The crystal structure of UDP-galactose 4'-epimerase from the protozoan parasite Trypanosoma brucei in complex with the cofactor NAD(+) and a fragment of the substrates, UDP, has been determined at 2.0 A resolution (1 A = 0.1 nm). This enzyme, recently proven to be essential for this pathogenic parasite, shares 33% sequence identity with the corresponding enzyme in the human host. Structural comparisons indicate that many of the protein-ligand interactions are conserved between the two enzymes. However, in the UDP-binding pocket there is a non-conservative substitution from Gly237 in the human enzyme to Cys266 in the T. brucei enzyme. Such a significant difference could be exploited by the structure-based design of selective inhibitors using the structure of the trypanosomatid enzyme as a template.

Prion-like domains (PLDs) are low complexity sequences found in RNA binding proteins associated with the neurodegenerative disorder amyotrophic lateral sclerosis. Recently, PLDs have been implicated in mediating gene regulation via liquid-phase transitions that drive ribonucleoprotein granule assembly. In this paper, we report many PLDs in proteins associated with paraspeckles, subnuclear bodies that form around long noncoding RNA. We mapped the interactome network of paraspeckle proteins, finding enrichment of PLDs. We show that one protein, RBM14, connects key paraspeckle subcomplexes via interactions mediated by its PLD. We further show that the RBM14 PLD, as well as the PLD of another essential paraspeckle protein, FUS, is required to rescue paraspeckle formation in cells in which their endogenous counterpart has been knocked down. Similar to FUS, the RBM14 PLD also forms hydrogels with amyloid-like properties. These results suggest a role for PLD-mediated liquid-phase transitions in paraspeckle formation, highlighting this nuclear body as an excellent model system for understanding the perturbation of such processes in neurodegeneration.

KARRIKIN INSENSITIVE 2 (KAI2) is an α/β hydrolase involved in seed germination and seedling development. It is essential for plant responses to karrikins, a class of butenolide compounds derived from burnt plant material that are structurally similar to strigolactone plant hormones. The mechanistic basis for the function of KAI2 in plant development remains unclear. We have determined the crystal structure of Arabidopsis thaliana KAI2 in space groups P2(1) 2(1) 2(1) (a =63.57 Å, b =66.26 Å, c =78.25 Å) and P2(1) (a =50.20 Å, b =56.04 Å, c =52.43 Å, β =116.12°) to 1.55 and 2.11 Å respectively. The catalytic residues are positioned within a large hydrophobic pocket similar to that of DAD2, a protein required for strigolactone response in Petunia hybrida. KAI2 possesses a second solvent-accessible pocket, adjacent to the active site cavity, which offers the possibility of allosteric regulation. The structure of KAI2 is consistent with its designation as a serine hydrolase, as well as previous data implicating the protein in karrikin and strigolactone signalling.

Homologous recombinational repair is an essential mechanism for repair of double-strand breaks in DNA. Recombinases of the RecA-fold family play a crucial role in this process, forming filaments that utilize ATP to mediate their interactions with single- and double-stranded DNA. The recombinase molecules present in the archaea (RadA) and eukaryota (Rad51) are more closely related to each other than to their bacterial counterpart (RecA) and, as a result, RadA makes a suitable model for the eukaryotic system. The crystal structure of Sulfolobus solfataricus RadA has been solved to a resolution of 3.2 A in the absence of nucleotide analogues or DNA, revealing a narrow filamentous assembly with three molecules per helical turn. As observed in other RecA-family recombinases, each RadA molecule in the filament is linked to its neighbour via interactions of a short beta-strand with the neighbouring ATPase domain. However, despite apparent flexibility between domains, comparison with other structures indicates conservation of a number of key interactions that introduce rigidity to the system, allowing allosteric control of the filament by interaction with ATP. Additional analysis reveals that the interaction specificity of the five human Rad51 paralogues can be predicted using a simple model based on the RadA structure.

