Human Rad51 Protein Requires Higher Concentrations of Calcium Ions for D-Loop Formation than for Oligonucleotide Strand Exchange.

Human Rad51 protein (HsRad51)-promoted DNA strand exchange, a crucial step in homologous recombination, is regulated by proteins and calcium ions. Both the activator protein Swi5/Sfr1 and Ca2+ ions stimulate different reaction steps and induce perpendicular DNA base alignment in the presynaptic complex. To investigate the role of base orientation in the strand exchange reaction, we examined the Ca2+ concentration dependence of strand exchange activities and structural changes in the presynaptic complex. Our results show that optimal D-loop formation (strand exchange with closed circular DNA) required Ca2+ concentrations greater than 5 mM, whereas 1 mM Ca2+ was sufficient for strand exchange between two oligonucleotides. Structural changes indicated by increased fluorescence intensity of poly(dεA) (a poly(dA) analog) reached a plateau at 1 mM Ca2+. Ca2+ > 2 mM was required for saturation of linear dichroism signal intensity at 260 nm, associated with rigid perpendicular DNA base orientation, suggesting a correlation with the stimulation of D-loop formation. Therefore, Ca2+ exerts two different effects. Thermal stability measurements suggest that HsRad51 binds two Ca2+ ions with KD values of 0.2 and 2.5 mM, implying that one step is stimulated by one Ca2+ bond and the other by two Ca2+ bonds. Our results indicate parallels between the Mg2+ activation of RecA and the Ca2+ activation of HsRad51.

The structure of POMGNT2 provides new insights into the mechanism to determine the functional O-mannosylation site on α-dystroglycan.

Defects in the O-mannosyl glycan of α-dystroglycan (α-DG) are associated with α-dystroglycanopathy, a group of congenital muscular dystrophies. While α-DG has many O-mannosylation sites, only the specific positions can be modified with the functional O-mannosyl glycan, namely, core M3-type glycan. POMGNT2 is a glycosyltransferase which adds ß1,4-linked GlcNAc to the O-mannose (Man) residue to acquire core M3-type glycan. Although it is assumed that POMGNT2 extends the specific O-Man residues around particular amino acid sequences, the details are not well understood. Here, we determined a series of crystal structures of POMGNT2 with and without the acceptor O-mannosyl peptides and identified the critical interactions between POMGNT2 and the acceptor peptide. POMGNT2 has an N-terminal catalytic domain and a C-terminal fibronectin type III (FnIII) domain and forms a dimer. The acceptor peptide is sandwiched between the two protomers. The catalytic domain of one protomer recognizes the O-mannosylation site (TPT motif), and the FnIII domain of the other protomer recognizes the C-terminal region of the peptide. Structure-based mutational studies confirmed that amino acid residues of the catalytic domain interacting with mannose or the TPT motif are essential for POMGNT2 enzymatic activity. In addition, the FnIII domain is also essential for the activity and it interacts with the peptide mainly by hydrophobic interaction. Our study provides the first atomic-resolution insights into specific acceptor recognition by the FnIII domain of POMGNT2. The catalytic mechanism of POMGNT2 is proposed based on the structure.

Crystal structures of the RNA triphosphatase from Trypanosoma cruzi provide insights into how it recognizes the 5'-end of the RNA substrate.

RNA triphosphatase catalyzes the first step in mRNA cap formation, hydrolysis of the terminal phosphate from the nascent mRNA transcript. The RNA triphosphatase from the protozoan parasite Trypanosoma cruzi, TcCet1, belongs to the family of triphosphate tunnel metalloenzymes (TTMs). TcCet1 is a promising antiprotozoal drug target because the mechanism and structure of the protozoan RNA triphosphatases are completely different from those of the RNA triphosphatases found in mammalian and arthropod hosts. Here, we report several crystal structures of the catalytically active form of TcCet1 complexed with a divalent cation and an inorganic tripolyphosphate in the active-site tunnel at 2.20-2.51 Å resolutions. The structures revealed that the overall structure, the architecture of the tunnel, and the arrangement of the metal-binding site in TcCet1 are similar to those in other TTM proteins. On the basis of the position of three sulfate ions that cocrystallized on the positively charged surface of the protein and results obtained from mutational analysis, we identified an RNA-binding site in TcCet1. We conclude that the 5'-end of the triphosphate RNA substrate enters the active-site tunnel directionally. The structural information reported here provides valuable insight into designing inhibitors that could specifically block the entry of the triphosphate RNA substrate into the TTM-type RNA triphosphatases of T. cruzi and related pathogens.

FAM3B/PANDER-Like Carbohydrate-Binding Domain in a Glycosyltransferase, POMGNT1.

