Takanori Matsuura

Crystal structure of the membrane fusion protein, MexA, of the multidrug transporter in Pseudomonas aeruginosa

The MexAB-OprM efflux pump of Pseudomonas aeruginosa is central to multidrug resistance of this organism, which infects immunocompromised hospital patients. The MexA, MexB, and OprM subunits were assumed to function as the membrane fusion protein, the body of the transporter, and the outer membrane channel protein, respectively. For better understanding of this important xenobiotic transporter, we show the x-ray crystallographic structure of MexA at a resolution of 2.40 Å. The global MexA structure showed unforeseen new features with a spiral assembly of six and seven protomers that were joined together at one end by a pseudo 2-fold image. The protomer showed a new protein structure with a tandem arrangement consisting of at least three domains and presumably one more. The rod domain had a long hairpin of twisted coiled-coil that extended to one end. The second domain adjacent to the rod α-helical domain was globular and constructed by a cluster of eight short β-sheets. The third domain located distal to the α-helical rod was globular and composed of seven short β-sheets and one short α-helix. The 13-mer was shaped like a woven rattan cylinder with a large internal tubular space and widely opened flared ends. The 6-mer and 7-mer had a funnel-like structure consisting of a tubular rod at one side and a widely opened flared funnel top at the other side. Based on these results, we constructed a model of the MexAB-OprM pump assembly. The three pairs of MexA dimers interacted with the periplasmic α-barrel domain of OprM via the α-helical hairpin, the second domain interacted with both MexB and OprM at their contact site, and the third and disordered domains probably interacted with the distal domain of MexB. In this fashion, the MexA subunit connected MexB and OprM, indicating that MexA is the membrane bridge protein.

Data Deposition and Annotation at the Worldwide Protein Data Bank

The Protein Data Bank (PDB) is the repository for three-dimensional structures of biological macromolecules, determined by experimental methods. The data in the archive is free and easily available via the Internet from any of the worldwide centers managing this global archive. These data are used by scientists, researchers, bioinformatics specialists, educators, students, and general audiences to understand biological phenomenon at a molecular level. Analysis of this structural data also inspires and facilitates new discoveries in science. This chapter describes the tools and methods currently used for deposition, processing, and release of data in the PDB. References to future enhancements are also included.

High-resolution X-ray analysis reveals binding of arginine to aromatic residues of lysozyme surface: implication of suppression of protein aggregation by arginine

While biotechnological applications of arginine (Arg) as a solution additive that prevents protein aggregation are increasing, the molecular mechanism of its effects remains unclear. In this study, we investigated the Arg-lysozyme complex by high-resolution crystallographic analysis. Three Arg molecules were observed to be in close proximity to aromatic amino acid residues of the protein surface, and their occupancies gradually increased with increasing Arg concentration. These interactions were mediated by electrostatic, hydrophobic and cation-π interactions with the surface residues. The binding of Arg decreased the accessible surface area of aromatic residues by 40%, but increased that of charged residues by 10%. These changes might prevent intermolecular hydrophobic interactions by shielding hydrophobic regions of the lysozyme surface, resulting in an increase in protein solubility.

Structural Diversity in the Cytoplasmic Region of G Protein-Gated Inward Rectifier K+ Channels

Inward rectifier K+ (Kir) channels can be functionally categorized into two groups: those that are constitutively active and those that are constitutively inactive, with examples such as Kir2.x and Kir3.x, respectively. Their cytoplasmic regions are thought to be critical for control of channel gating, but a structural basis for this hypothesis is not known. In this study, we report a structure for the cytoplasmic region of a G protein-gated Kir channel, Kir3.2, and compare it with those of Kir3.1 and Kir2.1 channels. The isolated cytoplasmic region of Kir3.2 forms a tetrameric assembly in solution and also in the crystal. While the secondary structure arrangement and the subunit interface of the Kir3.2 crystal structure are found to be nearly identical to those of Kir3.1 and Kir2.1, it is quite different at and around loops between βC- and βD-strands and between βH- and βI-strands. These structural elements are located at the interface with the plasma membrane. Therefore, these structural elements could associate with the Kir channel transmembrane helices and be involved in the regulation of Kir channel gating.

