Young Min Chi

Structural Basis for Antifreeze Activity of Ice-binding Protein from Arctic Yeast

Background: Ice-binding proteins improve the cold tolerance of cells by inhibiting ice growth and recrystallization. Results: Crystal structure and mutagenesis data of LeIBP suggests the B face as an ice-binding site. Conclusion: LeIBP structure adopts a β-helical fold and the aligned Thr/Ser/Ala residues are critical for ice binding. Significance: LeIBP structure can serve as a structural model for a large number of IBPs. Arctic yeast Leucosporidium sp. produces a glycosylated ice-binding protein (LeIBP) with a molecular mass of ∼25 kDa, which can lower the freezing point below the melting point once it binds to ice. LeIBP is a member of a large class of ice-binding proteins, the structures of which are unknown. Here, we report the crystal structures of non-glycosylated LeIBP and glycosylated LeIBP at 1.57- and 2.43-Å resolution, respectively. Structural analysis of the LeIBPs revealed a dimeric right-handed β-helix fold, which is composed of three parts: a large coiled structural domain, a long helix region (residues 96–115 form a long α-helix that packs along one face of the β-helix), and a C-terminal hydrophobic loop region (243PFVPAPEVV251). Unexpectedly, the C-terminal hydrophobic loop region has an extended conformation pointing away from the body of the coiled structural domain and forms intertwined dimer interactions. In addition, structural analysis of glycosylated LeIBP with sugar moieties attached to Asn185 provides a basis for interpreting previous biochemical analyses as well as the increased stability and secretion of glycosylated LeIBP. We also determined that the aligned Thr/Ser/Ala residues are critical for ice binding within the B face of LeIBP using site-directed mutagenesis. Although LeIBP has a common β-helical fold similar to that of canonical hyperactive antifreeze proteins, the ice-binding site is more complex and does not have a simple ice-binding motif. In conclusion, we could identify the ice-binding site of LeIBP and discuss differences in the ice-binding modes compared with other known antifreeze proteins and ice-binding proteins.

Structural studies of human brain-type creatine kinase complexed with the ADP-Mg2+-NO3- -creatine transition-state analogue complex

Creatine kinase is a member of the phosphagen kinase family, which catalyzes the reversible phosphoryl transfer reaction that occurs between ATP and creatine to produce ADP and phosphocreatine. Here, three structural aspects of human‐brain‐type‐creatine‐kinase (hBB‐CK) were identified by X‐ray crystallography: the ligand‐free‐form at 2.2 Å; the ADP–Mg2+, nitrate, and creatine complex (transition‐state‐analogue complex; TSAC); and the ADP–Mg2+‐complex at 2.0 Å. The structures of ligand‐bound hBB‐CK revealed two different monomeric states in a single homodimer. One monomer is a closed form, either bound to TSAC or the ADP–Mg2+‐complex, and the second monomer is an unliganded open form. These structural studies provide a detailed mechanism indicating that the binding of ADP–Mg2+ alone may trigger conformational changes in hBB‐CK that were not observed with muscle‐type‐CK.

Crystal structure of the response regulator spr1814 from Streptococcus pneumoniae reveals unique interdomain contacts among NarL family proteins.

Spr1814 belongs to the NarL/FixJ subfamily of signal transduction response regulators (RR), and has been predicted to regulate the neighboring ABC transporter, which translocates antibiotic molecules in Streptococcus pneumoniae. Here, we report the crystal structure of full-length unphosphorylated spr1814 at 1.7Å resolution. The asymmetric unit contains two spr1814 molecules, which display very different conformations. Through comparisons with other RRs structures, we concluded that one molecule adopts a general inactive conformation, whereas the other molecule adopts an intermediate conformation. The superposition of each molecule showed that rotational change of the effector domain occurred in intermediate conformational state, implying that domain rearrangement could occur upon phosphorylation.

Crystal structure of receiver domain of putative NarL family response regulator spr1814 from Streptococcus pneumoniae in the absence and presence of the phosphoryl analog beryllofluoride

Spr1814 of Streptococcus pneumoniae is a putative response regulator (RR) that has four-helix helix-turn-helix DNA-binding domain and belongs to the NarL family. The prototypical RR contains two domains, an N-terminal receiver domain linked to a variable effector domain. The receiver domain functions as a phosphorylation-activated switch and contains the typical doubly wound five-stranded α/β fold. Here, we report the crystal structure of the receiver domain of spr1814 (spr1814(R)) determined in the absence and presence of beryllofluoride as a phosphoryl analog. Based on the overall structure, spr1814(R) was shown to contain the typical fold similar with other structures of the receiver domain; however, an additional linker region connecting the receiver and DNA-binding domain was inserted into the dimer interface of spr1814(R), resulting in the formation of unique dimer interface. Upon phosphorylation, the conformational change of the linker region was observed and this suggests that domain rearrangement between the receiver domain and effector domain could occur in full-length spr1814.