Protein O-mannose ß1,2-N-acetylglucosaminyltransferase 1 (POMGNT1) is one of the gene products responsible for α-dystroglycanopathy, which is a type of congenital muscular dystrophy caused by O-mannosyl glycan defects. The originally identified function of POMGNT1 was as a glycosyltransferase that catalyzes the formation of the GlcNAcß1-2Man linkage of O-mannosyl glycan, but the enzyme function is not essential for α-dystroglycanopathy pathogenesis. Our recent study revealed that the stem domain of POMGNT1 has a carbohydrate-binding ability, which recognizes the GalNAcß1-3GlcNAc structure. This carbohydrate-binding activity is required for the formation of the ribitol phosphate (RboP)-3GalNAcß1-3GlcNAc structure by fukutin. This protocol describes methods to assess the carbohydrate-binding activity of the POMGNT1 stem domain.

Crystal structures of fukutin-related protein (FKRP), a ribitol-phosphate transferase related to muscular dystrophy.

α-Dystroglycan (α-DG) is a highly-glycosylated surface membrane protein. Defects in the O-mannosyl glycan of α-DG cause dystroglycanopathy, a group of congenital muscular dystrophies. The core M3 O-mannosyl glycan contains tandem ribitol-phosphate (RboP), a characteristic feature first found in mammals. Fukutin and fukutin-related protein (FKRP), whose mutated genes underlie dystroglycanopathy, sequentially transfer RboP from cytidine diphosphate-ribitol (CDP-Rbo) to form a tandem RboP unit in the core M3 glycan. Here, we report a series of crystal structures of FKRP with and without donor (CDP-Rbo) and/or acceptor [RboP-(phospho-)core M3 peptide] substrates. FKRP has N-terminal stem and C-terminal catalytic domains, and forms a tetramer both in crystal and in solution. In the acceptor complex, the phosphate group of RboP is recognized by the catalytic domain of one subunit, and a phosphate group on O-mannose is recognized by the stem domain of another subunit. Structure-based functional studies confirmed that the dimeric structure is essential for FKRP enzymatic activity.

Experimental phase determination with selenomethionine or mercury-derivatization in serial femtosecond crystallography.

Serial femtosecond crystallography (SFX) using X-ray free-electron lasers (XFELs) holds enormous potential for the structure determination of proteins for which it is difficult to produce large and high-quality crystals. SFX has been applied to various systems, but rarely to proteins that have previously unknown structures. Consequently, the majority of previously obtained SFX structures have been solved by the molecular replacement method. To facilitate protein structure determination by SFX, it is essential to establish phasing methods that work efficiently for SFX. Here, selenomethionine derivatization and mercury soaking have been investigated for SFX experiments using the high-energy XFEL at the SPring-8 Angstrom Compact Free-Electron Laser (SACLA), Hyogo, Japan. Three successful cases are reported of single-wavelength anomalous diffraction (SAD) phasing using X-rays of less than 1 Šwavelength with reasonable numbers of diffraction patterns (13 000, 60 000 and 11 000). It is demonstrated that the combination of high-energy X-rays from an XFEL and commonly used heavy-atom incorporation techniques will enable routine de novo structural determination of biomacromolecules.

X-ray structure of a protease-resistant mutant form of human galectin-9 having two carbohydrate recognition domains with a metal-binding site.

Galectin-9 (G9) is a tandem-repeat type ß-galactoside-specific animal lectin having N-terminal and C-terminal carbohydrate recognition domains (N-CRD and C-CRD, respectively) joined by a linker peptide that is involved in the immune system. G9 is divalent in glycan binding, and structural information about the spatial arrangement of the two CRDs is very important for elucidating its biological functions. As G9 is protease sensitive due to the long linker, the protease-resistant mutant form of G9 (G9Null) was developed by modification of the linker peptide, while retaining its biological functions. The X-ray structure of a mutant form of G9Null with the replacement of Arg221 by Ser (G9Null_R221S) having two CRDs was determined. The structure of G9Null_R221S was compact to associate the two CRDs in the back-to-back orientation with a large interface area, including hydrogen bonds and hydrophobic interactions. A metal ion was newly found in the galectin structure, possibly contributing to the stable structure of protein. The presented X-ray structure was thought to be one of the stable structures of G9, which likely occurs in solution. This was supported by structural comparisons with other tandem-repeated galectins and the analyses of protein thermostability by CD spectra measurements.

Carbohydrate-binding domain of the POMGnT1 stem region modulates O-mannosylation sites of α-dystroglycan.

