Nuclear Import Receptor Inhibits Phase Separation of FUS through Binding to Multiple Sites.

Liquid-liquid phase separation (LLPS) is believed to underlie formation of biomolecular condensates, cellular compartments that concentrate macromolecules without surrounding membranes. Physical mechanisms that control condensate formation/dissolution are poorly understood. The RNA-binding protein fused in sarcoma (FUS) undergoes LLPS in vitro and associates with condensates in cells. We show that the importin karyopherin-ß2/transportin-1 inhibits LLPS of FUS. This activity depends on tight binding of karyopherin-ß2 to the C-terminal proline-tyrosine nuclear localization signal (PY-NLS) of FUS. Nuclear magnetic resonance (NMR) analyses reveal weak interactions of karyopherin-ß2 with sequence elements and structural domains distributed throughout the entirety of FUS. Biochemical analyses demonstrate that most of these same regions also contribute to LLPS of FUS. The data lead to a model where high-affinity binding of karyopherin-ß2 to the FUS PY-NLS tethers the proteins together, allowing multiple, distributed weak intermolecular contacts to disrupt FUS self-association, blocking LLPS. Karyopherin-ß2 may act analogously to control condensates in diverse cellular contexts.

The apiosyltransferase celery UGT94AX1 catalyzes the biosynthesis of the flavone glycoside apiin.

Apiose is a unique branched-chain pentose found in plant glycosides and a key component of the cell wall polysaccharide pectin and other specialized metabolites. More than 1,200 plant-specialized metabolites contain apiose residues, represented by apiin, a distinctive flavone glycoside found in celery (Apium graveolens) and parsley (Petroselinum crispum) in the family Apiaceae. The physiological functions of apiin remain obscure, partly due to our lack of knowledge on apiosyltransferase during apiin biosynthesis. Here, we identified UGT94AX1 as an A. graveolens apiosyltransferase (AgApiT) responsible for catalyzing the last sugar modification step in apiin biosynthesis. AgApiT showed strict substrate specificity for the sugar donor, UDP-apiose, and moderate specificity for acceptor substrates, thereby producing various apiose-containing flavone glycosides in celery. Homology modeling of AgApiT with UDP-apiose, followed by site-directed mutagenesis experiments, identified unique Ile139, Phe140, and Leu356 residues in AgApiT, which are seemingly crucial for the recognition of UDP-apiose in the sugar donor pocket. Sequence comparison and molecular phylogenetic analysis of celery glycosyltransferases suggested that AgApiT is the sole apiosyltransferase-encoding gene in the celery genome. Identification of this plant apiosyltransferase gene will enhance our understanding of the physioecological functions of apiose and apiose-containing compounds.

Mechanism underlying liquid-to-solid phase transition in fused in sarcoma liquid droplets.

The RNA-binding protein fused in sarcoma (FUS) forms ribonucleoprotein granules via liquid-liquid phase separation (LLPS) in the cytoplasm. The phase separation of FUS accelerates aberrant liquid-solid phase separation and leads to the onset of familial amyotrophic lateral sclerosis (ALS). We previously found that FUS forms two types of liquid condensates in equilibrium, specifically LP-LLPS (i.e., normal type) and HP-LLPS (i.e., aberrant type), each with different partial molar volumes. However, it is unclear how liquid condensates are converted to the pathogenic solid phase. Here, we report a mechanism underlying the aberrant liquid-to-solid phase transition of FUS liquid condensates and the inhibition of this transition with small molecules. We found that the liquid condensate formed via HP-LLPS had greatly reduced dynamics, which is a common feature of aged wild-type FUS droplets and the droplet-like assembly of the ALS patient-type FUS variant. The longer FUS remained on the HP-LLPS, the harder it was to transform it into a mixed state (i.e., one-phase). These results indicate that liquid-to-solid phase transition, namely the aging of droplets, is accelerated with HP-LLPS. Interestingly, arginine suppressed the aging of droplets and HP-LLPS formation more strongly than LP-LLPS formation. These data indicate that the formation of HP-LLPS via the one-phase state or LP-LLPS is a pathway leading to irreversible solid aggregates. Dopamine and pyrocatechol also suppressed HP-LLPS formation. Our data highlight the potential of HP-LLPS to be used as a therapeutic target and arginine as a plausible drug candidate for ALS-causing FUS.

