Biomedical subjects

K Nikaido

Publications and source records attributed to K Nikaido.

At least 19 recordsLinked to original sources

PubMed2001

Purification and characterization of the membrane-bound complex of an ABC transporter, the histidine permease.

The bacterial histidine permease, an ABC transporter, from Salmonella typhimurium is composed of a membrane-bound complex, HisQMP2, comprising two hydrophobic subunits (HisQ and HisM), two copies of an ATP-hydrolyzing subunit, HisP, and a soluble receptor, HisJ. We describe the purification and characterization of HisQMP2 using a 6-histidines extension at the carboxy terminus of HisP [HisQMP2(his6)]. The purification is rapid and effective, giving a seven-fold purification with a yield of 85 and 98% purity. Two procedures are described differing in the detergent used (decanoylsucrose and octylglucoside, respectively) and in the presence of phospholipid. HisQMP2(his6) has ATPase and transport activities upon reconstitution into proteoliposomes (PLS). HisQMP2(his6) has a low level ATPase activity (intrinsic activity), which is stimulated to a different extent by the receptor--liganded and unliganded. Its pH optimum is 7.8-8.0, it requires a cation for activity and it displays cooperativity for ATP. The effect of various ATP analogs was analyzed. Determination of the molecular size of HisQMP2(his6) indicates that it is a monomer. The permeability properties of two kinds of reconstituted PLS preparations are described.

ATP-Binding Cassette Transporters↗

PubMed2000

Over-expression of cbaAB genes of Bacillus stearothermophilus produces a two-subunit SoxB-type cytochrome c oxidase with proton pumping activity.

We constructed expression plasmids containing cbaAB, the structural genes for the two-subunit cytochrome bo(3)-type cytochrome c oxidase (SoxB type) recently isolated from a Gram-positive thermophile Bacillus stearothermophilus. B. stearothermophilus cells transformed with the plasmids over-expressed an enzymatically active bo(3)-type cytochrome c oxidase protein composed of the two subunits, while the transformed Escherichia coli cells produced an inactive protein composed of subunit I without subunit II. The oxidase over-expressed in B. stearothermophilus was solubilized and purified. The oxidase contained protoheme IX and heme O, as the main low-spin heme and the high-spin heme, respectively. Analysis of the substrate specificity indicated that the high-affinity site is very specific for cytochrome c-551, a cytochrome c that is a membrane-bound lipoprotein of thermophilic Bacillus. The purified enzyme reconstituted into liposomal vesicles with cytochrome c-551 showed H(+) pumping activity, although the efficiency was lower than those of cytochrome aa(3)-type oxidases belonging to the SoxM-type.

Bacterial Proteins↗

PubMed1999

One intact ATP-binding subunit is sufficient to support ATP hydrolysis and translocation in an ABC transporter, the histidine permease.

The membrane-bound complex of the Salmonella typhimurium histidine permease, a member of the ABC transporters (or traffic ATPases) superfamily, is composed of two integral membrane proteins, HisQ and HisM, and two copies of an ATP-binding subunit, HisP, which hydrolyze ATP, thus supplying the energy for translocation. The three-dimensional structure of HisP has been resolved. Extensive evidence indicates that the HisP subunits form a dimer. We investigated the mechanism of action of such a dimer, both within the complex and in soluble form, by creating heterodimers between the wild type and mutant HisP proteins. The data strongly suggest that within the complex both subunits hydrolyze ATP and that one subunit is activated by the other. In a heterodimer containing one wild type and one hydrolysis defective subunit both hydrolysis and ligand translocation occur at half the rate of the wild type. Soluble HisP also hydrolyzes ATP if one subunit is inactive; its specific activity is identical to that of the wild type, indicating that only one of the subunits in a soluble dimer is involved in hydrolysis. We show that the activating ability varies depending on the nature of the substitution of a well conserved residue, His-211.

ATP-Binding Cassette Transporters↗

PubMed1998

Crystal structure of the ATP-binding subunit of an ABC transporter.

