Kimihiko Mizutani

Alternative structural state of transferrin. The crystallographic analysis of iron-loaded but domain-opened ovotransferrin N-lobe.

Transferrins bind Fe3+ very tightly in a closed interdomain cleft by the coordination of four protein ligands (Asp60, Tyr92, Tyr191, and His250 in ovotransferrin N-lobe) and of a synergistic anion, physiologically bidentate CO3 2−. Upon Fe3+uptake, transferrins undergo a large scale conformational transition: the apo structure with an opening of the interdomain cleft is transformed into the closed holo structure, implying initial Fe3+ binding in the open form. To solve the Fe3+-loaded, domain-opened structure, an ovotransferrin N-lobe crystal that had been grown as the apo form was soaked with Fe3+-nitrilotriacetate, and its structure was solved at 2.1 Å resolution. The Fe3+-soaked form showed almost exactly the same overall open structure as the iron-free apo form. The electron density map unequivocally proved the presence of an iron atom with the coordination by the two protein ligands of Tyr92-OH and Tyr191-OH. Other Fe3+ coordination sites are occupied by a nitrilotriacetate anion, which is stabilized through the hydrogen bonds with the peptide NH groups of Ser122, Ala123, and Gly124 and a side chain group of Thr117. There is, however, no clear interaction between the nitrilotriacetate anion and the synergistic anion binding site, Arg121.

High-throughput plasmid construction using homologous recombination in yeast: its mechanisms and application to protein production for X-ray crystallography

Homologous recombination is a system for repairing the broken genomes of living organisms by connecting two DNA strands at their homologous sequences. Today, homologous recombination in yeast is used for plasmid construction as a substitute for traditional methods using restriction enzymes and ligases. This method has various advantages over the traditional method, including flexibility in the position of DNA insertion and ease of manipulation. Recently, the author of this review reported the construction of plasmids by homologous recombination in the methanol-utilizing yeast Pichia pastoris, which is known to be an excellent expression host for secretory proteins and membrane proteins. The method enabled high-throughput construction of expression systems of proteins using P. pastoris; the constructed expression systems were used to investigate the expression conditions of membrane proteins and to perform X-ray crystallography of secretory proteins. This review discusses the mechanisms and applications of homologous recombination, including the production of proteins for X-ray crystallography. Graphical Abstract High-throughput construction of an expression system using Pichia pastoris: its application to production of a GFP-membrane protein fusion.

X-ray structures of transferrins and related proteins.

BACKGROUND Transferrins are a group of iron-binding proteins including serum transferrin, lactoferrin and ovotransferrin. SCOPE OF REVIEW The structures of transferrins are discussed. GENERAL SIGNIFICANCE The typical transferrin molecules are folded into two homologous lobes. X-ray crystallography revealed that each lobe is further divided into two similarly sized domains, and that an iron-binding site is contained within the inter-domain cleft. The six iron coordination sites are occupied by four residues and a bidentate carbonate anion. MAJOR CONCLUSIONS The structures of the apo- and holo-forms revealed that the transferrins undergo a large-scale conformational change upon the uptake and release of irons: domains rotate as rigid bodies around a screw axis passing through inter-domain contacts. The iron-release mechanism of transferrin N-lobe is also revealed by X-ray crystallography; two basic residues in two domains form an unusual hydrogen bond in neutral pH, and the bond should be broken and facilitate iron release at a low pH of the endosome. For ovotransferrin, the iron release kinetics of two lobes correspond well with the numbers of anion binding sites found in crystal structures. The structures of transferrins bound to other metals revealed that the flexibility of the transferrin structure allows the ability to bind to other metals. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.

Anion-mediated Fe3+ release mechanism in ovotransferrin C-lobe: a structurally identified SO4(2-) binding site and its implications for the kinetic pathway.