Pressure-Jump Kinetics of Liquid-Liquid Phase Separation: Comparison of Two Different Condensed Phases of the RNA-Binding Protein, Fused in Sarcoma.

The RNA-binding protein fused in sarcoma (FUS) undergoes liquid-liquid phase separation (LLPS) both in vivo and in vitro. Self-assembled liquid droplets of FUS transform into reversible hydrogels and into more irreversible and toxic aggregates. Although LLPS can be a precursor of irreversible aggregates, a generic method to study kinetics of the formation of LLPS has not been developed. Here, we demonstrated the pressure-jump kinetics of phase transition between the 1-phase state and FUS-LLPS states observed at low pressure (<2 kbar, LP-LLPS) and high pressure (>2 kbar, HP-LLPS) using high-pressure UV/vis spectroscopy. Absorbance (turbidity) changes were reproduced repeatedly using pressure cycles. The Johnson-Mehl-Avrami-Kolmogorov theory was used to understand droplet formation occurring via nucleation and growth. The Avrami exponent n, representing the dimensionality of growing droplets, and the reaction rate constant k were calculated. The HP-LLPS formation rate was ∼2-fold slower than that of LP-LLPS. The Avrami exponent obtained for both LLPS states could be explained by diffusion-limited growth. Nucleation and growth rates decreased during LP-LLPS formation (n = 0.51), and the nucleation rate decreased with a constant growth rate in HP-LLPS formation (n = 1.4). The HP-LLPS vanishing rate was ∼20-fold slower than that of LP-LLPS. This difference in vanishing rates indicates a stronger intermolecular interaction in HP-LLPS than in LP-LLPS, which might promote transformation into irreversible aggregates in the droplets. Further, direct transition from HP-LLPS to LP-LLPS was observed. This indicates that interconversion between LP-LLPS and HP-LLPS occurs in equilibrium. Formation of reversible liquid droplets, followed by phase transition into another liquid phase, could thus be part of the physiological maturation process of FUS-LLPS.

Expression, purification and crystallization of TGW6, which limits grain weight in rice.

Rice is the staple food for over half the world's population. Genes associated with rice yield include THOUSAND GRAIN WEIGHT 6 (TGW6), which negatively regulates the number of endosperm cells as well as grain weight. The 1-bp deletion allele of tgw6 cloned from the Indian landrace rice cultivar Kasalath, which has lost function, enhances both grain size and yield. TGW6 has been utilized as a target for breeding and genome editing to increase the yield of rice. In the present study, we describe an improved heterologous expression system of TGW6 in Escherichia coli to enable purification of the recombinant protein. The best expression was achieved using codon optimized TGW6 with a 30 amino acid truncation at the N-terminus (Δ30TGW6) in the Rosetta-gami 2(DE3) host strain. Furthermore, we found that calcium ions were critical for the purification of stable Δ30TGW6. Crystals of Δ30TGW6 were obtained using the sitting-drop vapor-diffusion method at 283 K, which diffracted X-rays to at least 2.6 Å resolution. Herein, we established an efficient procedure for the production and purification of TGW6 in sufficient quantities for structural and functional studies. Detailed information concerning the molecular mechanism of TGW6 will enable the design of more efficient ways to control the activity of the enzyme.

Dynamic Assembly/Disassembly of Staphylococcus aureus FtsZ Visualized by High-Speed Atomic Force Microscopy.

FtsZ is a key protein in bacterial cell division and is assembled into filamentous architectures. FtsZ filaments are thought to regulate bacterial cell division and have been investigated using many types of imaging techniques such as atomic force microscopy (AFM), but the time scale of the method was too long to trace the filament formation process. Development of high-speed AFM enables us to achieve sub-second time resolution and visualize the formation and dissociation process of FtsZ filaments. The analysis of the growth and dissociation rates of the C-terminal truncated FtsZ (FtsZt) filaments indicate the net growth and dissociation of FtsZt filaments in the growth and dissociation conditions, respectively. We also analyzed the curvatures of the full-length FtsZ (FtsZf) and FtsZt filaments, and the comparative analysis indicated the straight-shape preference of the FtsZt filaments than those of FtsZf. These findings provide insights into the fundamental dynamic behavior of FtsZ protofilaments and bacterial cell division.

