Observation of the Liquid-Liquid Phase Separation of FUS-LC Using Vacuum-Ultraviolet Circular Dichroism Spectroscopy.

To reveal the structural mechanism by which the low-complexity domain of the fused in sarcoma protein (FUS-LC) mediates liquid-liquid phase separation (LLPS), we conducted a vacuum-ultraviolet circular dichroism (VUV-CD) spectroscopic study, a technique to analyze the secondary structures of proteins. The VUV-CD measurements were performed at the BL12 VUV-CD station at the Hiroshima Synchrotron Radiation Center (HiSOR) in Japan. CD spectra were measured between 180 and 260 nm while controlling the temperature of samples from 37°C to 5°C to obtain the LLPS of FUS-LC. The CD spectrum obtained at 37°C exhibited a large negative peak at 195 nm and a small negative shoulder near 220 nm. The peak intensity around 195 nm decreased as the sample temperature decreased. The spectral changes originated from the LLPS formation.

Crystal Structure Determination of Ubiquitin by Fusion to a Protein That Forms a Highly Porous Crystal Lattice.

The protein crystallization process requires screening of a large number of conditions using a large quantity of high-purity protein, which makes crystal structure analysis difficult. Thus, the development of easy and versatile protein crystallization techniques is both extremely desirable and highly challenging. Here I demonstrate the crystallization and structure determination of ubiquitin by genetic fusion to the highly porous honeycomb lattice of R1EN. I successfully crystallized and collected X-ray data from three R1EN-ubiquitin constructs with various linker lengths under the same conditions as the original R1EN. The crystals diffracted to 1.7-2.4 Å resolution, and the ubiquitin structures were determined with results almost identical to the previously published structure. Moreover, the ubiquitin structure could be solved by molecular replacement using R1EN alone. This method may reduce the effort required for crystallization screening and is applicable to de novo protein structure determination.

Structural basis for simultaneous recognition of an O-glycan and its attached peptide of mucin family by immune receptor PILRα.

Paired Ig-like type 2 receptor α (PILRα) recognizes a wide range of O-glycosylated mucin and related proteins to regulate broad immune responses. However, the molecular characteristics of these recognitions are largely unknown. Here we show that sialylated O-linked sugar T antigen (sTn) and its attached peptide region are both required for ligand recognition by PILRα. Furthermore, we determined the crystal structures of PILRα and its complex with an sTn and its attached peptide region. The structures show that PILRα exhibits large conformational change to recognize simultaneously both the sTn O-glycan and the compact peptide structure constrained by proline residues. Binding and functional assays support this binding mode. These findings provide significant insight into the binding motif and molecular mechanism (which is distinct from sugar-recognition receptors) by which O-glycosylated mucin proteins with sTn modifications are recognized in the immune system as well as during viral entry.

Structural analysis of the TKB domain of ubiquitin ligase Cbl-b complexed with its small inhibitory peptide, Cblin.

Cbl-b is a RING-type ubiquitin ligase. Previously, we showed that Cbl-b-mediated ubiquitination and proteosomal degradation of IRS-1 contribute to muscle atrophy caused by unloading stress. The phospho-pentapeptide DGpYMP (Cblin) mimics Tyr612-phosphorylated IRS-1 and inhibits the Cbl-b-mediated ubiquitination and degradation of IRS-1 in vitro and in vivo. In this study, we confirmed the direct interaction between Cblin and the TKB domain of Cbl-b using NMR. Moreover, we showed that the shortened tripeptide GpYM also binds to the TKB domain. To elucidate the inhibitory mechanism of Cblin, we solved the crystal structure of the TKB-Cblin complex at a resolution of 2.5 Å. The pY in Cblin inserts into a positively charged pocket in the TKB domain via hydrogen-bond networks and hydrophobic interactions. Within this complex, the Cblin structure closely resembles the TKB-bound form of another substrate-derived phosphopeptide, Zap-70-derived phosphopeptide. These peptides lack the conserved intrapeptidyl hydrogen bond between pY and a conserved residue involved in TKB-domain binding. Instead of the conserved interaction, these peptides specifically interact with the TKB domain. Based on this binding mode of Cblin to the TKB domain, we can design drugs against unloading-mediated muscle atrophy.