The differential properties of anion-mediated Fe3+ release between the N- and C-lobes of transferrins have been a focus in transferrin biochemistry. The structural and kinetic characteristics for isolated lobe have, however, been documented with the N-lobe only. Here we demonstrate for the first time the quantitative Fe3+ release kinetics and the anion-binding structure for the isolated C-lobe of ovotransferrin. In the presence of pyrophosphate, sulfate, and nitrilotriacetate anions, the C-lobe released Fe3+ with a decelerated rate in a single exponential progress curve, and the observed first order rate constants displayed a hyperbolic profile as a function of the anion concentration. The profile was consistent with a newly derived single-pathway Fe3+ release model in which the holo form is converted depending on the anion concentration into a “mixed ligand” intermediate that releases Fe3+. The apo C-lobe was crystallized in ammonium sulfate solution, and the structure determined at 2.3 Å resolution demonstrated the existence of a single bound SO 4 2 − in the interdomain cleft, which interacts directly with Thr461-OG1, Tyr431-OH, and His592-NE2 and indirectly with Tyr524-OH. The latter three groups are Fe3+-coordinating ligands, strongly suggesting the facilitated Fe3+ release upon the anion occupation at this site. The SO 4 2 − binding structure supported the single-pathway kinetic model.

High-throughput construction of expression system using yeast Pichia pastoris, and its application to membrane proteins

The well-established method for high-throughput construction of an expression system of the yeast Saccharomyces cerevisiae uses homologous recombination between an expression plasmid and a target gene (with homologous regions of the plasmid on both ends added by PCR). This method has been widely used for membrane proteins using plasmids containing GFP, and has been successfully used to investigate the cellular localization and solubilization conditions of the proteins. Although the methanol-utilizing yeast Pichia pastoris is known as an excellent expression host, a method for high-throughput construction of an expression system like that in S. cerevisiae has not been reported. In this study, we have attempted to construct expression systems via homologous recombination in P. pastoris. The insertion of genes into a plasmid could be easily checked by colony-PCR. Expression systems for seven membrane proteins of medaka fish (Oryzias latipes) and yeast (S. cerevisiae) were constructed, and the expression of proteins was analyzed by fluorescence spectra, fluorescence microscopy, and SDS-PAGE (in-gel fluorescence detection).

Structural and Functional Characterization of Recombinant Human Serum Transferrin Secreted from Pichia pastoris

Serum transferrin is an iron-binding glycoprotein with a bilobal structure. It binds iron ions in the blood serum and delivers them into target cells via transferrin receptor. We identified structural and functional characteristics of recombinant human transferrin which is produced in the yeast Pichia pastoris. Using the signal sequence of the α factor of the yeast Saccharomyces cerevisiae, high-level secretion was obtained, up to 30 mg/l of culture medium. Correct processing at designed sites was confirmed by N-terminal sequence analysis. Carbohydrate modification was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis after digestion with endo-β-N-acetylglucosaminidase H. Reflecting the secondary structure, the circular dichroism spectrum of the recombinant protein was indistinguishable from that of serum transferrin. Consequently, the recombinant product had an iron binding function just as the serum specimen has: two Fe3+ sites existed in a recombinant transferrin molecule, as estimated by titration analysis using visible absorption, fluorescence spectra, and electrophoretic behavior in urea denaturing polyacrylamide gel electrophoresis (PAGE).

Crystal Structure of Pyridoxamine-Pyruvate Aminotransferase from Mesorhizobium loti MAFF303099

Pyridoxamine-pyruvate aminotransferase (PPAT; EC 2.6.1.30) is a pyridoxal 5′-phosphate-independent aminotransferase and catalyzes reversible transamination between pyridoxamine and pyruvate to form pyridoxal and l-alanine. The crystal structure of PPAT from Mesorhizobium loti has been solved in space group P43212 and was refined to an R factor of 15.6% (Rfree = 20.6%) at 2.0Å resolution. In addition, the structures of PPAT in complexes with pyridoxamine, pyridoxal, and pyridoxyl-l-alanine have been refined to R factors of 15.6, 15.4, and 14.5% (Rfree = 18.6, 18.1, and 18.4%) at 1.7, 1.7, and 2.0Å resolution, respectively. PPAT is a homotetramer and each subunit is composed of a large N-terminal domain, consisting of seven β-sheets and eight α-helices, and a smaller C-terminal domain, consisting of three β-sheets and four α-helices. The substrate pyridoxal is bound through an aldimine linkage to Lys-197 in the active site. The α-carboxylate group of the substrate amino/keto acid is hydrogen-bonded to Arg-336 and Arg-345. The structures revealed that the bulky side chain of Glu-68 interfered with the binding of the phosphate moiety of pyridoxal 5′-phosphate and made PPAT specific to pyridoxal. The reaction mechanism of the enzyme is discussed based on the structures and kinetics results.