Replication of human cytomegalovirus (CMV) requires the expression of the viral mitochondria-localized inhibitor of apoptosis (vMIA). vMIA inhibits apoptosis by recruiting Bax to mitochondria, resulting in its neutralization. We show that vMIA decreases cell size, reduces actin polymerization, and induces cell rounding. As compared with vMIA-expressing CMV, vMIA-deficient CMV, which replicates in fibroblasts expressing the adenoviral apoptosis suppressor E1B19K, induces less cytopathic effects. These vMIA effects can be separated from its cell death-inhibitory function because vMIA modulates cellular morphology in Bax-deficient cells. Expression of vMIA coincided with a reduction in the cellular adenosine triphosphate (ATP) level. vMIA selectively inhibited one component of the ATP synthasome, namely, the mitochondrial phosphate carrier. Exposure of cells to inhibitors of oxidative phosphorylation produced similar effects, such as an ATP level reduced by 30%, smaller cell size, and deficient actin polymerization. Similarly, knockdown of the phosphate carrier reduced cell size. Our data suggest that the cytopathic effect of CMV can be explained by vMIA effects on mitochondrial bioenergetics.

A class of long noncoding RNAs (lncRNAs) has architectural functions in nuclear body construction; however, specific RNA domains dictating their architectural functions remain uninvestigated. Here, we identified the domains of the architectural NEAT1 lncRNA that construct paraspeckles. Systematic deletion of NEAT1 portions using CRISPR/Cas9 in haploid cells revealed modular domains of NEAT1 important for RNA stability, isoform switching, and paraspeckle assembly. The middle domain, containing functionally redundant subdomains, was responsible for paraspeckle assembly. Artificial tethering of the NONO protein to a NEAT1_2 mutant lacking the functional subdomains rescued paraspeckle assembly, and this required the NOPS dimerization domain of NONO. Paraspeckles exhibit phase-separated properties including susceptibility to 1,6-hexanediol treatment. RNA fragments of the NEAT1_2 subdomains preferentially bound NONO/SFPQ, leading to phase-separated aggregates in vitro. Thus, we demonstrate that the enrichment of NONO dimers on the redundant NEAT1_2 subdomains initiates construction of phase-separated paraspeckles, providing mechanistic insights into lncRNA-based nuclear body formation.

Constrained, cyclic peptides encoded by plant genes represent a new generation of drug leads. Evolution has repeatedly recruited the Cys-protease asparaginyl endopeptidase (AEP) to perform their head-to-tail ligation. These macrocyclization reactions use the substrates amino terminus instead of water to deacylate, so a peptide bond is formed. How solvent-exposed plant AEPs macrocyclize is poorly understood. Here we present the crystal structure of an active plant AEP from the common sunflower, Helianthus annuus. The active site contained electron density for a tetrahedral intermediate with partial occupancy that predicted a binding mode for peptide macrocyclization. By substituting catalytic residues we could alter the ratio of cyclic to acyclic products. Moreover, we showed AEPs from other species lacking cyclic peptides can perform macrocyclization under favorable pH conditions. This structural characterization of AEP presents a logical framework for engineering superior enzymes that generate macrocyclic peptide drug leads.

Human pancreatic islet amyloid polypeptide (hIAPP) and beta amyloid (Aβ) can accumulate in Type 2 diabetes (T2D) and Alzheimer's disease (AD) brains and evidence suggests that interaction between the two amyloidogenic proteins can lead to the formation of heterocomplex aggregates. However, the structure and consequences of the formation of these complexes remains to be determined. The main objective of this study was to characterise the different types and morphology of Aβ-hIAPP heterocomplexes and determine if formation of such complexes exacerbate neurotoxicity. We demonstrate that hIAPP promotes Aβ oligomerization and formation of small oligomer and large aggregate heterocomplexes. Co-oligomerized Aβ42-hIAPP mixtures displayed distinct amorphous structures and a 3-fold increase in neuronal cell death as compared to Aβ and hIAPP alone. However, in contrast to hIAPP, non-amyloidogenic rat amylin (rIAPP) reduced oligomer Aβ-mediated neuronal cell death. rIAPP exhibited reductions in Aβ induced neuronal cell death that was independent of its ability to interact with Aβ and form heterocomplexes; suggesting mediation by other pathways. Our findings reveal distinct effects of IAPP peptides in modulating Aβ aggregation and toxicity and provide new insight into the potential pathogenic effects of Aβ-IAPP hetero-oligomerization and development of IAPP based therapies for AD and T2D.

Despite their location at the cell surface, several receptor tyrosine kinases (RTK) are also found in the nucleus, as either intracellular domains or full length proteins. However, their potential nuclear functions remain poorly understood. Here we find that a fraction of full length Colony Stimulating Factor-1 Receptor (CSF-1R), an RTK involved in monocyte/macrophage generation, migrates to the nucleus upon CSF-1 stimulation in human primary monocytes. Chromatin-immunoprecipitation identifies the preferential recruitment of CSF-1R to intergenic regions, where it co-localizes with H3K4me1 and interacts with the transcription factor EGR1. When monocytes are differentiated into macrophages with CSF-1, CSF-1R is redirected to transcription starting sites, colocalizes with H3K4me3, and interacts with ELK and YY1 transcription factors. CSF-1R expression and chromatin recruitment is modulated by small molecule CSF-1R inhibitors and altered in monocytes from chronic myelomonocytic leukemia patients. Unraveling this dynamic non-canonical CSF-1R function suggests new avenues to explore the poorly understood functions of this receptor and its ligands.