ABC transporters (also known as traffic ATPases) form a large family of proteins responsible for the translocation of a variety of compounds across membranes of both prokaryotes and eukaryotes. The recently completed Escherichia coli genome sequence revealed that the largest family of paralogous E. coli proteins is composed of ABC transporters. Many eukaryotic proteins of medical significance belong to this family, such as the cystic fibrosis transmembrane conductance regulator (CFTR), the P-glycoprotein (or multidrug-resistance protein) and the heterodimeric transporter associated with antigen processing (Tap1-Tap2). Here we report the crystal structure at 1.5 A resolution of HisP, the ATP-binding subunit of the histidine permease, which is an ABC transporter from Salmonella typhimurium. We correlate the details of this structure with the biochemical, genetic and biophysical properties of the wild-type and several mutant HisP proteins. The structure provides a basis for understanding properties of ABC transporters and of defective CFTR proteins.

ATP-Binding Cassette Transporters↗

PubMed1998

The cbaAB genes for bo3-type cytochrome c oxidase in Bacillus stearothermophilus.

Structural genes were cloned for cytochrome bo3-type cytochrome c oxidase recently isolated from a Gram-positive thermophile Bacillus stearothermophilus. Sequencing and Northern blotting analyses indicated that the two genes cbaA and cbaB composed an operon encoding for subunits I and II, respectively, and that the oxidase was SoxB-type. They are the first genes for a SoxB-type cytochrome c oxidase whose natural substrate is known.

Amino Acid Sequence↗

PubMed1997

Purification and characterization of HisP, the ATP-binding subunit of a traffic ATPase (ABC transporter), the histidine permease of Salmonella typhimurium. Solubility, dimerization, and ATPase activity.

The nucleotide-binding subunit, HisP, of the histidine permease, a traffic ATPase (ABC transporter), has been purified as a soluble protein and characterized. Addition of a 6-histidine extension (HisP(His6)) allows a rapid and effective metal affinity purification, giving a 30-fold purification with a yield of 50%. HisP(his6) is indistinguishable from underivatized HisP when incorporated into the permease membrane-bound complex, HisQMP2. Purified HisP(his6) has a strong tendency to precipitate; 5 mM ATP and 20% glycerol maintain it in solution at a high protein concentration. HisP(his6) is active as a dimer, binds ATP with a Kd value of 205 microM, and hydrolyzes it at a rate comparable to that of HisQMP2; in contrast to the latter, it does not display cooperativity for ATP. HisP(his6) has been characterized with respect to substrate and inhibitor specificity and various physico-chemical characteristics. Its pH optimum is 7 and it requires a cation for activity, with Co2+ and Mn2+ being more effective than Mg2+ at lower concentrations but inhibitory in the higher concentration range. In contrast to the intact complex, HisP(his6) is not inhibited by vanadate but is inhibited by N-ethylmaleimide. Neither the soluble receptor, HisJ, nor the transport substrate, histidine, has any effect on the activity.

ATP-Binding Cassette Transporters↗

PubMed1996

Protein globularization during folding. A study by synchrotron small-angle X-ray scattering.

Various conformational states of polypeptide chains were investigated by synchrotron small-angle X-ray scattering (SAXS). SAXS patterns of proteins and model polypeptides in globular states (native and "molten globule") and in non-globular states (unfolded protein as well as randomly coiled, partially alpha-helical and partially beta-structural synthetic polypeptides) were analyzed in terms of Guinier and Kratky plots. Large differences in the SAXS pattern have been found between globular and non-globular conformations of the polypeptide chains, and they have been interpreted in terms of differences in the shape and size of the globular and non-globular scatterers with the same molecular mass. The equilibrium and time-resolved unfolding curves of bovine carbonic anhydrase and yeast phosphoglycerate kinase were monitored by integrated SAXS intensity, and were found to be coincident with the curves measured by other physicochemical techniques, such as tryptophan fluorescence and peptide circular dichroism spectra. The intermolecular association of the protein "molten globule"-like intermediates accumulated during the guanidine hydrochloride-induced unfolding of bovine carbonic anhydrase has been investigated by various SAXS parameters. It has been shown that the integrated SAXS intensity is much less sensitive to the protein intermolecular association than the zero angle intensity and the radius of gyration. We propose the integrated SAXS intensity as a global parameter which is particularly appropriate for fast kinetic studies of protein coil to globule transitions. Time-resolved refolding curves of the above proteins were monitored by the integrated SAXS intensity to investigate the globularization process in protein folding. Two fast kinetic processes for bovine carbonic anhydrase and two fast (each within two seconds) as well as two slow (within 500 seconds) kinetic processes for yeast phosphoglycerate kinase have been recorded. The kinetic processes reflect both protein intramolecular globularization and its intermolecular association.