Structural and Kinetic Analysis of Free Methionine-R-sulfoxide Reductase from Staphylococcus aureus CONFORMATIONAL CHANGES DURING CATALYSIS AND IMPLICATIONS FOR THE CATALYTIC AND INHIBITORY MECHANISMS

Free methionine-R-sulfoxide reductase (fRMsr) reduces free methionine R-sulfoxide back to methionine, but its catalytic mechanism is poorly understood. Here, we have determined the crystal structures of the reduced, substrate-bound, and oxidized forms of fRMsr from Staphylococcus aureus. Our structural and biochemical analyses suggest the catalytic mechanism of fRMsr in which Cys102 functions as the catalytic residue and Cys68 as the resolving Cys that forms a disulfide bond with Cys102. Cys78, previously thought to be a catalytic Cys, is a non-essential residue for catalytic function. Additionally, our structures provide insights into the enzyme-substrate interaction and the role of active site residues in substrate binding. Structural comparison reveals that conformational changes occur in the active site during catalysis, particularly in the loop of residues 97–106 containing the catalytic Cys102. We have also crystallized a complex between fRMsr and isopropyl alcohol, which acts as a competitive inhibitor for the enzyme. This isopropyl alcohol-bound structure helps us to understand the inhibitory mechanism of fRMsr. Our structural and enzymatic analyses suggest that a branched methyl group in alcohol seems important for competitive inhibition of the fRMsr due to its ability to bind to the active site.

Characterization of pneumolysin from Streptococcus pneumoniae, interacting with carbohydrate moiety and cholesterol as a component of cell membrane.

The cytolytic mechanism of cholesterol-dependent cytolysins (CDCs) requires the presence of cholesterol in the target cell membrane. Membrane cholesterol was thought to serve as the common receptor for these toxins, but not all CDCs require cholesterol for binding. One member of this toxin family, pneumolysin (PLY) is a major virulence factor of Streptococcus pneumoniae, and the mechanism via which PLY binds to its putative receptor or cholesterol on the cell membrane is still poorly understood. Here, we demonstrated that PLY interacted with carbohydrate moiety and cholesterol as a component of the cell membrane, using the inhibitory effect of hemolytic activity. The hemolytic activity of PLY was inhibited by cholesterol-MβCD, which is in a 3β configuration at the C3-hydroxy group, but is not in a 3α-configuration. In the interaction between PLY and carbohydrate moiety, the mannose showed a dose-dependent increase in the inhibition of PLY hemolytic activity. The binding ability of mannose with truncated PLYs, as determined by the pull-down assay, showed that mannose might favor binding to domain 4 rather than domains 1-3. These studies provide a new model for the mechanism of cellular recognition by PLY, as well as a foundation for future investigations into whether non-sterol molecules can serve as receptors for other members of the CDC family of toxins.

Kinetic and Structural Characterization for Cofactor Preference of Succinic Semialdehyde Dehydrogenase from Streptococcus pyogenes

The γ-Aminobutyric acid (GABA) that is found in prokaryotic and eukaryotic organisms has been used in various ways as a signaling molecule or a significant component generating metabolic energy under conditions of nutrient limitation or stress, through GABA catabolism. Succinic semialdehyde dehydrogenase (SSADH) catalyzes the oxidation of succinic semialdehyde to succinic acid in the final step of GABA catabolism. Here, we report the catalytic properties and two crystal structures of SSADH from Streptococcus pyogenes (SpSSADH) regarding its cofactor preference. Kinetic analysis showed that SpSSADH prefers NADP+ over NAD+ as a hydride acceptor. Moreover, the structures of SpSSADH were determined in an apo-form and in a binary complex with NADP+ at 1.6 Å and 2.1 Å resolutions, respectively. Both structures of SpSSADH showed dimeric conformation, containing a single cysteine residue in the catalytic loop of each subunit. Further structural analysis and sequence comparison of SpSSADH with other SSADHs revealed that Ser158 and Tyr188 in SpSSADH participate in the stabilization of the 2’-phosphate group of adenine-side ribose in NADP+. Our results provide structural insights into the cofactor preference of SpSSADH as the gram-positive bacterial SSADH.

Crystal structure of fatty acid-CoA racemase from Mycobacterium tuberculosis H37Rv.