Human α-L-iduronidase uses its own N-glycan as a substrate-binding and catalytic module.

N-glycosylation is a major posttranslational modification that endows proteins with various functions. It is established that N-glycans are essential for the correct folding and stability of some enzymes; however, the actual effects of N-glycans on their activities are poorly understood. Here, we show that human α-l-iduronidase (hIDUA), of which a dysfunction causes accumulation of dermatan/heparan sulfate leading to mucopolysaccharidosis type I, uses its own N-glycan as a substrate binding and catalytic module. Structural analysis revealed that the mannose residue of the N-glycan attached to N372 constituted a part of the substrate-binding pocket and interacted directly with a substrate. A deglycosylation study showed that enzyme activity was highly correlated with the N-glycan attached to N372. The kinetics of native and deglycosylated hIDUA suggested that the N-glycan is also involved in catalytic processes. Our study demonstrates a previously unrecognized function of N-glycans.

Observation of the controlled assembly of preclick components in the in situ click chemistry generation of a chitinase inhibitor.

The Huisgen cycloaddition of azides and alkynes, accelerated by target biomolecules, termed "in situ click chemistry," has been successfully exploited to discover highly potent enzyme inhibitors. We have previously reported a specific Serratia marcescens chitinase B (SmChiB)-templated syn-triazole inhibitor generated in situ from an azide-bearing inhibitor and an alkyne fragment. Several in situ click chemistry studies have been reported. Although some mechanistic evidence has been obtained, such as X-ray analysis of [protein]-["click ligand"] complexes, indicating that proteins act as both mold and template between unique pairs of azide and alkyne fragments, to date, observations have been based solely on "postclick" structural information. Here, we describe crystal structures of SmChiB complexed with an azide ligand and an O-allyl oxime fragment as a mimic of a click partner, revealing a mechanism for accelerating syn-triazole formation, which allows generation of its own distinct inhibitor. We have also performed density functional theory calculations based on the X-ray structure to explore the acceleration of the Huisgen cycloaddition by SmChiB. The density functional theory calculations reasonably support that SmChiB plays a role by the cage effect during the pretranslation and posttranslation states of selective syn-triazole click formation.

Structural and clinical implications of amino acid substitutions in α-L-iduronidase: insight into the basis of mucopolysaccharidosis type I.

Allelic mutations, predominantly missense ones, of the α-l-iduronidase (IDUA) gene cause mucopolysaccharidosis type I (MPS I), which exhibits heterogeneous phenotypes. These phenotypes are basically classified into severe, intermediate, and attenuated types. We previously examined the structural changes in IDUA due to MPS I by homology modeling, but the reliability was limited because of the low sequence identity. In this study, we built new structural models of mutant IDUAs due to 57 amino acid substitutions that had been identified in 27 severe, 1 severe-intermediate, 13 intermediate, 1 attenuated-intermediate and 15 attenuated type MPS I patients based on the crystal structure of human IDUA, which was recently determined by us. The structural changes were examined by calculating the root-mean-square distances (RMSD) and the number of atoms influenced by the amino acid replacements. The results revealed that the structural changes of the enzyme protein tended to be correlated with the severity of the disease. Then we focused on the structural changes resulting from amino acid replacements in the immunoglobulin-like domain and adjacent region, of which the structure had been missing in the IDUA model previously built. Coloring of atoms influenced by an amino acid substitution was performed in each case and the results revealed that the structural changes occurred in a region far from the active site of IDUA, suggesting that they affected protein folding. Structural analysis is thus useful for elucidation of the basis of MPS I.

