta}2, {gamma}2, and {delta}2), two glycosidic torsion angles ({chi}1 and {chi}2) and the pseudo-torsion angle . Focusing on dinucleotide steps present in both interface and non-interface regions, we performed density-based clustering using selected backbone torsion angles to identify recurrent conformational states. We identify 28 distinct RNA dinucleotide conformers containing at least ten members each. Among these, eight conformers represent previously unreported nucleotide conformers (NtCs), including the transitional and the non-canonical states AB06, AB07, BB21, BB22, OP32, OP33, IC08 and IC09. Several of these conformers are preferentially enriched at protein-binding interfaces, suggesting their involvement in local conformational adaptation during protein-RNA recognition. The newly identified conformers span transitional A-B geometries, distorted B-like states, open conformations and compact intercalated structures, highlighting the remarkable structural plasticity of RNA in ribonucleoprotein complexes. Overall, this study expands the current understanding of RNA conformational space and provides a refined RNA dinucleotide conformer library for protein-RNA complexes. These findings will facilitate the identification of novel RNA structural motifs and improved RNA structural modeling, docking protein-RNA complexes and deep learning-based prediction frameworks for describing RNA tertiary structures.

The Rho family of small GTPases plays a critical role in regulating actin cytoskeleton dynamics during endocytic processes in E. histolytica, including phagocytosis, pinocytosis, and trogocytosis. These proteins act as molecular switches, transitioning between inactive GDP-bound and active GTP-bound states, with guanine nucleotide exchange factors (GEFs) catalyzing this transition. Among the GEFs, EhFP10--a FYVE-domain-containing protein harbouring Dbl homology (DH) and pleckstrin homology (PH) domain was observed in phagocytosis along with seven functionally characterized Rho GTPases (EhRho1, EhRho2, EhRho4, EhRho5, EhRho6, EhRho8, and EhRho13). To study the specificity of FP10, a combination of GEF activity, binding affinity, and molecular dynamics simulations was used to characterize the interactions between EhFP10 and seven Rho GTPases systematically. The results revealed EhRho2 as the most specific and high-affinity interactor of EhFP10, with the highest nucleotide exchange rate and lowest dissociation constant (KD = 0.58 {micro}M). Structural modeling, sequence alignment, and interaction mapping further demonstrated that EhRho2 retains critical contact residues--such as Glu33, Arg4, and Leu69--that are variably absent in other isoforms, correlating with decreased GEF responsiveness. Molecular dynamics simulations and cross-correlation analyses supported the presence of a stable and coordinated interaction interface in the EhFP10-EhRho2 complex, distinguishing it from less active complexes. These findings indicate a highly selective GEF-GTPase module in E. histolytica, analogous to those in higher eukaryotes. The results uncover a potential regulatory mechanism specific to pathogenic amoebae and present EhFP10-EhRho2 as a novel therapeutic target for disrupting cytoskeleton-mediated processes crucial to virulence.

O-fucosylation plays an essential role in controlling protein folding, secretion and protein-protein interactions within the extracellular space. Recently, we identified a new form of protein O-fucosylation occurring on the N-terminal Elastin Microfibril Interaction (EMI) domain of several secreted proteins, mediated by two previously uncharacterized protein O-fucosyltransferases, POFUT3 (FUT10) and POFUT4 (FUT11). As all POFUT enzymes (POFUT1-4) are highly specific for the three-dimensional (3D) structure of their substrate protein domains, we postulated that structural homologues of these domains in other proteins may also be O-fucosylated. Here, we employed iterative protein structural homology searches as a novel strategy for identifying EMI-like domains that may serve as potential substrates for POFUT3/4. We discovered that microfibrillar-associated protein 2 and 5 (MFAP2/MFAP5) contain EMI-like domains and are O-fucosylated at high stoichiometry in human tissues. Unexpectedly, we showed that only POFUT3 is both necessary and sufficient for MFAP2/MFAP5 O-fucosylation, despite POFUT4 also having strong protein-protein interactions with MFAP2/MFAP5. Finally, we determined that O-fucosylation of MFAP2/MFAP5 is required for their efficient secretion, similar to other EMI domain-containing proteins. Together, these data demonstrate the power of sensitive structural homology analysis in identifying new enzyme-substrate relationships and protein-protein interactions.