COX6A2 variants cause a muscle-specific cytochrome c oxidase deficiency.

OBJECTIVE: Cytochrome c oxidase (COX) deficiency is a major mitochondrial respiratory chain defect that has vast genetic and phenotypic heterogeneity. This study aims to identify novel causative genes of COX deficiency with only striated muscle-specific symptoms. METHODS: Whole exome sequencing was performed in 2 unrelated individuals who were diagnosed with congenital myopathy and presented COX deficiency in muscle pathology. We assessed the COX6A2 variants using measurements of enzymatic activities and assembly of mitochondrial respiratory chain complexes in the samples from the patients and knockout mice. RESULTS: Both patients presented muscle weakness and hypotonia in 4 limbs along with facial muscle weakness. One patient had cardiomyopathy. Neither patient exhibited involvement from other organs. Whole exome sequencing identified biallelic missense variants in COX6A2, which is expressed only in the skeletal muscle and heart. The variants detected were homozygous c.117C > A (p.Ser39Arg) and compound heterozygous c.117C > A (p.Ser39Arg) and c.127T > C (p.Cys43Arg). We found specific reductions in complex IV activities in the skeletal muscle of both individuals. Assembly of complex IV and its supercomplex formation were impaired in the muscle. INTERPRETATION: This study indicates that biallelic variants in COX6A2 cause a striated muscle-specific form of COX deficiency. ANN NEUROL 2019;86:193-202.

Structural Basis for the Serratia marcescens Lipase Secretion System: Crystal Structures of the Membrane Fusion Protein and Nucleotide-Binding Domain.

Serratia marcescens secretes a lipase, LipA, through a type I secretion system (T1SS). The T1SS for LipA, the Lip system, is composed of an inner membrane ABC transporter with its nucleotide-binding domains (NBD), LipB, a membrane fusion protein, LipC, and an outer membrane channel protein, LipD. Passenger protein secreted by this system has been functionally and structurally characterized well, but relatively little information about the transporter complex is available. Here, we report the crystallographic studies of LipC without the membrane anchor region, LipC-, and the NBD of LipB (LipB-NBD). LipC- crystallographic analysis has led to the determination of the structure of the long α-helical and lipoyl domains, but not the area where it interacts with LipB, suggesting that the region is flexible without LipB. The long α-helical domain has three α-helices, which interacts with LipD in the periplasm. LipB-NBD has the common overall architecture and ATP hydrolysis activity of ABC transporter NBDs. Using the predicted models of full-length LipB and LipD, the overall structural insight into the Lip system is discussed.

THOUSAND-GRAIN WEIGHT 6, which is an IAA-glucose hydrolase, preferentially recognizes the structure of the indole ring.

An indole-3-acetic acid (IAA)-glucose hydrolase, THOUSAND-GRAIN WEIGHT 6 (TGW6), negatively regulates the grain weight in rice. TGW6 has been used as a target for breeding increased rice yield. Moreover, the activity of TGW6 has been thought to involve auxin homeostasis, yet the details of this putative TGW6 activity remain unclear. Here, we show the three-dimensional structure and substrate preference of TGW6 using X-ray crystallography, thermal shift assays and fluorine nuclear magnetic resonance (19F NMR). The crystal structure of TGW6 was determined at 2.6 Å resolution and exhibited a six-bladed ß-propeller structure. Thermal shift assays revealed that TGW6 preferably interacted with indole compounds among the tested substrates, enzyme products and their analogs. Further analysis using 19F NMR with 1,134 fluorinated fragments emphasized the importance of indole fragments in recognition by TGW6. Finally, docking simulation analyses of the substrate and related fragments in the presence of TGW6 supported the interaction specificity for indole compounds. Herein, we describe the structure and substrate preference of TGW6 for interacting with indole fragments during substrate recognition. Uncovering the molecular details of TGW6 activity will stimulate the use of this enzyme for increasing crop yields and contributes to functional studies of IAA glycoconjugate hydrolases in auxin homeostasis.

Structural basis for inhibition of xyloglucan-specific endo-ß-1,4-glucanase (XEG) by XEG-protein inhibitor.

