Michikazu Tanio

Trivalent recognition unit of innate immunity system: crystal structure of trimeric human M-ficolin fibrinogen-like domain.

Ficolins are a kind of pathogen-recognition molecule in the innate immune systems. To investigate the discrimination mechanism between self and non-self by ficolins, we determined the crystal structure of the human M-ficolin fibrinogen-like domain (FD1), which is the ligand-binding domain, at 1.9Å resolution. Although the FD1 monomer shares a common fold with the fibrinogen γ fragment and tachylectin-5A, the Asp-282–Cys-283 peptide bond, which is the predicted ligand-binding site on the C-terminal P domain, is a normal trans bond, unlike the cases of the other two proteins. The trimeric formation of FD1 results in the separation of the three P domains, and the spatial arrangement of the three predicted ligand-binding sites on the trimer is very similar to that of the trimeric collectin, indicating that such an arrangement is generally required for pathogen-recognition. The ligand binding study of FD1 in solution indicated that the recombinant protein binds to N-acetyl-d-glucosamine and the peptide Gly-Pro-Arg-Pro and suggested that the ligand-binding region exhibits a conformational equilibrium involving cis-trans isomerization of the Asp-282–Cys-283 peptide bond. The crystal structure and the ligand binding study of FD1 provide an insight of the self- and non-self discrimination mechanism by ficolins.

A structure-based mechanism for benzalacetone synthase from Rheum palmatum

Benzalacetone synthase (BAS), a plant-specific type III polyketide synthase (PKS), catalyzes a one-step decarboxylative condensation of malonyl-CoA and 4-coumaroyl-CoA to produce the diketide benzalacetone. We solved the crystal structures of both the wild-type and chalcone-producing I207L/L208F mutant of Rheum palmatum BAS at 1.8 Å resolution. In addition, we solved the crystal structure of the wild-type enzyme, in which a monoketide coumarate intermediate is covalently bound to the catalytic cysteine residue, at 1.6 Å resolution. This is the first direct evidence that type III PKS utilizes the cysteine as the nucleophile and as the attachment site for the polyketide intermediate. The crystal structures revealed that BAS utilizes an alternative, novel active-site pocket for locking the aromatic moiety of the coumarate, instead of the chalcone synthase’s coumaroyl-binding pocket, which is lost in the active-site of the wild-type enzyme and restored in the I207L/L208F mutant. Furthermore, the crystal structures indicated the presence of a putative nucleophilic water molecule which forms hydrogen bond networks with the Cys-His-Asn catalytic triad. This suggested that BAS employs novel catalytic machinery for the thioester bond cleavage of the enzyme-bound diketide intermediate and the final decarboxylation reaction to produce benzalacetone. These findings provided a structural basis for the functional diversity of the type III PKS enzymes.

Expression, purification and crystallization of a human tau-tubulin kinase 2 that phosphorylates tau protein

Tau-tubulin kinase 2 (TTBK2) is a Ser/Thr kinase that putatively phosphorylates residues Ser208 and Ser210 (numbered according to a 441-residue human tau isoform) in tau protein. Functional analyses revealed that a recombinant kinase domain (residues 1-331) of human TTBK2 expressed in insect cells with a baculovirus overexpression system retains kinase activity for tau protein. The kinase domain of TTBK2 was crystallized using the hanging-drop vapour-diffusion method. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 55.6, b = 113.7, c = 117.3 A, alpha = beta = gamma = 90.0 degrees. Diffraction data were collected to 2.9 A resolution using synchrotron radiation at BL24XU of SPring-8.

Binding site of C-reactive protein on M-ficolin

The binding abilities of human C-reactive protein (CRP) with the C-terminal fibrinogen-like (FBG) domain and the full-length form of human M-ficolin were investigated by pull-down and zonal affinity chromatography analyses. Pull-down assays using an N-acetyl-D-glucosamine (GlcNAc)-agarose column demonstrated that CRP binds to the trimeric FBG domains, and that the GlcNAc-binding ability of the FBG domain is unaffected by CRP binding. Interestingly, the full-length M-ficolin, comprising the N-terminal collagen-like (COL) and C-terminal FBG domains, displayed lower affinity for CRP, and the monomeric FBG domain showed virtually no binding to CRP, as qualitatively judged by zonal affinity chromatography using a GlcNAc column. These results indicated that CRP binding requires the trimeric form of the FBG domain, and that the presence of the COL domain reduces the interaction between CRP and M-ficolin. In addition, pull-down assays using a histidine-tag affinity column demonstrated that neither the full-length M-ficolin nor the trimeric FBG domains, immobilized through their C-terminal histidine tags, showed any affinity for CRP, indicating that the CRP binding site is located near Ala326 at the C-terminus of M-ficolin, spatially close to a neck region (around Pro115) between the FBG and COL domains. From these findings, we concluded that CRP binding is enhanced by conformational bending at the neck region of M-ficolin, to avoid steric hindrance by the COL domain. Such a situation may be generated by oligomeric M-ficolin binding to surfaces with widely distributed ligands, such as pathogens.