Centrosome dysfunction is linked to developmental disorders affecting brain and body size, including microcephaly and primordial dwarfism. However, the cellular mechanisms underlying these rare conditions remain poorly understood. In this study, we investigate a rare variant of the centrosome-associated protein Pericentrin, which was discovered in a single family with Majewski/microcephalic osteodysplastic primordial dwarfism type II (MOPD II). Unlike the majority of pathogenic PCNT variants that cause severe protein truncation, the p.Lys3154del variant ({Delta}K3154) involves a single amino acid deletion in the proteins only conserved functional domain, providing a unique opportunity to explore PCNT function in MOPD II. To model PCNT{Delta}K3154, we examined the effects of Drosophila Pericentrin-like protein (PLP) carrying an orthologous deletion (Plp{Delta}R). Our results show that plp{Delta}R animals exhibit smaller tissues that recapitulate MOPD II phenotypes. Behavioral assays revealed defects in climbing and mechanosensation, suggesting impaired sensory cilia function. We also found that Plp{Delta}R cells exhibit accelerated mitosis, increased apoptosis, and reduced pericentriolar material recruitment. In silico structural modeling, yeast two-hybrid, and co-immunoprecipitation experiments show that Plp{Delta}R produces a protein that disrupts PLP dimerization and PLP interaction with Asterless, another centrosome protein. Overall, modeling the human MOPD II patient variant PCNT{Delta}K3154 in Drosophila reveals how a single amino acid deletion affects biological processes from the molecular level to the organismal level. Our work offers new insights into the defective cellular mechanisms underlying MOPD II in patients with the PCNT{Delta}K3154 variant, potentially linking the etiology of the disease in these individuals to the loss of a single protein-protein interaction.

Structure Integration with Function, Taxonomy and Sequences (SIFTS) provides residue-level mappings between UniProt Knowledgebase sequences and Protein Data Bank structures and has historically been generated through internal Protein Data Bank in Europe (PDBe) pipelines. Here, PDBe-SIFTS is presented as a fully open-source, locally deployable implementation of this mapping framework. The pipeline combines fast, scalable sequence search using MMseqs2, an improved bounded scoring scheme for ranking candidate mappings, and residue-level mapping refinement based on backbone connectivity. PDBe-SIFTS is distributed as a Python package with command-line tools for 1) building a sequence search database, 2) identifying the best sequence-structure match, 3) one-to-one mapping at the residue level, and 4) generating SIFTS annotations in PDBx/mmCIF format. Benchmarking on the complete Protein Data Bank archive showed that MMseqs2 reduced archive-scale UniProtKB searches from hours with BLASTP to minutes, approximately 22-36 times faster, while curated mappings were recovered at top rank in 93.1% of cases. The remaining discrepancies mainly involved biologically ambiguous cases such as highly conserved proteins, chimeric constructs, or closely related orthologs. These results show that PDBe-SIFTS enables fast mapping, improving structural coherence in residue-level alignments while delivering the most up-to-date and accurate mappings, comparable to expert curation. Tool: https://github.com/PDBeurope/SIFTS Quick start notebook with example: https://github.com/PDBeurope/SIFTS/tree/master/notebooks Broader audience statementMatching protein sequences to their three-dimensional structures, and mapping annotations across both, is essential for understanding protein function, interactions, and molecular mechanisms. This integrated view enables richer interpretation of biological data and underpins advances in drug discovery, disease research, and protein engineering. PDBe-SIFTS provides an open and functional framework for structure-sequence mapping, allowing researchers and databases to run, inspect, and extend these mappings locally, while benefiting from faster searches, transparent scoring, and structurally informed residue-level alignments. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/721839v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@5e6ea6org.highwire.dtl.DTLVardef@1b2754dorg.highwire.dtl.DTLVardef@1334f9forg.highwire.dtl.DTLVardef@1b083a1_HPS_FORMAT_FIGEXP M_FIG C_FIG

Synucleinopathies are a group of neurodegenerative diseases characterized by the presence of misfolded -synuclein inclusions which cause progressive disease by spreading throughout the brain in a prion-like manner. Throughout the neurodegenerative disease field, the ability of a single protein to give rise to multiple distinct clinical disorders is explained by the strain hypothesis, or the idea that the misfolded protein conformation determines the resulting disease. This was initially shown using transmission studies in cell lines and mouse models; more recently cryo-electron microscopy (cryo-EM) validated this idea by identifying distinct -synuclein filament folds in brain tissues from patients with Parkinsons disease, multiple system atrophy (MSA), and juvenile-onset synucleinopathy. However, very little is known about the -synuclein filament structures that form in animal models of these disorders, and thus their relevance to human disease and suitability as models for therapeutic development remains a question. Here we report the first atomic resolution cryo-EM structures of -synuclein fibrils from an MSA patient sample before and after transmission to a transgenic mouse model of disease. Our findings indicate that while distinct adaptations occur during fibril replication in the mouse host, key structural facets are maintained, validating the merits of this transmission model for supporting preclinical research on MSA.