Animals↗

PubMed1996

Liganded and unliganded receptors interact with equal affinity with the membrane complex of periplasmic permeases, a subfamily of traffic ATPases.

The histidine-binding protein, HisJ, is the soluble receptor for the periplasmic histidine permease of Salmonella typhimurium. The receptor binds the substrate in the periplasm, interacts with the membrane-bound complex, transmits a transmembrane signal to hydrolyze ATP, and releases the ligand for translocation. HisJ, like other periplasmic receptors, has two lobes that are apart in the unliganded structure (open conformation) and drawn close together in the liganded structure (closed conformation), burying deeply the ligand. Such receptors are postulated to interact with the membrane-bound complex with high affinity in their liganded conformation, and, upon substrate translocation, to undergo a reduction in affinity and therefore be released. Here we show that in contrast to the current postulate, liganded and unliganded receptors have equal affinity for the membrane-bound complex. The affinity is measured both by chemical cross-linking and co-sedimentation procedures. An ATPase activity assay is also used to demonstrate the interaction of unliganded receptor with the membrane-bound complex. These findings support a new model for the transport mechanism, in which the soluble receptor functions independently of the commonly accepted high-low affinity switch.

ATP-Binding Cassette Transporters↗

PubMed1994

The histidine-binding protein undergoes conformational changes in the absence of ligand as analyzed with conformation-specific monoclonal antibodies.

The periplasmic histidine-binding protein, HisJ, and the lysine-, arginine-, ornithine-binding protein (LAO) are receptors for histidine transport via the histidine permease of Salmonella typhimurium. The receptors have similar structures, being composed of two lobes held together by two peptide segments, with the ligand-binding site located in a cleft between the lobes. The two lobes are far apart in the unliganded structure (open conformation) and are drawn close together in the liganded structure (closed conformation). The tight binding of the ligand via protein side chains as well as the peptide backbone from both lobes stabilizes the closed conformation (Oh, B.-H., Pandit, J., Kang, C.-H., Nikaido, K., Gokcen, S., Ames, G. F.-L., and Kim, S.-H. (1993) J. Biol. Chem. 268, 11348-11353; Oh, B.-H., Kang, C.-H., De Bondt, H., Kim, S.-H., Nikaido, K., Joshi, A., and Ames, G. F.-L. (1994) J. Biol. Chem. 269, 4135-4143). In this study two conformation-specific monoclonal antibodies (mAbs) that trap the protein in the closed empty form have been characterized and used to provide evidence that HisJ can assume the closed empty form in the absence of ligand. Several pieces of evidence were provided to demonstrate that these mAbs are specific for HisJ in the closed form. Histidine improves the interaction of these mAbs with immobilized HisJ. The mAbs inhibit both the exchange and the dissociation of histidine from HisJ, indicating that they are able to trap the protein in the closed liganded form. The characterization of the epitopes of the conformation-specific mAbs shows that they include residues that are located in both lobes and that are far apart in the open form but close to each other in the closed form, so that the mAbs must be sensitive to their spatial orientation. Two mAbs that are not conformation-specific according to these criteria were also identified.

Antibodies, Monoclonal↗

PubMed1994

The bacterial periplasmic histidine-binding protein. structure/function analysis of the ligand-binding site and comparison with related proteins.

Bacterial periplasmic binding proteins are initial receptors in the process of active transport across cell membranes and/or chemotaxis. Among them, the histidine-binding protein (HisJ) has been extensively studied from the biochemical, physiological, and genetic points of view. The three-dimensional crystal structure of the histidine-binding protein complexed with histidine has been determined at 2.5-A resolution by the molecular replacement method using a probe structure the previously solved lysine-liganded structure of the lysine-, arginine-, ornithine-binding protein (LAO), which shares 70% sequence identity with HisJ. The structure is bi-lobate; the two lobes, one bigger than the other, are connected by two short strands and are in contact with each other (closed) enclosing the histidine. Charged, polar, and non-polar side chains, as well as the peptide backbone, are involved in tight binding of the histidine. The bound histidine is involved in eight direct hydrogen bonds, six with the bigger lobe and two with the smaller lobe, in one potential water-mediated hydrogen bond with the bigger lobe, as well as in ionic interactions. The HisJ residues surrounding the ligand are the same as the LAO residues interacting with lysine, except for residue 52 which is leucine in HisJ and phenylalanine in LAO. The Leu-52 in HisJ makes a hydrophobic interaction with the imidazole ring of histidine. Of seven mutations affecting the ligand-binding site, five are located in the ligand-binding site, one in a connecting strand, and one at the domains interface. Based on comparisons among related binding proteins, the specific interactions between the ligands and the respective binding protein residues are predicted for the glutamine-binding protein and the opines-binding protein.

