Synthesis of seleno-fucose compounds and their application to the X-ray structural determination of carbohydrate-lectin complexes using single/multi-wavelength anomalous dispersion phasing.

Selenium-incorporated fucoses (seleno-fucoses) differing in the position of the seleno-substituent were synthesized and applied to the X-ray structural determination of a carbohydrate-lectin complex using single/multi-wavelength anomalous dispersion (SAD/MAD) phasing. The hydroxyl groups at the C-1, -2, -3 and -4 position of fucose were individually substituted with a methylseleno group via a transacetalization reaction using MeSeCH2OBn or by an SN2 reaction with TolSe- equivalents to afford the corresponding MeSe-fucose. The three-dimensional structures of a fucose-binding lectin complexed with several of these MeSe-fucoses have been determined by SAD/MAD phasing by utilizing the diffraction of selenium in the bound MeSe-fucoses.

Structural basis for Arf6-MKLP1 complex formation on the Flemming body responsible for cytokinesis.

A small GTPase, Arf6, is involved in cytokinesis by localizing to the Flemming body (the midbody). However, it remains unknown how Arf6 contributes to cytokinesis. Here, we demonstrate that Arf6 directly interacts with mitotic kinesin-like protein 1 (MKLP1), a Flemming body-localizing protein essential for cytokinesis. The crystal structure of the Arf6-MKLP1 complex reveals that MKLP1 forms a homodimer flanked by two Arf6 molecules, forming a 2:2 heterotetramer containing an extended ß-sheet composed of 22 ß-strands that spans the entire heterotetramer, suitable for interaction with a concave membrane surface at the cleavage furrow. We show that, during cytokinesis, Arf6 is first accumulated around the cleavage furrow and, prior to abscission, recruited onto the Flemming body via interaction with MKLP1. We also show by structure-based mutagenesis and siRNA-mediated knockdowns that the complex formation is required for completion of cytokinesis. A model based on these results suggests that the Arf6-MKLP1 complex plays a crucial role in cytokinesis by connecting the microtubule bundle and membranes at the cleavage plane.

Six independent fucose-binding sites in the crystal structure of Aspergillus oryzae lectin.

The crystal structure of AOL (a fucose-specific lectin of Aspergillus oryzae) has been solved by SAD (single-wavelength anomalous diffraction) and MAD (multi-wavelength anomalous diffraction) phasing of seleno-fucosides. The overall structure is a six-bladed ß-propeller similar to that of other fucose-specific lectins. The fucose moieties of the seleno-fucosides are located in six fucose-binding sites. Although the Arg and Glu/Gln residues bound to the fucose moiety are common to all fucose-binding sites, the amino-acid residues involved in fucose binding at each site are not identical. The varying peak heights of the seleniums in the electron density map suggest that each fucose-binding site has a different carbohydrate binding affinity.

Expanded potential of seleno-carbohydrates as a molecular tool for X-ray structural determination of a carbohydrate-protein complex with single/multi-wavelength anomalous dispersion phasing.

Seleno-lactoses have been successfully synthesized as candidates for mimicking carbohydrate ligands for human galectin-9 N-terminal carbohydrate recognition domain (NCRD). Selenium was introduced into the mono- or di-saccharides using p-methylselenobenzoic anhydride (Tol2Se) as a novel selenating reagent. The TolSe-substituted monosaccharides were converted into selenoglycosyl donors or acceptors, which were reacted with coupling partners to afford seleno-lactoses. The seleno-lactoses were converted to the target compounds. The structure of human galectin-9 NCRD co-crystallized with 6-MeSe-lactose was determined with single/multi-wavelength anomalous dispersion (SAD/MAD) phasing and was similar to that of the co-crystal with natural lactose.

Structural basis for membrane binding specificity of the Bin/Amphiphysin/Rvs (BAR) domain of Arfaptin-2 determined by Arl1 GTPase.