Glycan-shielded homodimer structure and dynamical features of the canine distemper virus hemagglutinin relevant for viral entry and efficient vaccination.

Canine distemper virus (CDV) belongs to morbillivirus, including measles virus (MeV) and rinderpest virus, which causes serious immunological and neurological disorders in carnivores, including dogs and rhesus monkeys, as recently reported, but their vaccines are highly effective. The attachment glycoprotein hemagglutinin (CDV-H) at the CDV surface utilizes signaling lymphocyte activation molecule (SLAM) and Nectin-4 (also called poliovirus-receptor-like-4; PVRL4) as entry receptors. Although fusion models have been proposed, the molecular mechanism of morbillivirus fusion entry is poorly understood. Here, we determined the crystal structure of the globular head domain of CDV-H vaccine strain at 3.2 Å resolution, revealing that CDV-H exhibits a highly tilted homodimeric form with a six-bladed ß-propeller fold. While the predicted Nectin-4-binding site is well conserved with that of MeV-H, that of SLAM is similar but partially different, which is expected to contribute to host specificity. Five N-linked sugars covered a broad area of the CDV-H surface to expose receptor-binding sites only, supporting the effective production of neutralizing antibodies. These features are common to MeV-H, although the glycosylation sites are completely different. Furthermore, real-time observation using high-speed atomic force microscopy revealed highly mobile features of the CDV-H dimeric head via the connector region. These results suggest that sugar-shielded tilted homodimeric structure and dynamic conformational changes are common characteristics of morbilliviruses and ensure effective fusion entry and vaccination.

Structure-guided identification of a new catalytic motif of oligosaccharyltransferase.

Asn-glycosylation is widespread not only in eukaryotes but also in archaea and some eubacteria. Oligosaccharyltransferase (OST) catalyzes the co-translational transfer of an oligosaccharide from a lipid donor to an asparagine residue in nascent polypeptide chains. Here, we report that a thermophilic archaeon, Pyrococcus furiosus OST is composed of the STT3 protein alone, and catalyzes the transfer of a heptasaccharide, containing one hexouronate and two pentose residues, onto peptides in an Asn-X-Thr/Ser-motif-dependent manner. We also determined the 2.7-A resolution crystal structure of the C-terminal soluble domain of Pyrococcus STT3. The structure-based multiple sequence alignment revealed a new motif, DxxK, which is adjacent to the well-conserved WWDYG motif in the tertiary structure. The mutagenesis of the DK motif residues in yeast STT3 revealed the essential role of the motif in the catalytic activity. The function of this motif may be related to the binding of the pyrophosphate group of lipid-linked oligosaccharide donors through a transiently bound cation. Our structure provides the first structural insights into the formation of the oligosaccharide-asparagine bond.

Crystallization, X-ray diffraction analysis and SIRAS phasing of human α-L-iduronidase.

Human lysosomal α-L-iduronidase, whose deficiency causes mucopolysaccharidosis type I, was crystallized using sodium/potassium tartrate and polyethylene glycol 3350 as a precipitant. Using synchrotron radiation, a native data set was collected from a single crystal at 100 K to 2.3 Šresolution. The crystal belonged to space group R3 with unit-cell dimensions of a=b=259.22, c=71.83 Å. To obtain the phase information, mercury-derivative crystals were prepared and a single-wavelength anomalous dispersion (SAD) data set was collected at the Hg peak wavelength. Phase calculation with the single isomorphous replacement with anomalous scattering (SIRAS) method successfully yielded an interpretable electron-density map.

Crystal structure of the PX domain of Vps17p from Saccharomyces cerevisiae.