The nuclear receptor superfamily is comprised of ligand-regulated transcription factors that contain an intrinsically disordered domain at the amino-terminal end, known as the N-terminal domain (NTD). While this poorly conserved domain is known to possess ligand-independent activation function (AF-1), few NTD functions are conserved between nuclear receptors (NRs). Identified roles in other receptors include androgen receptor (AR), estrogen receptor (ER) and mineralocorticoid receptor (MR). Here, we aim to define the function of the NTD of the farnesoid X receptor (FXR), a crucial regulator of lipid and bile acid metabolism. We show that the NTD engages in interdomain contact with other FXR domains. We also observe that the NTD interacts directly with coregulator proteins. Using mutagenesis, mammalian two-hybrid assays and molecular dynamics simulations, we identify and validate a novel SXXLF motif in the NTD which mediates interactions with both coregulators and the ligand binding domain. Mutation of the motif induces large changes in conformational and allosteric coupling in FXR. Our study identifies a new nuclear receptor-interacting motif that modulates the transcriptional activity of FXR. Graphical AbstractFXR-NTD regulates transcriptional activity through interdomain communication with the LBD and is also involved in co-activator recruitment. The SENLF motif is the first defined functional element within the FXR-NTD and mediates both NTD-LBD interaction and selective co-activator engagements to drive NTD-mediated transcriptional activity. O_FIG O_LINKSMALLFIG WIDTH=135 HEIGHT=200 SRC="FIGDIR/small/724725v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@5a37aorg.highwire.dtl.DTLVardef@2fa9e1org.highwire.dtl.DTLVardef@13a19daorg.highwire.dtl.DTLVardef@1775ed2_HPS_FORMAT_FIGEXP M_FIG C_FIG

Citrullination is a charge-modifying post-translational modification whereby proteinogenic arginine is converted to the non-coded amino acid citrulline by calcium-activated protein arginine deiminases (PADs; EC 3.5.3.15). The five known PAD enzymes in humans (PADs 1, 2, 3, 4, and 6) are differentially expressed and have distinct targets, including histones. While some PAD histone citrullination sites are known, a comprehensive investigation of all histone tail arginines targeted by catalytically active PADs 1-4 is lacking. Here, we sought to identify PAD citrullination sites in histone tails, both within histone peptides and in reconstituted nucleosomes. Toward this objective, we utilized a real-time 1H-15N NMR spectroscopy-based assay. By monitoring both arginine and citrulline backbone amide peak intensities over time, we identified sites of citrullination in 15N-labeled histone tails within peptides and reconstituted nucleosome core particles. We found that PADs 1, 2, and 4 citrullinate all directly observable histone tail arginines to varying degrees. This is distinct from PAD3, which only moderately citrullinates H2A and H4 arginine residues and does not modify H3 tail arginines. Together, these data suggest a level of histone arginine specificity by each PAD. Furthermore, histone tail citrullination is altered within nucleosomes compared to isolated peptides, which we interpret to reflect changes in conformation and accessibility. We speculate that citrullination increases nucleosomal histone tail dynamics, with implications for crosstalk between sites of histone citrullination and other important sites of regulation by PTMs (including lysines) within and between tails.

Mycobacterium tuberculosis fatty acid synthase I (Mtb FAS-I) is a multifunctional hexameric complex essential for fatty acid (FA) synthesis. The need of a hexameric structure for activity of the complex in Mtb remains elusive. Here, we model a conformation of the functionally active complex with acyl carrier protein (ACP) at ketoacyl synthase (KS). Our model reveals a crucial cross-dome dependence in the mechanism of FA synthesis at the condensation step. Using molecular dynamics simulation, we identify key ACP and KS residues that mantain persistent interactions. ACPs phosphopantetheine (PPT) arm adopts several conformations while accessing KSs catalytic pocket, including two distinct conformations that correlate with volumes of ACP and KS pockets. A PHE residue, reported as a gatekeeper of the KS pocket in other species, also shows open and closed orientations in our simulation. Our results provide crucial insights that are essential for a mechanistic undersanding of the Mtb FAS-I complex.