Histidine-regulated activity of M-ficolin.

Human M-ficolin is a pathogen-associated molecular recognition molecule in the innate immune system, and it binds to some sugars, such as GlcNAc (N-acetylglucosamine), on pathogen surfaces. From previous structural and functional studies of the FD1 (M-ficolin fibrinogen-like domain), we proposed that the ligand-binding region of FD1 exists in a conformational equilibrium between active and non-active states depending on three groups with a pK(a) of 6.2, which are probably histidine residues, and suggested that the 2-state conformational equilibrium as well as the trimer formation contributes to the discrimination mechanism between self and non-self of FD1 [Tanio, M., Kondo, S., Sugio, S. and Kohno, T. (2007) J. Biol. Chem. 282, 3889-3895]. To investigate the origins of the pH dependency, mutational analyses were performed on FD1 expressed by Brevibacillus choshinensis. The GlcNAc binding study of a series of single histidine mutants of FD1 demonstrated that His(251), His(284) and His(297) are required for the activity, and thus we concluded that the three histidines are the origins of the pH dependency of FD1. Monomeric mutants of FD1 show weaker affinity for the ligand than the trimeric wild-type, indicating that trimer formation confers high avidity for the ligand. In addition, analyses of the GlcNAc association and dissociation of FD1 provided evidence that FD1 always exchanges between the active and non-active states with the pH-dependent populations in solution. The biological roles of the histidine-regulated conformational equilibrium of M-ficolin are discussed in terms of the self and non-self discrimination mechanism.

Amino acid-selective isotope labeling of proteins for nuclear magnetic resonance study: proteins secreted by Brevibacillus choshinensis.

Here we report the first application of amino acid-type selective (AATS) isotope labeling of a recombinant protein secreted by Brevibacillus choshinensis for a nuclear magnetic resonance (NMR) study. To prepare the 15N-AATS-labeled protein, the transformed B. choshinensis was cultured in 15N-labeled amino acid-containing C.H.L. medium, which is commonly used in the Escherichia coli expression system. The analyses of the 1H-15N heteronuclear single quantum coherence (HSQC) spectra of the secreted proteins with a 15N-labeled amino acid demonstrated that alanine, arginine, asparagine, cysteine, glutamine, histidine, lysine, methionine, and valine are suitable for selective labeling, although acidic and aromatic amino acids are not suitable. The 15N labeling for glycine, isoleucine, leucine, serine, and threonine resulted in scrambling to specific amino acids. These results indicate that the B. choshinensis expression system is an alternative tool for AATS labeling of recombinant proteins, especially secretory proteins, for NMR analyses.

Trimeric structure and conformational equilibrium of M-ficolin fibrinogen-like domain

The crystal structure and ligand-binding study of M-ficolin provides an insight into its discrimination mechanism between self and non-self.

Overexpression, purification and preliminary crystallographic analysis of human M-ficolin fibrinogen-like domain.

Ficolins, which are comprised of a collagen-like domain and a fibrinogen-like domain, are a kind of pattern-recognition molecule for pathogens in the innate immunity system. To investigate the molecular mechanism of the discrimination between self and non-self by ficolins, human M-ficolin fibrinogen-like domain (FD1), which contains the ligand-binding site, was overexpressed in Pichia pastoris, purified and crystallized using the vapour-diffusion method at 293 K. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 55.16, b = 117.45, c = 55.19 angstroms, beta = 99.88 degrees, and contain three molecules per asymmetric unit. An X-ray data set was collected to 1.9 angstroms resolution using synchrotron radiation at beamline BL24XU at the SPring-8 facility in Japan.

Crystallization and preliminary crystallographic analysis of a plant type III polyketide synthase that produces benzalacetone

Benzalacetone synthase (BAS) from Rheum palmatum is a plant-specific type III polyketide synthase that catalyzes the one-step decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce the diketide 4-(4-hydroxyphenyl)-but-3-en-2-one. Recombinant BAS expressed in Escherichia coli was crystallized by the sitting-drop vapour-diffusion method. The crystals belong to space group P2(1), with unit-cell parameters a = 54.6, b = 89.6, c = 81.1 A, alpha = gamma = 90.0, beta = 100.5 degrees . Diffraction data were collected to 1.8 A resolution using synchrotron radiation at BL24XU of SPring-8.