The structure determination of the PX (phox homology) domain of the Saccharomyces cerevisiae Vps17p protein presented a challenging case for molecular replacement because it has noncrystallographic symmetry close to a crystallographic axis. The combination of diffraction-quality crystals grown under microgravity on the International Space Station and a highly accurate template structure predicted by AlphaFold2 provided the key to successful crystal structure determination. Although the structure of the Vps17p PX domain is seen in many PX domains, no basic residues are found around the canonical phosphatidylinositol phosphate (PtdIns-P) binding site, suggesting an inability to bind PtdIns-P molecules.

Effects of targeting signal mutations in a mitochondrial presequence on the spatial distribution of the conformational ensemble in the binding site of Tom20.

The 20-kDa TOM (translocase of outer mitochondrial membrane) subunit, Tom20, is the first receptor of the protein import pathway into mitochondria. Tom20 recognizes the mitochondrial targeting signal embedded in the presequences attached to mature mitochondrial proteins, as an N-terminal extension. Consequently, ~1,000 different mitochondrial proteins are sorted into the mitochondrial matrix, and distinguished from non-mitochondrial proteins. We previously reported the MPRIDE (multiple partial recognitions in dynamic equilibrium) mechanism to explain the structural basis of the promiscuous recognition of presequences by Tom20. A subset of the targeting signal features is recognized in each pose of the presequence in the binding state, and all of the features are collectively recognized in the dynamic equilibrium between the poses. Here, we changed the volumes of the hydrophobic side chains in the targeting signal, while maintaining the binding affinity. We tethered the mutated presequences to the binding site of Tom20 and placed them in the crystal contact-free space (CCFS) created in the crystal lattice. The spatial distributions of the mutated presequences were visualized as smeared electron densities in the low-pass filtered difference maps obtained by X-ray crystallography. The mutated presequence ensembles shifted their positions in the binding state to accommodate the larger side chains, thus providing positive evidence supporting the use of the MPRIDE mechanism in the promiscuous recognition by Tom20.

Crystallographic snapshots of Tom20-mitochondrial presequence interactions with disulfide-stabilized peptides.

Most mitochondrial proteins are synthesized in the cytosol and imported into mitochondria. The Tom20 protein, residing on the mitochondrial surface, recognizes the N-terminal presequences of precursor proteins. We previously determined the crystal structures of the Tom20-presequence complex. The successful crystallization involved tethering the presequence to Tom20 through an intermolecular disulfide bond with an optimized linker. In this work, we assessed the tethering method. The intermolecular disulfide bond was cleaved in crystal with a reducing agent. The pose (i.e., conformation and position) of the presequence was identical to the previously determined pose. In another experiment, a longer linker than the optimized length was used for the tethering. The perturbation of the tether changed the pose slightly, but the interaction mode was preserved. These results argue against the forced interaction of the presequence by its covalent attachment to Tom20. Second, as an alternative method referred to as "molecular stiffening", we introduced a disulfide bond within the presequence peptide to restrict the freedom of the peptide in the unbound states. One presequence analogue exhibited over 100-fold higher affinity than its linear counterpart and generated cocrystals with Tom20. One of the two crystallographic snapshots revealed a known pose previously determined by the tethering method, and the other snapshot depicted a new pose. These results confirmed and extended the dynamic, multiple bound state model of the Tom20-presequence interactions and also demonstrated the validity of the molecular tethering and stiffening techniques in studies of transient protein-peptide interactions.

Comparative structural biology of eubacterial and archaeal oligosaccharyltransferases.

Oligosaccharyltransferase (OST) catalyzes the transfer of an oligosaccharide from a lipid donor to an asparagine residue in nascent polypeptide chains. In the bacterium Campylobacter jejuni, a single-subunit membrane protein, PglB, catalyzes N-glycosylation. We report the 2.8 A resolution crystal structure of the C-terminal globular domain of PglB and its comparison with the previously determined structure from the archaeon Pyrococcus AglB. The two distantly related oligosaccharyltransferases share unexpected structural similarity beyond that expected from the sequence comparison. The common architecture of the putative catalytic sites revealed a new catalytic motif in PglB. Site-directed mutagenesis analyses confirmed the contribution of this motif to the catalytic function. Bacterial PglB and archaeal AglB constitute a protein family of the catalytic subunit of OST along with STT3 from eukaryotes. A structure-aided multiple sequence alignment of the STT3/PglB/AglB protein family revealed three types of OST catalytic centers. This novel classification will provide a useful framework for understanding the enzymatic properties of the OST enzymes from Eukarya, Archaea, and Bacteria.