Introduction. The classic catabolic pathway by which fatty acids are degraded is -oxidation, and a mitochondrial as well as a peroxisomal -oxidation pathway is known. Very long-chain fatty acids, 2-methyl-branched fatty acids, the side-chains of bile acid intermediates, and eicosanoids are mainly handled by the peroxisomal pathway, whereas shortand medium-chain fatty acids are oxidized mainly in mitochondria. Phytanic acid and other 3-methyl-branched fatty acids cannot undergo -oxidation, because the 3-methyl group prevents the formation of a 3-keto substituent in the dehydrogenation step. Therefore, 3-methyl-branched fatty acids first undergo -oxidation. In the case of phytanic acid, this results in the generation of 2-methyl-branched pristanic acid (2,6,10,14tetramethyl pentadecanoic acid), which is then shortened to 4,8-dimethyl nonanoic acid via peroxisomal -oxidation. The dimethyl fatty acid is then degraded further via mitochondrial -oxidation. The -oxidation of fatty acids is not a stereoselective process, so that after -oxidation of branched-chain fatty acids, both (R,R,R)and (S,R,R)isomers are formed. However, the -oxidation system is stereoselective because only the (2S)-isomer is accepted as substrate by branched-chain acyl–coenzyme A (CoA) oxidase, the first enzyme of the -oxidation system. Thus, the (R,R,R)-isomers of the metabolites of -oxidation need to be converted to their (S,R,R)-isomers by enzymatic racemization prior to further degradation. The fatty acid-CoA racemase (FAR) encoded by the far gene of Mycobacterium tuberculosis H37Rv converts several (2R)-branched-chain fatty acid-CoA’s to their (2S)stereoisomers in the metabolism of fatty acids. FAR is thought to be involved in this racemization prior to the stereospecific -oxidation, although this has not yet been demonstrated experimentally. Bhaumik et al. found three genes—mcr, far, and Rv3727—in the M. tuberculosis genome that encode proteins that are homologous to mammalian -methylacyl-CoA racemase (AMACR), which is a mitochondrial and peroxisomal enzyme that is essential in the -oxidation of bile acid intermediates and branchedchain fatty acids. Among them, only one crystal structure of -methylacyl-CoA racemase (MCR) from M. tuberculosis relative to mammalian AMACR has been reported to date. Thus, the FAR from M. tuberculosis was taken as a model protein to provide the structural information of another protein corresponding to mammalian AMACR. Moreover, regarding the strong sequence similarity of AMACR with CoA transferases of family III, the question arises whether this enzyme also catalyzes CoA trans-

Effects of the Polysaccharide from the Sporophyll of Brown Alga Undaria Pinnatifida on Serum Lipid Profile and Fat Tissue Accumulation in Rats Fed a High-Fat Diet

We investigated the effects of the polysaccharide from the sporophyll of a selected brown alga Undaria pinnatifida on serum lipid profile, fat tissue accumulation, and gastrointestinal transit time in rats fed a high-fat diet. The algal polysaccharide (AP) was prepared by the treatment of multiple cellulase-producing fungi Trichoderma reesei and obtained from the sporophyll with a yield of 38.7% (dry basis). The AP was mostly composed of alginate and fucoidan (up to 89%) in a ratio of 3.75:1. The AP was added to the high-fat diet in concentrations of 0.6% and 1.7% and was given to male Sprague-Dawley rats (5-wk-old) for 5 wk. The 1.7% AP addition notably reduced body weight gain and fat tissue accumulation, and it improved the serum lipid profile, including triglycerides, total cholesterol, and very low-density lipoprotein-cholesterol. The effects were associated with increased feces weight and shortened gastrointestinal transit time. In addition, the lipid peroxidation of the liver was decreased in both groups.

Crystallization and preliminary X-ray crystallographic studies of the ice-binding protein from the Arctic [correction of Aantarctic] yeast Leucosporidium sp. AY30.

Freezing is dangerous to cellular organisms because it causes an increase in the concentration of ions and other solutes in the plasma, denatures biomolecules and ruptures cell membranes. Some cold-adapted organisms can survive at subzero temperatures by producing proteins that bind to and inhibit the growth of ice crystals. To better understand the structure and function of these proteins, the ice-binding protein from Leucosporidium sp. AY30 (LeIBP) was overexpressed, purified and crystallized. The native crystal belonged to space group P4(3)2(1)2, with unit-cell parameters a=b=98.05, c=106.13 Å. Since LeIBP lacks any cysteine or methionine residues, two leucine residues (Leu69 and Leu155) were substituted by methionine residues in order to obtain selenomethionine-substituted LeIBP for use in multiple-wavelength anomalous diffraction (MAD) phasing. The selenomethionine-substituted mutant crystallized in the same space group as the native protein.