The dystrophin glycoprotein complex, which connects the cell membrane to the basement membrane, is essential for a variety of biological events, including maintenance of muscle integrity. An O-mannose-type GalNAc-ß1,3-GlcNAc-ß1,4-(phosphate-6)-Man structure of α-dystroglycan (α-DG), a subunit of the complex that is anchored to the cell membrane, interacts directly with laminin in the basement membrane. Reduced glycosylation of α-DG is linked to some types of inherited muscular dystrophy; consistent with this relationship, many disease-related mutations have been detected in genes involved in O-mannosyl glycan synthesis. Defects in protein O-linked mannose ß1,2-N-acetylglucosaminyltransferase 1 (POMGnT1), a glycosyltransferase that participates in the formation of GlcNAc-ß1,2-Man glycan, are causally related to muscle-eye-brain disease (MEB), a congenital muscular dystrophy, although the role of POMGnT1 in postphosphoryl modification of GalNAc-ß1,3-GlcNAc-ß1,4-(phosphate-6)-Man glycan remains elusive. Our crystal structures of POMGnT1 agreed with our previous results showing that the catalytic domain recognizes substrate O-mannosylated proteins via hydrophobic interactions with little sequence specificity. Unexpectedly, we found that the stem domain recognizes the ß-linked GlcNAc of O-mannosyl glycan, an enzymatic product of POMGnT1. This interaction may recruit POMGnT1 to a specific site of α-DG to promote GlcNAc-ß1,2-Man clustering and also may recruit other enzymes that interact with POMGnT1, e.g., fukutin, which is required for further modification of the GalNAc-ß1,3-GlcNAc-ß1,4-(phosphate-6)-Man glycan. On the basis of our findings, we propose a mechanism for the deficiency in postphosphoryl modification of the glycan observed in POMGnT1-KO mice and MEB patients.

Regulation of the vitamin D receptor by vitamin D lactam derivatives.

The active metabolite of vitamin D3 , 1α,25-dihydroxyvitamin D3 , acts as a ligand for the vitamin D receptor (VDR) and activates VDR-mediated gene expression. Recently, we characterized 1α,25-dihydroxyvitamin D3 -26,23-lactams (DLAMs), which mimic vitamin D3 metabolites, as noncalcemic VDR ligands that barely activate the receptor. In this study, we present structural insights onto the regulation of VDR function by DLAMs. X-ray crystallographic analysis revealed that DLAMs induced a large conformational change in the loop region between helices H6 and H7 in the VDR ligand-binding domain. Our structural analysis suggests that targeting of the loop region may be a new mode of VDR regulation.

Novel helical assembly in arginine methyltransferase 8.

Protein arginine methyltransferase 8 (PRMT8) is unique among PRMTs, as it is specifically expressed in brain and localized to the plasma membrane via N-terminal myristoylation. Here, we describe the crystal structure of human PRMT8 (hPRMT8) at 3.0-Å resolution. The crystal structure of hPRMT8 exhibited a novel helical assembly. Biochemical, biophysical and mutagenesis experiments demonstrated that hPRMT8 forms an octamer in solution. This octameric structure is necessary for proper localization to the plasma membrane and efficient methyltransferase activity. The helical assembly might be a relevant quaternary form for hPRMT1, which is the predominant PRMT in mammalian cells and most closely related to hPRMT8.

A conserved island of BAG6/Scythe is related to ubiquitin domains and participates in short hydrophobicity recognition.

BAG6 (also called Scythe) interacts with the exposed hydrophobic regions of newly synthesized proteins and escorts them to the degradation machinery through mechanisms that remain to be elucidated. In this study, we provide evidence that BAG6 physically interacts with the model defective protein substrate CL1 in a manner that depends directly on its short hydrophobicity. We found that the N terminus of BAG6 contains an evolutionarily conserved island tentatively designated the BAG6 ubiquitin-linked domain. Partial deletion of this domain in the BAG6 N-terminal fragment abolished in cell recognition of polyubiquitinated polypeptides as well as the hydrophobicity-mediated recognition of the CL1 degron in cell and in vitro. These observations suggest a mechanism whereby the BAG6 ubiquitin-linked domain provides a platform for discriminating substrates with shorter hydrophobicity stretches as a signal for defective proteins.

Structure of a BAG6 (Bcl-2-associated athanogene 6)-Ubl4a (ubiquitin-like protein 4a) complex reveals a novel binding interface that functions in tail-anchored protein biogenesis.