Microorganisms such as plant pathogens secrete glycoside hydrolases (GHs) to digest the polysaccharide chains of plant cell walls. The degradation of cell walls by these enzymes is a crucial step for nutrition and invasion. To protect the cell wall from these enzymes, plants secrete glycoside hydrolase inhibitor proteins (GHIPs). Xyloglucan-specific endo-ß-1,4-glucanase (XEG), a member of GH family 12 (GH12), could be a great threat to many plants because xyloglucan is a major component of the cell wall in most plants. Understanding the inhibition mechanism of XEG by GHIP is therefore of great importance in the field of plant defense, but to date the mechanism and specificity of GHIPs remain unclear. We have determined the crystal structure of XEG in complex with extracellular dermal glycoprotein (EDGP), a carrot GHIP that inhibits XEG. The structure reveals that the conserved arginines of EDGP intrude into the active site of XEG and interact with the catalytic glutamates of the enzyme. We have also determined the crystal structure of the XEG-xyloglucan complex. These structures show that EDGP closely mimics the XEG-xyloglucan interaction. Although EDGP shares structural similarity to a wheat GHIP (Triticum aestivum xylanase inhibitor-IA (TAXI-IA)) that inhibits GH11 family xylanases, the arrangement of GH and GHIP in the XEG-EDGP complex is distinct from that in the xylanase-TAXI-IA complex. Our findings imply that plants have evolved structures of GHIPs to inhibit different GH family members that attack their cell walls.

Structures of a FtsZ single protofilament and a double-helical tube in complex with a monobody.

FtsZ polymerizes into protofilaments to form the Z-ring that acts as a scaffold for accessory proteins during cell division. Structures of FtsZ have been previously solved, but detailed mechanistic insights are lacking. Here, we determine the cryoEM structure of a single protofilament of FtsZ from Klebsiella pneumoniae (KpFtsZ) in a polymerization-preferred conformation. We also develop a monobody (Mb) that binds to KpFtsZ and FtsZ from Escherichia coli without affecting their GTPase activity. Crystal structures of the FtsZ-Mb complexes reveal the Mb binding mode, while addition of Mb in vivo inhibits cell division. A cryoEM structure of a double-helical tube of KpFtsZ-Mb at 2.7 Å resolution shows two parallel protofilaments. Our present study highlights the physiological roles of the conformational changes of FtsZ in treadmilling that regulate cell division.

Purification, crystallization and X-ray diffraction study of extracellular dermal glycoprotein from carrot and the inhibition complex that it forms with an endo-ß-glucanase from Aspergillus aculeatus.

Extracellular dermal glycoprotein (EDGP) may play an important role in the plant defence system of the carrot (Daucus carota) as it has inhibitory activity against endo-ß-glucanase produced by invading pathogens. Here, EDGP and the inhibition complex that it forms with FI-CMCase, a carboxyl methyl cellulase from Aspergillus aculeatus, were successfully crystallized. The hexagonal crystal of EDGP belonged to space group P6(2), with unit-cell parameters a=b=130.4, c=44.5 Å, γ=120°. The monoclinic crystal of the complex of EDGP with FI-CMCase belonged to space group C2, with unit-cell parameters a=169.5, b=143.0, c=63.0 Å, ß=110.9°.

Purification, crystallization and X-ray diffraction study of basic 7S globulin from soybean.

Basic 7S globulin (Bg7S) is expressed by soybeans in response to biotic or abiotic stress. Bg7S is capable of binding to a 4 kDa protein which is supposedly involved in cell proliferation. Bg7S is widely found not only in legumes, but also in other plants; however, its function is still unclear. Here, Bg7S was successfully crystallized. Orthorhombic and monoclinic crystals of Bg7S were obtained under different conditions and belonged to space groups P2(1)2(1)2, with unit-cell parameters a=111.9, b=130.1, c=287.8 Å, and P2(1), with unit-cell parameters a=85.3, b=137.6, c=162.1 Å, ß=91.2°, respectively.

Karyopherin-ßs play a key role as a phase separation regulator.