Bacterial Proteins↗

PubMed1994

The chaperonin GroEL does not recognize apo-alpha-lactalbumin in the molten globule state.

We investigate here the interaction between GroEL and two kinds of non-native alpha-lactalbumin. alpha-Lactalbumin is a Ca(2+)-binding protein which assumes a molten globule state in the absence of Ca2+ (apo-alpha-lactalbumin) at neutral pH. Our results, obtained by molecular-sieve chromatography and hydrogen-exchange measurements, show that apo-alpha-lactalbumin in this molten globule state is not bound to GroEL either in the absence or in the presence of KCl. On the other hand, we show by molecular-sieve chromatography that alpha-lactalbumin, in which the four disulphide bonds are fully reduced, is bound to GroEL when 50 mM KCl is present. The results demonstrate that the protein state recognized by GroEL is more unfolded and expanded than the typical molten globule state of alpha-lactalbumin.

Animals↗

PubMed1993

Three-dimensional structures of the periplasmic lysine/arginine/ornithine-binding protein with and without a ligand.

Many proteins exhibit a large-scale movement of rigid globular domains. Among these, bacterial periplasmic binding proteins involved in substrate transport, or transport and chemotaxis, can be used as prototypes for understanding the mechanism of the movement. Such movements have been found to be associated with specific functions, such as substrate binding, catalysis, and recognition by other biomolecules. We have determined the three-dimensional structures of the lysine/arginine/ornithine-binding protein (LAO) from Salmonella typhimurium with and without lysine by x-ray crystallographic methods at 1.8- and 1.9-A resolution, respectively. The structures are composed of two lobes held together by two short connecting strands. The two lobes are far apart in the unliganded structure, but in contact with each other in the lysine-liganded structure. The large movement of the lobes is a consequence of a 52 degrees rotation of a single backbone torsion angle in the first connecting strand and of distributed smaller changes of three backbone torsion angles of the second connecting strand. The absence of contact between the lysine and the connecting strands suggests that the ligand does not induce the conformational change directly. We instead propose that the unliganded protein undergoes a dynamic change between an "open" and a "closed" conformation and that the role of the ligand is to stabilize the closed conformation. We discuss the nature of a surface area which might be recognized by the membrane-bound complex of these amino acids transport systems.

Amino Acid Sequence↗

PubMed1992

Purification and characterization of the periplasmic lysine-, arginine-, ornithine-binding protein (LAO) from Salmonella typhimurium.

The lysine-, arginine-, ornithine-binding protein (LAO) from Salmonella typhimurium has been purified to homogeneity and characterized. The dissociation constants (KD) were determined by equilibrium dialysis assay to be 14, 15, and 29 nM for L-arginine, L-lysine, and L-ornithine respectively. L-Histidine was found to be a relatively good ligand (KD, 500 nM). Methods have been developed for the separation of liganded from unliganded LAO, for the estimation of bound ligand, and for unliganding LAO. Liganded and unliganded LAO are shown to have distinct UV spectra. The UV spectrum also varies with the nature of the substrate. Inhibition studies with substrate analogs yielded information useful for understanding the nature of the ligand-binding pocket.

Arginine↗

PubMed1991

Identification and characterization of porins in Pseudomonas aeruginosa.

Earlier studies have shown that the major porin species in Pseudomonas aeruginosa outer membrane is protein F (OprF), which produces channels wider than those produced by Escherichia coli porins. In contrast, Yoshihara and Nakae ((1989) J. Biol. Chem. 264, 6297-6301) reported that protein F has no pore-forming activity as measured by the flux of L-arabinose, and that the channels in P. aeruginosa outer membrane, being produced by proteins C, "D," and "E," are much narrower than E. coli porin channels. In this study, we followed the protein purification scheme of Yoshihara and Nakae as closely as possible, and found that protein F had a specific activity for pore formation similar to that of proteins D1, D2, and E2. Furthermore, proteoliposome reconstitution assays showed conclusively that the channels formed by protein F, as well as by unfractionated outer membranes, allowed the diffusion of a tetrasaccharide, stachyose, at a significant rate, indicating that these channels are much larger than E. coli porin channels. It appears likely that in the study of Yoshihara and Nakae protein F was inadvertently inactivated during purification. We further suggest a hypothesis that resolves the apparent conflict between the presence of large diameter channels and the low permeability of the outer membrane in P. aeruginosa.