Global conformational change associated with the two-step reaction catalyzed by Escherichia coli lipoate-protein ligase A.

Lipoate-protein ligase A (LplA) catalyzes the attachment of lipoic acid to lipoate-dependent enzymes by a two-step reaction: first the lipoate adenylation reaction and, second, the lipoate transfer reaction. We previously determined the crystal structure of Escherichia coli LplA in its unliganded form and a binary complex with lipoic acid (Fujiwara, K., Toma, S., Okamura-Ikeda, K., Motokawa, Y., Nakagawa, A., and Taniguchi, H. (2005) J Biol. Chem. 280, 33645-33651). Here, we report two new LplA structures, LplA.lipoyl-5'-AMP and LplA.octyl-5'-AMP.apoH-protein complexes, which represent the post-lipoate adenylation intermediate state and the pre-lipoate transfer intermediate state, respectively. These structures demonstrate three large scale conformational changes upon completion of the lipoate adenylation reaction: movements of the adenylate-binding and lipoate-binding loops to maintain the lipoyl-5'-AMP reaction intermediate and rotation of the C-terminal domain by about 180 degrees . These changes are prerequisites for LplA to accommodate apoprotein for the second reaction. The Lys(133) residue plays essential roles in both lipoate adenylation and lipoate transfer reactions. Based on structural and kinetic data, we propose a reaction mechanism driven by conformational changes.

Crystal structure of aminomethyltransferase in complex with dihydrolipoyl-H-protein of the glycine cleavage system: implications for recognition of lipoyl protein substrate, disease-related mutations, and reaction mechanism.

Aminomethyltransferase, a component of the glycine cleavage system termed T-protein, reversibly catalyzes the degradation of the aminomethyl moiety of glycine attached to the lipoate cofactor of H-protein, resulting in the production of ammonia, 5,10-methylenetetrahydrofolate, and dihydrolipoate-bearing H-protein in the presence of tetrahydrofolate. Several mutations in the human T-protein gene are known to cause nonketotic hyperglycinemia. Here, we report the crystal structure of Escherichia coli T-protein in complex with dihydrolipoate-bearing H-protein and 5-methyltetrahydrofolate, a complex mimicking the ternary complex in the reverse reaction. The structure of the complex shows a highly interacting intermolecular interface limited to a small area and the protein-bound dihydrolipoyllysine arm inserted into the active site cavity of the T-protein. Invariant Arg(292) of the T-protein is essential for complex assembly. The structure also provides novel insights in understanding the disease-causing mutations, in addition to the disease-related impairment in the cofactor-enzyme interactions reported previously. Furthermore, structural and mutational analyses suggest that the reversible transfer of the methylene group between the lipoate and tetrahydrofolate should proceed through the electron relay-assisted iminium intermediate formation.

Crystal structure of thymine DNA glycosylase conjugated to SUMO-1.

Members of the small ubiquitin-like modifier (SUMO) family can be covalently attached to the lysine residue of a target protein through an enzymatic pathway similar to that used in ubiquitin conjugation, and are involved in various cellular events that do not rely on degradative signalling via the proteasome or lysosome. However, little is known about the molecular mechanisms of SUMO-modification-induced protein functional transfer. During DNA mismatch repair, SUMO conjugation of the uracil/thymine DNA glycosylase TDG promotes the release of TDG from the abasic (AP) site created after base excision, and coordinates its transfer to AP endonuclease 1, which catalyses the next step in the repair pathway. Here we report the crystal structure of the central region of human TDG conjugated to SUMO-1 at 2.1 A resolution. The structure reveals a helix protruding from the protein surface, which presumably interferes with the product DNA and thus promotes the dissociation of TDG from the DNA molecule. This helix is formed by covalent and non-covalent contacts between TDG and SUMO-1. The non-covalent contacts are also essential for release from the product DNA, as verified by mutagenesis.