Structure of β-1,4-mannanase from the common sea hare Aplysia kurodai at 1.05 Å resolution

β-1,4-Mannanase (EC 3.2.1.78) catalyzes the hydrolysis of β-1,4-glycosidic bonds within mannan, a major constituent group of the hemicelluloses. Bivalves and gastropods possess β-1,4-mannanase and may degrade mannan in seaweed and/or phytoplankton to obtain carbon and energy using the secreted enzymes in their digestive systems. In the present study, the crystal structure of AkMan, a gastropod β-1,4-mannanase prepared from the common sea hare Aplysia kurodai, was determined at 1.05 Å resolution. This is the first report of the three-dimensional structure of a gastropod β-1,4-mannanase. The structure was compared with bivalve β-1,4-mannanase and the roles of residues in the catalytic cleft were investigated. No obvious binding residue was found in subsite +1 and the substrate-binding site was exposed to the molecular surface, which may account for the enzymatic properties of mannanases that can digest complex substrates such as glucomannan and branched mannan.

Structural and functional characterization of recombinant medaka fish alpha-amylase expressed in yeast Pichia pastoris.

The medaka fish α-amylase was expressed and purified. The expression systems were constructed using methylotrophic yeast Pichia pastoris, and the recombinant proteins were secreted into the culture medium. Purified recombinant α-amylase exhibited starch hydrolysis activity. The optimal pH, denaturation temperature, and K(M) and V(max) values were determined; chloride ions were essential for enzyme activity. The purified protein was also crystallized and examined by X-ray crystallography. The structure has the (α/β)(8) barrel fold, as do other known α-amylases, and the overall structure is very similar to the structure of vertebrate (human and pig) α-amylases. A novel expression plasmid was developed. Using this plasmid, high-throughput construction of an expression system by homologous recombination in P. pastoris cells, previously reported for membrane proteins, was successfully applied to the secretory protein.

Crystal structures of protein-glutaminase and its pro forms converted into enzyme-substrate complex

Background: Protein glutaminase (PG) catalyzes deamination of Gln residues in proteins. Results: The structures of mature and pro forms and a pro form mutant reveal that the side chain of Gln-47 of mutant A47Q mimics the protein substrate of PG. Conclusion: Gln-47 of A47Q forms an S-acyl covalent intermediate with the catalytic Cys. Significance: PG shares a common catalytic mechanism with transglutaminase and cysteine protease. Protein glutaminase, which converts a protein glutamine residue to a glutamate residue, is expected to be useful as a new food-processing enzyme. The crystal structures of the mature and pro forms of the enzyme were refined at 1.15 and 1.73 Å resolution, respectively. The overall structure of the mature enzyme has a weak homology to the core domain of human transglutaminase-2. The catalytic triad (Cys-His-Asp) common to transglutaminases and cysteine proteases is located in the bottom of the active site pocket. The structure of the recombinant pro form shows that a short loop between S2 and S3 in the proregion covers and interacts with the active site of the mature region, mimicking the protein substrate of the enzyme. Ala-47 is located just above the pocket of the active site. Two mutant structures (A47Q-1 and A47Q-2) refined at 1.5 Å resolution were found to correspond to the enzyme-substrate complex and an S-acyl intermediate. Based on these structures, the catalytic mechanism of protein glutaminase is proposed.