Paraspeckles are subnuclear structures that form around nuclear paraspeckle assembly transcript 1 (NEAT1) long noncoding RNA (lncRNA). Recently, paraspeckles were shown to be functional nuclear bodies involved in stress responses and the development of specific organs. Paraspeckle formation is initiated by transcription of the NEAT1 chromosomal locus and proceeds in conjunction with NEAT1 lncRNA biogenesis and a subsequent assembly step involving >40 paraspeckle proteins (PSPs). In this study, subunits of SWItch/Sucrose NonFermentable (SWI/SNF) chromatin-remodeling complexes were identified as paraspeckle components that interact with PSPs and NEAT1 lncRNA. EM observations revealed that SWI/SNF complexes were enriched in paraspeckle subdomains depleted of chromatin. Knockdown of SWI/SNF components resulted in paraspeckle disintegration, but mutation of the ATPase domain of the catalytic subunit BRG1 did not affect paraspeckle integrity, indicating that the essential role of SWI/SNF complexes in paraspeckle formation does not require their canonical activity. Knockdown of SWI/SNF complexes barely affected the levels of known essential paraspeckle components, but markedly diminished the interactions between essential PSPs, suggesting that SWI/SNF complexes facilitate organization of the PSP interaction network required for intact paraspeckle assembly. The interactions between SWI/SNF components and essential PSPs were maintained in NEAT1-depleted cells, suggesting that SWI/SNF complexes not only facilitate interactions between PSPs, but also recruit PSPs during paraspeckle assembly. SWI/SNF complexes were also required for Satellite III lncRNA-dependent formation of nuclear stress bodies under heat-shock conditions. Our data suggest the existence of a common mechanism underlying the formation of lncRNA-dependent nuclear body architectures in mammalian cells.

SFPQ, (a.k.a. PSF), is a human tumor suppressor protein that regulates many important functions in the cell nucleus including coordination of long non-coding RNA molecules into nuclear bodies. Here we describe the first crystal structures of Splicing Factor Proline and Glutamine Rich (SFPQ), revealing structural similarity to the related PSPC1/NONO heterodimer and a strikingly extended structure (over 265 Å long) formed by an unusual anti-parallel coiled-coil that results in an infinite linear polymer of SFPQ dimers within the crystals. Small-angle X-ray scattering and transmission electron microscopy experiments show that polymerization is reversible in solution and can be templated by DNA. We demonstrate that the ability to polymerize is essential for the cellular functions of SFPQ: disruptive mutation of the coiled-coil interaction motif results in SFPQ mislocalization, reduced formation of nuclear bodies, abrogated molecular interactions and deficient transcriptional regulation. The coiled-coil interaction motif thus provides a molecular explanation for the functional aggregation of SFPQ that directs its role in regulating many aspects of cellular nucleic acid metabolism.

Aberrant KRAS signaling is a driver of many cancers and yet remains an elusive target for drug therapy. The nuclease hypersensitive element of the KRAS promoter has been reported to form secondary DNA structures called G-quadruplexes (G4s) which may play important roles in regulating KRAS expression, and has spurred interest in structural elucidation studies of the KRAS G-quadruplexes. Here, we report the first high-resolution crystal structure (1.6 Å) of a KRAS G-quadruplex as a 5'-head-to-head dimer with extensive poly-A π-stacking interactions observed across the dimer. Molecular dynamics simulations confirmed that the poly-A π-stacking interactions are also maintained in the G4 monomers. Docking and molecular dynamics simulations with two G4 ligands that display high stabilization of the KRAS G4 indicated the poly-A loop was a binding site for these ligands in addition to the 5'-G-tetrad. Given sequence and structural variability in the loop regions provide the opportunity for small-molecule targeting of specific G4s, we envisage this high-resolution crystal structure for the KRAS G-quadruplex will aid in the rational design of ligands to selectively target KRAS.