BAG6 is an essential protein that functions in two distinct biological pathways, ubiquitin-mediated protein degradation of defective polypeptides and tail-anchored (TA) transmembrane protein biogenesis in mammals, although its structural and functional properties remain unknown. We solved a crystal structure of the C-terminal heterodimerization domains of BAG6 and Ubl4a and characterized their interaction biochemically. Unexpectedly, the specificity and structure of the C terminus of BAG6, which was previously classified as a BAG domain, were completely distinct from those of the canonical BAG domain. Furthermore, the tight association of BAG6 and Ubl4a resulted in modulation of Ubl4a protein stability in cells. Therefore, we propose to designate the Ubl4a-binding region of BAG6 as the novel BAG-similar (BAGS) domain. The structure of Ubl4a, which interacts with BAG6, is similar to the yeast homologue Get5, which forms a homodimer. These observations indicate that the BAGS domain of BAG6 promotes the TA protein biogenesis pathway in mammals by the interaction with Ubl4a.

Swi5-Sfr1 protein stimulates Rad51-mediated DNA strand exchange reaction through organization of DNA bases in the presynaptic filament.

The Swi5-Sfr1 heterodimer protein stimulates the Rad51-promoted DNA strand exchange reaction, a crucial step in homologous recombination. To clarify how this accessory protein acts on the strand exchange reaction, we have analyzed how the structure of the primary reaction intermediate, the Rad51/single-stranded DNA (ssDNA) complex filament formed in the presence of ATP, is affected by Swi5-Sfr1. Using flow linear dichroism spectroscopy, we observe that the nucleobases of the ssDNA are more perpendicularly aligned to the filament axis in the presence of Swi5-Sfr1, whereas the bases are more randomly oriented in the absence of Swi5-Sfr1. When using a modified version of the natural protein where the N-terminal part of Sfr1 is deleted, which has no affinity for DNA but maintained ability to stimulate the strand exchange reaction, we still observe the improved perpendicular DNA base orientation. This indicates that Swi5-Sfr1 exerts its activating effect through interaction with the Rad51 filament mainly and not with the DNA. We propose that the role of a coplanar alignment of nucleobases induced by Swi5-Sfr1 in the presynaptic Rad51/ssDNA complex is to facilitate the critical matching with an invading double-stranded DNA, hence stimulating the strand exchange reaction.

Conformational change of a unique sequence in a fungal galectin from Agrocybe cylindracea controls glycan ligand-binding specificity.

A fungal galectin from Agrocybe cylindracea (ACG) exhibits broad binding specificity for ß-galactose-containing glycans. We determined the crystal structures of wild-type ACG and the N46A mutant, with and without glycan ligands. From these structures and a saccharide-binding analysis of the N46A mutant, we revealed that a conformational change of a unique insertion sequence containing Asn46 provides two binding modes for ACG, and thereby confers broad binding specificity. We propose that the unique sequence provides these two distinct glycan-binding modes by an induced-fit mechanism.

Structural basis for vitamin D receptor agonism by novel non-secosteroidal ligands.

Non-secosteroidal ligands for vitamin D receptor (VDR) have been developed for the agonist with non-calcemic profiles. Here, we provide the structural mechanism of VDR agonism by novel non-secosteroidal ligands. All ligands had the similar efficacy, while two had the higher potency. Crystallographic analyses revealed that all ligands interacted with helix H10 and the loop between helices H6 and H7 in a similar manner, but also that the two ligands with higher potency had different interaction modes. This study suggests that distinct ligand potency depend upon differences in the formation and rearrangement of hydrogen-bond networks induced by each ligand.

Characterisation of an intrinsically disordered protein complex of Swi5-Sfr1 by ion mobility mass spectrometry and small-angle X-ray scattering.

It is now recognized that intrinsically disordered proteins (IDPs) play important roles as hubs in intracellular networks, and their structural characterisation is of significance. However, due to their highly dynamic features, it is challenging to investigate the structures of IDPs solely by conventional methods. In the present study, we demonstrate a novel method to characterise protein complexes using electrospray ionization ion mobility mass spectrometry (ESI-IM-MS) in combination with small-angle X-ray scattering (SAXS). This method enables structural characterisation even of proteins that have difficulties in crystallisation. With this method, we have characterised the Schizosaccharomyces pombe Swi5-Sfr1 complex, which is expected to have a long disordered region at the N-terminal portion of Sfr1. ESI-IM-MS analysis of the Swi5-Sfr1 complex revealed that its experimental collision cross-section (CCS) had a wide distribution, and the CCS values of the most dominant ions were ∼56% of the theoretically calculated value based on the SAXS low-resolution model, suggesting a significant size reduction in the gas phase. The present study demonstrates that the newly developed method for calculation of the theoretical CCSs of the SAXS low-resolution models of proteins allows accurate evaluation of the experimental CCS values of IDPs provided by ESI-IM-MS by comparing with the low-resolution solution structures. Furthermore, it was revealed that the combination of ESI-IM-MS and SAXS is a promising method for structural characterisation of protein complexes that are unable to crystallise.