Crystal structure of inhibitor-bound human MSPL that can activate high pathogenic avian influenza.

Infection of certain influenza viruses is triggered when its HA is cleaved by host cell proteases such as proprotein convertases and type II transmembrane serine proteases (TTSP). HA with a monobasic motif is cleaved by trypsin-like proteases, including TMPRSS2 and HAT, whereas the multibasic motif found in high pathogenicity avian influenza HA is cleaved by furin, PC5/6, or MSPL. MSPL belongs to the TMPRSS family and preferentially cleaves [R/K]-K-K-R↓ sequences. Here, we solved the crystal structure of the extracellular region of human MSPL in complex with an irreversible substrate-analog inhibitor. The structure revealed three domains clustered around the C-terminal α-helix of the SPD. The inhibitor structure and its putative model show that the P1-Arg inserts into the S1 pocket, whereas the P2-Lys and P4-Arg interacts with the Asp/Glu-rich 99-loop that is unique to MSPL. Based on the structure of MSPL, we also constructed a homology model of TMPRSS2, which is essential for the activation of the SARS-CoV-2 spike protein and infection. The model may provide the structural insight for the drug development for COVID-19.

Crystal structure of measles virus hemagglutinin provides insight into effective vaccines.

Measles still remains a major cause of childhood morbidity and mortality worldwide. Measles virus (MV) vaccines are highly successful, but the mechanism underlying their efficacy has been unclear. Here we report the crystal structure of the MV attachment protein, hemagglutinin, responsible for MV entry. The receptor-binding head domain exhibits a cubic-shaped beta-propeller structure and forms a homodimer. N-linked sugars appear to mask the broad regions and cause the two molecules forming the dimer to tilt oppositely toward the horizontal plane. Accordingly, residues of the putative receptor-binding site, highly conserved among MV strains, are strategically positioned in the unshielded area of the protein. These conserved residues also serve as epitopes for neutralizing antibodies, ensuring the serological monotype, a basis for effective MV vaccines. Our findings suggest that sugar moieties in the MV hemagglutinin critically modulate virus-receptor interaction as well as antiviral antibody responses, differently from sugars of the HIV gp120, which allow for immune evasion.

P219L substitution in human D-amino acid oxidase impacts the ligand binding and catalytic efficiency.

Human D-amino acid oxidase (DAO) is a flavoenzyme that is implicated in neurodegenerative diseases. We investigated the impact of replacement of proline with leucine at Position 219 (P219L) in the active site lid of human DAO on the structural and enzymatic properties, because porcine DAO contains leucine at the corresponding position. The turnover numbers (kcat) of P219L were unchanged, but its Km values decreased compared with wild-type, leading to an increase in the catalytic efficiency (kcat/Km). Moreover, benzoate inhibits P219L with lower Ki value (0.7-0.9 µM) compared with wild-type (1.2-2.0 µM). Crystal structure of P219L in complex with flavin adenine dinucleotide (FAD) and benzoate at 2.25 Å resolution displayed conformational changes of the active site and lid. The distances between the H-bond-forming atoms of arginine 283 and benzoate and the relative position between the aromatic rings of tyrosine 224 and benzoate were changed in the P219L complex. Taken together, the P219L substitution leads to an increase in the catalytic efficiency and binding affinity for substrates/inhibitors due to these structural changes. Furthermore, an acetic acid was located near the adenine ring of FAD in the P219L complex. This study provides new insights into the structure-function relationship of human DAO.