Recent studies have revealed that cells utilize liquid-liquid phase separation (LLPS) as a mechanism in assembly of membrane-less organelles, such as RNP granules. The nucleus is a well-known membrane-bound organelle surrounded by the nuclear envelope; the nuclear pore complex on the nuclear envelope likely applies LLPS in the central channel to facilitate selective biological macromolecule exchange. Karyopherin-ß family proteins exclusively pass through the central channel with cargos by dissolving the phase separated hydrogel formed by the phenylalanine-glycine (FG) repeats-containing nucleoporins. Karyopherin-ßs also exhibit dissolution activity for the phase separation of cargo proteins. Many cargos, including RNA-binding proteins containing intrinsically disordered regions (IDRs), undergo phase separation; however, aberrant phase separation is linked to fatal neurodegenerative diseases. Multiple weak interactions between karyopherin-ßs and phase separation-prone proteins, such as FG repeats-containing nucleoporins or IDR-containing karyopherin-ß cargos, are likely to be important for passing through the nuclear pore complex and maintaining the soluble state of cargo, respectively. In this review, we discuss how karyopherin-ßs regulate phase separation to function.

Pressure and Temperature Phase Diagram for Liquid-Liquid Phase Separation of the RNA-Binding Protein Fused in Sarcoma.

Liquid-liquid phase separation (LLPS) of proteins and nucleic acids to form membraneless cellular compartments is considered to be involved in various biological functions. The RNA-binding protein fused in sarcoma (FUS) undergoes LLPS in vivo and in vitro. Here, we investigated the effects of pressure and temperature on the LLPS of FUS by high-pressure microscopy and high-pressure UV/vis spectroscopy. The phase-separated condensate of FUS was obliterated with increasing pressure but was observed again at a higher pressure. We generated a pressure-temperature phase diagram that describes the phase separation of FUS and provides a general understanding of the thermodynamic properties of self-assembly and phase separation of proteins. FUS has two types of condensed phases, observed at low pressure (LP-LLPS) and high pressure (HP-LLPS). The HP-LLPS state was more condensed and exhibited lower susceptibility to dissolution by 1,6-hexanediol and karyopherin-ß2 than the LP-LLPS state. Moreover, molecular dynamic simulations revealed that electrostatic interactions were destabilized, whereas cation-π, π-π, and hydrophobic interactions were stabilized in HP-LLPS. When cation-π, π-π, and hydrophobic interactions were transiently stabilized in the cellular environment, the phase transition to HP-LLPS occurred; this might be correlated to the aberrant enrichment of cytoplasmic ribonucleoprotein granules, leading to amyotrophic lateral sclerosis.

Distinct RNA polymerase transcripts direct the assembly of phase-separated DBC1 nuclear bodies in different cell lines.

The mammalian cell nucleus is a highly organized organelle that contains membrane-less structures referred to as nuclear bodies (NBs). Some NBs carry specific RNA types that play architectural roles in their formation. Here, we show two types of RNase-sensitive DBC1-containing NBs, DBC1 nuclear body (DNB) in HCT116 cells and Sam68 nuclear body (SNB) in HeLa cells, that exhibit phase-separated features and are constructed using RNA polymerase I or II transcripts in a cell type-specific manner. We identified additional protein components present in DNB by immunoprecipitation-mass spectrometry, some of which (DBC1 and heterogeneous nuclear ribonucleoprotein L [HNRNPL]) are required for DNB formation. The rescue experiment using the truncated HNRNPL mutants revealed that two RNA-binding domains and intrinsically disordered regions of HNRNPL play significant roles in DNB formation. All these domains of HNRNPL promote in vitro droplet formation, suggesting the need for multivalent interactions between HNRNPL and RNA as well as proteins in DNB formation.

Insertion loop-mediated folding propagation governs efficient maturation of hyperthermophilic Tk-subtilisin at high temperatures.

The serine protease Tk-subtilisin from the hyperthermophilic archaeon Thermococcus kodakarensis possesses three insertion loops (IS1-IS3) on its surface, as compared to its mesophilic counterparts. Although IS1 and IS2 are required for maturation of Tk-subtilisin at high temperatures, the role of IS3 remains unknown. Here, CD spectroscopy revealed that IS3 deletion arrested Tk-subtilisin folding at an intermediate state, in which the central nucleus was formed, but the subsequent folding propagation into terminal subdomains did not occur. Alanine substitution of the aspartate residue in IS3 disturbed the intraloop hydrogen-bonding network, as evidenced by crystallographic analysis, resulting in compromised folding at high temperatures. Taking into account the high conservation of IS3 across hyperthermophilic homologues, we propose that the presence of IS3 is important for folding of hyperthermophilic subtilisins in high-temperature environments.