Membrane-sculpting BAR (Bin/Amphiphysin/Rvs) domains form a crescent-shaped homodimer that can sense and induce membrane curvature through its positively charged concave face. We have recently shown that Arfaptin-2, which was originally identified as a binding partner for the Arf and Rac1 GTPases, binds to Arl1 through its BAR domain and is recruited onto Golgi membranes. There, Arfaptin-2 induces membrane tubules. Here, we report the crystal structure of the Arfaptin-2 BAR homodimer in complex with two Arl1 molecules bound symmetrically to each side, leaving the concave face open for membrane association. The overall structure of the Arl1·Arfaptin-2 BAR complex closely resembles that of the PX-BAR domain of sorting nexin 9, suggesting similar mechanisms underlying BAR domain targeting to specific organellar membranes. The Arl1·Arfaptin-2 BAR structure suggests that one of the two Arl1 molecules competes with Rac1, which binds to the concave face of the Arfaptin-2 BAR homodimer and may hinder its membrane association.

Structural basis of preferential binding of fucose-containing saccharide by the Caenorhabditis elegans galectin LEC-6.

Galectins are a group of lectins that can bind carbohydrate chains containing ß-galactoside units. LEC-6, a member of galectins of Caenorhabditis elegans, binds fucose-containing saccharides. We solved the crystal structure of LEC-6 in complex with galactose-ß1,4-fucose (Galß1-4Fuc) at 1.5 Å resolution. The overall structure of the protein and the identities of the amino-acid residues binding to the disaccharide are similar to those of other galectins. However, further structural analysis and multiple sequence alignment between LEC-6 and other galectins indicate that a glutamic acid residue (Glu67) is important for the preferential binding between LEC-6 and the fucose moiety of the Galß1-4Fuc unit. Frontal affinity chromatography analysis indicated that the affinities of E67D and E67A mutants for Galß1-4Fuc are lower than that of wild-type LEC-6. Furthermore, the affinities of Glu67 mutants for an endogenous oligosaccharide, which contains a Galß1-4Fuc unit, are drastically reduced relative to that of the wild-type protein. We conclude that the Glu67 in the oligosaccharide-binding site assists the recognition of the fucose moiety by LEC-6.

Platform for the rapid construction and evaluation of GPCRs for crystallography in Saccharomyces cerevisiae.

BACKGROUND: Recent successes in the determination of G-protein coupled receptor (GPCR) structures have relied on the ability of receptor variants to overcome difficulties in expression and purification. Therefore, the quick screening of functionally expressed stable receptor variants is vital. RESULTS: We developed a platform using Saccharomyces cerevisiae for the rapid construction and evaluation of functional GPCR variants for structural studies. This platform enables us to perform a screening cycle from construction to evaluation of variants within 6-7 days. We firstly confirmed the functional expression of 25 full-length class A GPCRs in this platform. Then, in order to improve the expression level and stability, we generated and evaluated the variants of the four GPCRs (hADRB2, hCHRM2, hHRH1 and hNTSR1). These stabilized receptor variants improved both functional activity and monodispersity. Finally, the expression level of the stabilized hHRH1 in Pichia pastoris was improved up to 65 pmol/mg from negligible expression of the functional full-length receptor in S. cerevisiae at first screening. The stabilized hHRH1 was able to be purified for use in crystallization trials. CONCLUSIONS: We demonstrated that the S. cerevisiae system should serve as an easy-to-handle and rapid platform for the construction and evaluation of GPCR variants. This platform can be a powerful prescreening method to identify a suitable GPCR variant for crystallography.

Advanced method for high-throughput expression of mutated eukaryotic membrane proteins in Saccharomyces cerevisiae.

Crystallization of eukaryotic membrane proteins is a challenging, iterative process. The protein of interest is often modified in an attempt to improve crystallization and diffraction results. To accelerate this process, we took advantage of a GFP-fusion yeast expression system that uses PCR to direct homologous recombination and gene cloning. We explored the possibility of employing more than one PCR fragment to introduce various mutations in a single step, and found that when up to five PCR fragments were co-transformed into yeast, the recombination frequency was maintained as the number of fragments was increased. All transformants expressed the model membrane protein, while the resulting plasmid from each clone contained the designed mutations only. Thus, we have demonstrated a technique allowing the expression of mutant membrane proteins within 5 days, combining a GFP-fusion expression system and yeast homologous recombination.