Mechanistic insights into the activation of Rad51-mediated strand exchange from the structure of a recombination activator, the Swi5-Sfr1 complex.

Rad51 forms a helical filament on single-stranded DNA and promotes strand exchange between two homologous DNA molecules during homologous recombination. The Swi5-Sfr1 complex interacts directly with Rad51 and stimulates strand exchange. Here we describe structural and functional aspects of the complex. Swi5 and the C-terminal core domain of Sfr1 form an essential activator complex with a parallel coiled-coil heterodimer joined firmly together via two previously uncharacterized leucine-zipper motifs and a bundle. The resultant coiled coil is sharply kinked, generating an elongated crescent-shaped structure suitable for transient binding within the helical groove of the Rad51 filament. The N-terminal region of Sfr1, meanwhile, has an interface for binding of Rad51. Our data suggest that the snug fit resulting from the complementary geometry of the heterodimer activates the Rad51 filament and that the N-terminal domain of Sfr1 plays a role in the efficient recruitment of the Swi5-Sfr1 complex to the Rad51 filaments.

Peroxisome proliferator-activated receptors (PPARs) have multiple binding points that accommodate ligands in various conformations: phenylpropanoic acid-type PPAR ligands bind to PPAR in different conformations, depending on the subtype.

Human peroxisome proliferator-activated receptors (hPPARs) are ligand-dependent transcription factors that control various biological responses, and there are three subtypes: hPPARα, hPPARδ, and hPPARγ. We report here that α-substituted phenylpropanoic acid-type hPPAR agonists with similar structure bind to the hPPAR ligand binding domain (LBD) in different conformations, depending on the receptor subtype. These results might indicate that hPPAR ligand binding pockets have multiple binding points that can be utilized to accommodate structurally flexible hPPAR ligands.

Fission yeast Swi5-Sfr1 protein complex, an activator of Rad51 recombinase, forms an extremely elongated dogleg-shaped structure.

In eukaryotes, DNA strand exchange is the central reaction of homologous recombination, which is promoted by Rad51 recombinases forming a right-handed nucleoprotein filament on single-stranded DNA, also known as a presynaptic filament. Accessory proteins known as recombination mediators are required for the formation of the active presynaptic filament. One such mediator in the fission yeast Schizosaccharomyces pombe is the Swi5-Sfr1 complex, which has been identified as an activator of Rad51 that assists in presynaptic filament formation and stimulates its strand exchange reaction. Here, we determined the 1:1 binding stoichiometry between the two subunits of the Swi5-Sfr1 complex using analytical ultracentrifugation and electrospray ionization mass spectrometry. Small-angle x-ray scattering experiments revealed that the Swi5-Sfr1 complex displays an extremely elongated dogleg-shaped structure in solution, which is consistent with its exceptionally high frictional ratio (f/f(0)) of 2.0 ± 0.2 obtained by analytical ultracentrifugation. Furthermore, we determined a rough topology of the complex by comparing the small-angle x-ray scattering-based structures of the Swi5-Sfr1 complex and four Swi5-Sfr1-Fab complexes, in which the Fab fragments of monoclonal antibodies were specifically bound to experimentally determined sites of Sfr1. We propose a model for how the Swi5-Sfr1 complex binds to the Rad51 filament, in which the Swi5-Sfr1 complex fits into the groove of the Rad51 filament, leading to an active and stable presynaptic filament.

Expression, purification and crystallization of Swi5 and the Swi5-Sfr1 complex from fission yeast.

The assembly of the presynaptic filament of recombinases represents the most important step in homologous recombination. The formation of the filament requires assistance from mediator proteins. Swi5 and Sfr1 have been identified as mediators in fission yeast and these proteins form a complex that stimulates strand exchange. Here, the expression, purification and crystallization of Swi5 and its complex with an N-terminally truncated form of Sfr1 (DeltaN180Sfr1) are presented. Analytical ultracentrifugation of the purified samples showed that Swi5 and the protein complex exist as tetramers and heterodimers in solution, respectively. Swi5 was crystallized in two forms belonging to space groups C2 and R3 and the crystals diffracted to 2.7 A resolution. Swi5-DeltaN180Sfr1 was crystallized in space group P2(1)2(1)2 and the crystals diffracted to 2.3 A resolution. The crystals of Swi5 and Swi5-DeltaN180Sfr1 are likely to contain one tetramer and two heterodimers in the asymmetric unit, respectively.