In Staphylococcus aureus, most multiresistance plasmids lack conjugation or mobilization genes for horizontal transfer. However, most are mobilizable due to carriage of origin-of-transfer (oriT) sequences mimicking those of conjugative plasmids related to pWBG749. pWBG749-family plasmids have diverged to carry five distinct oriT subtypes and non-conjugative plasmids have been identified that contain mimics of each. The relaxasome accessory factor SmpO, encoded by each conjugative plasmid, determines specificity for its cognate oriT. Here we characterized the binding of SmpO proteins to each oriT. SmpO proteins predominantly formed tetramers in solution and bound 5'-GNNNNC-3' sites within each oriT. Four of the five SmpO proteins specifically bound their cognate oriT. An F7K substitution in pWBG749 SmpO switched oriT-binding specificity in vitro. In vivo, the F7K substitution reduced but did not abolish self-transfer of pWBG749. Notably, the substitution broadened the oriT subtypes that were mobilized. Thus, this substitution represents a potential evolutionary intermediate with promiscuous DNA-binding specificity that could facilitate a switch between oriT specificities. Phylogenetic analysis suggests pWBG749-family plasmids have switched oriT specificity more than once during evolution. We hypothesize the convergent evolution of oriT specificity in distinct branches of the pWBG749-family phylogeny reflects indirect selection pressure to mobilize plasmids carrying non-cognate oriT-mimics.

Despite the great advances in autophagy research in the last years, the specific functions of the four mammalian Atg4 proteases (ATG4A-D) remain unclear. In yeast, Atg4 mediates both Atg8 proteolytic activation, and its delipidation. However, it is not clear how these two roles are distributed along the members of the ATG4 family of proteases. We show that these two functions are preferentially carried out by distinct ATG4 proteases, being ATG4D the main delipidating enzyme. In mammalian cells, ATG4D loss results in accumulation of membrane-bound forms of mATG8s, increased cellular autophagosome number and reduced autophagosome average size. In mice, ATG4D loss leads to cerebellar neurodegeneration and impaired motor coordination caused by alterations in trafficking/clustering of GABAA receptors. We also show that human gene variants of ATG4D associated with neurodegeneration are not able to fully restore ATG4D deficiency, highlighting the neuroprotective role of ATG4D in mammals.

RNA-binding proteins of the DBHS (Drosophila Behavior Human Splicing) family, NONO, SFPQ, and PSPC1 have numerous roles in genome stability and transcriptional and posttranscriptional regulation. Critical to DBHS activity is their recruitment to distinct subnuclear locations, for example, paraspeckle condensates, where DBHS proteins bind to the long noncoding RNA NEAT1 in the first essential step in paraspeckle formation. To carry out their diverse roles, DBHS proteins form homodimers and heterodimers, but how this dimerization influences DBHS localization and function is unknown. Here, we present an inducible GFP-NONO stable cell line and use it for live-cell 3D-structured illumination microscopy, revealing paraspeckles with dynamic, twisted elongated structures. Using siRNA knockdowns, we show these labeled paraspeckles consist of GFP-NONO/endogenous SFPQ dimers and that GFP-NONO localization to paraspeckles depends on endogenous SFPQ. Using purified proteins, we confirm that partner swapping between NONO and SFPQ occurs readily in vitro. Crystallographic analysis of the NONO-SFPQ heterodimer reveals conformational differences to the other DBHS dimer structures, which may contribute to partner preference, RNA specificity, and subnuclear localization. Thus overall, our study suggests heterodimer partner availability is crucial for NONO subnuclear distribution and helps explain the complexity of both DBHS protein and paraspeckle dynamics through imaging and structural approaches.

The recombination directionality factors from Mesorhizobium spp. (RdfS) are involved in regulating the excision and transfer of integrative and conjugative elements. Here, solution small-angle X-ray scattering, and crystallization and preliminary structure solution of RdfS from Mesorhizobium japonicum R7A are presented. RdfS crystallizes in space group P212121, with evidence of eightfold rotational crystallographic/noncrystallographic symmetry. Initial structure determination by molecular replacement using ab initio models yielded a partial model (three molecules), which was completed after manual inspection revealed unmodelled electron density. The finalized crystal structure of RdfS reveals a head-to-tail polymer forming left-handed superhelices with large solvent channels. Additionally, RdfS has significant disorder in the C-terminal region of the protein, which is supported by the solution scattering data and the crystal structure. The steps taken to finalize structure determination, as well as the scattering and crystallographic characteristics of RdfS, are discussed.