Hexameric ring structure of the ATPase domain of the membrane-integrated metalloprotease FtsH from Thermus thermophilus HB8.

FtsH is a cytoplasmic membrane-integrated, ATP-dependent metalloprotease, which processively degrades both cytoplasmic and membrane proteins in concert with unfolding. The FtsH protein is divided into the N-terminal transmembrane region and the larger C-terminal cytoplasmic region, which consists of an ATPase domain and a protease domain. We have determined the crystal structures of the Thermus thermophilus FtsH ATPase domain in the nucleotide-free and AMP-PNP- and ADP-bound states, in addition to the domain with the extra preceding segment. Combined with the mapping of the putative substrate binding region, these structures suggest that FtsH internally forms a hexameric ring structure, in which ATP binding could cause a conformational change to facilitate transport of substrates into the protease domain through the central pore.

A new structure determination method of lectins using a selenium-containing sugar ligand.

Phase determination is essential for solving X-ray crystal structures of proteins and their complexes. Conventional phase-determination methods using heavy atoms (Pt, Au, Hg, etc.) or the selenium (Se) atom are routinely utilized in structure determination of protein crystals. Here, we describe an alternative phase-determination method for proteins such as lectins in which a Se-containing glycan is used as a ligand. In this technique, the Se atoms are simply introduced into the protein crystal as a complex, and the phase of the protein can be determined using anomalous signals from the Se-containing sugar.

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.

Structure of a central stalk subunit F of prokaryotic V-type ATPase/synthase from Thermus thermophilus.

The crystal structure of subunit F of vacuole-type ATPase/synthase (prokaryotic V-ATPase) was determined to of 2.2 A resolution. The subunit reveals unexpected structural similarity to the response regulator proteins that include the Escherichia coli chemotaxis response regulator CheY. The structure was successfully placed into the low-resolution EM structure of the prokaryotic holo-V-ATPase at a location indicated by the results of crosslinking experiments. The crystal structure, together with the single-molecule analysis using fluorescence resonance energy transfer, showed that the subunit F exhibits two conformations, a 'retracted' form in the absence and an 'extended' form in the presence of ATP. Our results postulated that the subunit F is a regulatory subunit in the V-ATPase.

Stabilization of FtsH-unfolded protein complex by binding of ATP and blocking of protease.

The function of an ATP-dependent membrane protease FtsH was investigated using the enzyme from Thermus thermophilus HB8. An FtsH mutant with replacement of Glu-419 in the zinc-binding motif by Cys lost the activity to digest casein, a model unfolded protein, and the small ATPase activity of this mutant was no longer stimulated by casein. In the presence of ATP or ATPgammaS, but not ADP, a mutant FtsH-unfolded protein complex was isolated, indicating that ATP binding, but not ATP hydrolysis, is required for FtsH to form a stable complex with an unfolded protein. The FtsH without mutation at Glu-419 did not produce a stable complex with casein in the presence of any nucleotides tested and therefore it appears that blocking proteolysis also contributes to stabilization of the complex.

Crystal structure of a central stalk subunit C and reversible association/dissociation of vacuole-type ATPase.

The vacuole-type ATPases (V-ATPases) exist in various intracellular compartments of eukaryotic cells to regulate physiological processes by controlling the acidic environment. The crystal structure of the subunit C of Thermus thermophilus V-ATPase, homologous to eukaryotic subunit d of V-ATPases, has been determined at 1.95-A resolution and located into the holoenzyme complex structure obtained by single particle analysis as suggested by the results of subunit cross-linking experiments. The result shows that V-ATPase is substantially longer than the related F-type ATPase, due to the insertion of subunit C between the V(1) (soluble) and the V(o) (membrane bound) domains. Subunit C, attached to the V(o) domain, seems to have a socket like function in attaching the central-stalk subunits of the V(1) domain. This architecture seems essential for the reversible association/dissociation of the V(1) and the V(o) domains, unique for V-ATPase activity regulation.