Mechanism of karyopherin-ß2 binding and nuclear import of ALS variants FUS(P525L) and FUS(R495X).

Mutations in the RNA-binding protein FUS cause familial amyotropic lateral sclerosis (ALS). Several mutations that affect the proline-tyrosine nuclear localization signal (PY-NLS) of FUS cause severe juvenile ALS. FUS also undergoes liquid-liquid phase separation (LLPS) to accumulate in stress granules when cells are stressed. In unstressed cells, wild type FUS resides predominantly in the nucleus as it is imported by the importin Karyopherin-ß2 (Kapß2), which binds with high affinity to the C-terminal PY-NLS of FUS. Here, we analyze the interactions between two ALS-related variants FUS(P525L) and FUS(R495X) with importins, especially Kapß2, since they are still partially localized to the nucleus despite their defective/missing PY-NLSs. The crystal structure of the Kapß2·FUS(P525L)PY-NLS complex shows the mutant peptide making fewer contacts at the mutation site, explaining decreased affinity for Kapß2. Biochemical analysis revealed that the truncated FUS(R495X) protein, although missing the PY-NLS, can still bind Kapß2 and suppresses LLPS. FUS(R495X) uses its C-terminal tandem arginine-glycine-glycine regions, RGG2 and RGG3, to bind the PY-NLS binding site of Kapß2 for nuclear localization in cells when arginine methylation is inhibited. These findings suggest the importance of the C-terminal RGG regions in nuclear import and LLPS regulation of ALS variants of FUS that carry defective PY-NLSs.

Structure-function studies of ultrahigh molecular weight isoprenes provide key insights into their biosynthesis.

Some plant trans-1,4-prenyltransferases (TPTs) produce ultrahigh molecular weight trans-1,4-polyisoprene (TPI) with a molecular weight of over 1.0 million. Although plant-derived TPI has been utilized in various industries, its biosynthesis and physiological function(s) are unclear. Here, we identified three novel Eucommia ulmoides TPT isoforms-EuTPT1, 3, and 5, which synthesized TPI in vitro without other components. Crystal structure analysis of EuTPT3 revealed a dimeric architecture with a central hydrophobic tunnel. Mutation of Cys94 and Ala95 on the central hydrophobic tunnel no longer synthesizd TPI, indicating that Cys94 and Ala95 were essential for forming the dimeric architecture of ultralong-chain TPTs and TPI biosynthesis. A spatiotemporal analysis of the physiological function of TPI in E. ulmoides suggested that it is involved in seed development and maturation. Thus, our analysis provides functional and mechanistic insights into TPI biosynthesis and uncovers biological roles of TPI in plants.

C9orf72-derived arginine-rich poly-dipeptides impede phase modifiers.

Nuclear import receptors (NIRs) not only transport RNA-binding proteins (RBPs) but also modify phase transitions of RBPs by recognizing nuclear localization signals (NLSs). Toxic arginine-rich poly-dipeptides from C9orf72 interact with NIRs and cause nucleocytoplasmic transport deficit. However, the molecular basis for the toxicity of arginine-rich poly-dipeptides toward NIRs function as phase modifiers of RBPs remains unidentified. Here we show that arginine-rich poly-dipeptides impede the ability of NIRs to modify phase transitions of RBPs. Isothermal titration calorimetry and size-exclusion chromatography revealed that proline:arginine (PR) poly-dipeptides tightly bind karyopherin-ß2 (Kapß2) at 1:1 ratio. The nuclear magnetic resonances of Kapß2 perturbed by PR poly-dipeptides partially overlapped with those perturbed by the designed NLS peptide, suggesting that PR poly-dipeptides target the NLS binding site of Kapß2. The findings offer mechanistic insights into how phase transitions of RBPs are disabled in C9orf72-related neurodegeneration.