Coxiella burnetii is a Gram-negative intracellular pathogen that causes the debilitating disease Q fever, which affects both animals and humans. The only available human vaccine, Q-Vax, is effective but has a high risk of severe adverse reactions, limiting its use as a countermeasure to contain outbreaks. Therefore, it is essential to identify new drug targets to treat this infection. Macrophage infectivity potentiator (Mip) proteins catalyse the folding of proline-containing proteins through their peptidyl prolyl cis-trans isomerase (PPIase) activity and have been shown to play an important role in the virulence of several pathogenic bacteria. To date the role of the Mip protein in C. burnetii pathogenesis has not been investigated. This study demonstrates that CbMip is likely to be an essential protein in C. burnetii. The pipecolic acid derived compounds, SF235 and AN296, which have shown utility in targeting other Mip proteins from pathogenic bacteria, demonstrate inhibitory activities against CbMip. These compounds were found to significantly inhibit intracellular replication of C. burnetii in both HeLa and THP-1 cells. Furthermore, SF235 and AN296 were also found to exhibit antibiotic properties against both the virulent (Phase I) and avirulent (Phase II) forms of C. burnetii Nine Mile Strain in axenic culture. Comparative proteomics, in the presence of AN296, revealed alterations in stress responses with H2O2 sensitivity assays validating that Mip inhibition increases the sensitivity of C. burnetii to oxidative stress. In addition, SF235 and AN296 were effective in vivo and significantly improved the survival of Galleria mellonella infected with C. burnetii. These results suggest that unlike in other bacteria, Mip in C. burnetii is required for replication and that the development of more potent inhibitors against CbMip is warranted and offer potential as novel therapeutics against this pathogen.

Horizontal gene transfer is tightly regulated in bacteria. Often only a fraction of cells become donors even when regulation of horizontal transfer is coordinated at the cell population level by quorum sensing. Here, we reveal the widespread 'domain of unknown function' DUF2285 represents an 'extended-turn' variant of the helix-turn-helix domain that participates in both transcriptional activation and antiactivation to initiate or inhibit horizontal gene transfer. Transfer of the integrative and conjugative element ICEMlSymR7A is controlled by the DUF2285-containing transcriptional activator FseA. One side of the DUF2285 domain of FseA has a positively charged surface which is required for DNA binding, while the opposite side makes critical interdomain contacts with the N-terminal FseA DUF6499 domain. The QseM protein is an antiactivator of FseA and is composed of a DUF2285 domain with a negative surface charge. While QseM lacks the DUF6499 domain, it can bind the FseA DUF6499 domain and prevent transcriptional activation by FseA. DUF2285-domain proteins are encoded on mobile elements throughout the proteobacteria, suggesting regulation of gene transfer by DUF2285 domains is a widespread phenomenon. These findings provide a striking example of how antagonistic domain paralogues have evolved to provide robust molecular control over the initiation of horizontal gene transfer.

The pentatricopeptide repeat (PPR) protein family is a large family of RNA-binding proteins that is characterized by tandem arrays of a degenerate 35-amino-acid motif which form an α-solenoid structure. PPR proteins influence the editing, splicing, translation and stability of specific RNAs in mitochondria and chloroplasts ZEA MAYS: PPR10 is amongst the best studied PPR proteins, where sequence-specific binding to two RNA transcripts, ATPH: and PSAJ, HAS BEEN DEMONSTRATED TO FOLLOW: a recognition code where the identity of two amino acids per repeat determines the base-specificity. A recently solved ZmPPR10: PSAJ: complex crystal structure suggested a homodimeric complex with considerably fewer sequence-specific protein-RNA contacts than inferred PREVIOUSLY: Here we describe the solution structure of the ZmPPR10: ATPH: complex using size-exclusion chromatography-coupled synchrotron small-angle X-ray scattering (SEC-SY-SAXS). Our results support prior evidence that PPR10 binds RNA as a monomer, and that it does so in a manner that is commensurate with a canonical and predictable RNA-binding mode across much of the RNA-protein interface.

The majority of peroxisomal matrix proteins destined for translocation into the peroxisomal lumen are recognised via a C-terminal Peroxisomal Target Signal type 1 by the cycling receptor Pex5p. The only structure to date of Pex5p in complex with a cargo protein is that of the C-terminal cargo-binding domain of the receptor with sterol carrier protein 2, a small, model peroxisomal protein. In this study, we have tested the contribution of a second, ancillary receptor-cargo binding site, which was found in addition to the characterised Peroxisomal Target Signal type 1.