Arabinose↗

PubMed1990

Structure of DNA polymerase alpha-primase complexes from mammalian cells analyzed by using monoclonal antibodies.

The molecular masses of two of the four DNA polymerase alpha-primase complex subunit peptides from various mammalian cells have been compared through the use of specific monoclonal antibodies. One monoclonal antibody (E4) binds to 77-kDa peptide from HeLa cells and cognate peptides from other mammalian cells (monkey, mouse, bovine, Indian muntjac, and hamster). Another monoclonal antibody (A5) binds the 180-kDa type peptide and its degradation product (160-kDa peptide) of the mammalian DNA polymerase alpha-primase complexes. Neither of these antibodies reacts with DNA polymerase alpha-primase complex from chicken cells. Comparative immunoblot analysis indicates that the molecular masses of the two main peptides of DNA polymerase alpha-primase complex isolated from the various mammalian sources are in excellent agreement with each other, except for the 77-kDa type peptide from bovine and Indian muntjac cells which was found to be significantly smaller (68 kDa) in these cases. The small molecular mass of bovine 77-kDa type peptide is not attributable to the action of a protease which may be present in the extract of bovine cells.

Animals↗

PubMed1989

Crystallization and preliminary X-ray studies of HisJ and LAO periplasmic proteins from Salmonella typhimurium.

Two periplasmic binding proteins, HisJ and LAO, which are involved in histidine and arginine transport, respectively, have been crystallized. Preliminary X-ray diffraction studies of the HisJ and LAO crystals show that both belong to the orthorhombic space group P2(1)2(1)2(1) and have unit cell dimensions of a = 39.26 A, b = 66.17 A, c = 88.33 A and a = 36.08 A, b = 78.34 A, c = 102.02 A, respectively. Both HisJ and LAO crystals diffract beyond 2.0 A resolution.

Bacterial Proteins↗

PubMed1989

Reconstitution of periplasmic transport in inside-out membrane vesicles. Energization by ATP.

The periplasmic histidine permease of Salmonella typhimurium has been reconstituted in inside-out vesicles (IOV) of Escherichia coli by disrupting the cells with a French press in the presence of a high concentration of the periplasmic histidine-binding protein, HisJ. Efflux from IOV, which is equivalent to uptake in whole cells, is induced by ATP. The reconstituted system depends on the presence of the membrane-bound permease proteins, HisQ, HisM, and HisP, and does not function if reconstitution is performed in the presence of a mutant HisJ protein, HisJ5625, that can bind histidine normally but can't interact properly with the membrane complex. Efflux is not induced by the nonhydrolyzable ATP analog, adenyl-5'-yl imidodiphosphate, supporting the contention that ATP hydrolysis is necessary. 8-Azido ATP inactivates IOV, indicating that the ATP effect occurs through the HisP protein, which has previously been shown to be modified by 8-azido ATP (Hobson, A., Weatherwax, R., and Ames, G.F.-L. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 733-7337). The estimated Km of the vesicles for ATP is about 200 microM. Vanadate, an inhibitor of phosphohydrolase enzymes, inhibits ATP-induced efflux. We conclude that ATP is likely to be the proximal energy source for periplasmic permeases.

Adenosine Triphosphate↗

PubMed1988

Formaldehyde and photoactivatable cross-linking of the periplasmic binding protein to a membrane component of the histidine transport system of Salmonella typhimurium.

The histidine permease of Salmonella typhimurium consists of four protein components, one located in the periplasm and three in the cytoplasmic membrane. Genetic evidence indicated that the periplasmic protein interacts with the membrane proteins during transport. We have utilized two different methods to demonstrate that the periplasmic protein cross-links specifically to one of the membrane components, the Q protein. Formaldehyde, a water-soluble permeant molecule was used in vivo. Sulfosuccinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate, a photoactivatable cross-linking reagent, was used in vitro in a reconstituted membrane vesicle system. Furthermore, we show that a mutant periplasmic protein, capable of binding substrate but not transporting it, is defective in cross-linking to the membrane protein, indicating this interaction to be a crucial step in the mechanism of transport.

ATP-Binding Cassette Transporters↗