A Structural Determinant for the Control of PIP2 Sensitivity in G Protein-gated Inward Rectifier K+ Channels

Inward rectifier K+ (Kir) channels are activated by phosphatidylinositol-(4,5)-bisphosphate (PIP2), but G protein-gated Kir (KG) channels further require either G protein βγ subunits (Gβγ) or intracellular Na+ for their activation. To reveal the mechanism(s) underlying this regulation, we compared the crystal structures of the cytoplasmic domain of KG channel subunit Kir3.2 obtained in the presence and the absence of Na+. The Na+-free Kir3.2, but not the Na+-plus Kir3.2, possessed an ionic bond connecting the N terminus and the CD loop of the C terminus. Functional analyses revealed that the ionic bond between His-69 on the N terminus and Asp-228 on the CD loop, which are known to be critically involved in Gβγ- and Na+-dependent activation, lowered PIP2 sensitivity. The conservation of these residues within the KG channel family indicates that the ionic bond is a character that maintains the channels in a closed state by controlling the PIP2 sensitivity.

Fluorescent visualisation of the hypothalamic oxytocin neurones activated by cholecystokinin-8 in rats expressing c-fos-enhanced green fluorescent protein and oxytocin-monomeric red fluorescent protein 1 fusion transgenes.

The up‐regulation of c‐fos gene expression is widely used as a marker of neuronal activation elicited by various stimuli. Anatomically precise observation of c‐fos gene products can be achieved at the RNA level by in situ hybridisation or at the protein level by immunocytochemistry. Both of these methods are time and labour intensive. We have developed a novel transgenic rat system that enables the trivial visualisation of c‐fos expression using an enhanced green fluorescent protein (eGFP) tag. These rats express a transgene consisting of c‐fos gene regulatory sequences that drive the expression of a c‐fos‐eGFP fusion protein. In c‐fos‐eGFP transgenic rats, robust nuclear eGFP fluorescence was observed in osmosensitive brain regions 90 min after i.p. administration of hypertonic saline. Nuclear eGFP fluorescence was also observed in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) 90 min after i.p. administration of cholecystokinin (CCK)‐8, which selectively activates oxytocin (OXT)‐secreting neurones in the hypothalamus. In double transgenic rats that express c‐fos‐eGFP and an OXT‐monomeric red fluorescent protein 1 (mRFP1) fusion gene, almost all mRFP1‐positive neurones in the SON and PVN expressed nuclear eGFP fluorescence 90 min after i.p. administration of CCK‐8. It is possible that not only a plane image, but also three‐dimensional reconstruction image may identify cytoplasmic vesicles in an activated neurone at the same time.

Crystal Structure of a Lysine Biosynthesis Enzyme, LysX, from Thermus thermophilus HB8

The thermophilic bacterium Thermus thermophilus synthesizes lysine through the alpha-aminoadipate pathway, which uses alpha-aminoadipate as a biosynthetic intermediate of lysine. LysX is the essential enzyme in this pathway, and is believed to catalyze the acylation of alpha-aminoadipate. We have determined the crystal structures of LysX and its complex with ADP at 2.0A and 2.38A resolutions, respectively. LysX is composed of three alpha+beta domains, each composed of a four to five-stranded beta-sheet core flanked by alpha-helices. The C-terminal and central domains form an ATP-grasp fold, which is responsible for ATP binding. LysX has two flexible loop regions, which are expected to play an important role in substrate binding and protection. In spite of the low level of sequence identity, the overall fold of LysX is surprisingly similar to that of other ATP-grasp fold proteins, such as D-Ala:D-Ala ligase, PurT-encoded glycinamide ribonucleotide transformylase, glutathione synthetase, and synapsin I. In particular, they share a similar spatial arrangement of the amino acid residues around the ATP-binding site. This observation strongly suggests that LysX is an ATP-utilizing enzyme that shares a common evolutionary ancestor with other ATP-grasp fold proteins possessing a carboxylate-amine/thiol ligase activity.

Fluorescent Visualisation of Oxytocin in the Hypothalamo-neurohypophysial/-spinal Pathways After Chronic Inflammation in Oxytocin-Monomeric Red Fluorescent Protein 1 Transgenic Rats

Oxytocin (OXT) is a well‐known neurohypophysial hormone that is synthesised in the paraventricular (PVN) and supraoptic nuclei (SON) of the hypothalamus. The projection of magnocellular neurosecretory cells, which synthesise OXT and arginine vasopressin in the PVN and SON, to the posterior pituitary plays an essential role in mammalian labour and lactation through its peripheral action. However, previous studies have shown that parvocellular OXTergic cells in the PVN, which project to the medulla and spinal cord, are involved in various physiological functions (e.g. sensory modulation and autonomic). In the present study, we examined OXT expression in the PVN, SON and spinal cord after chronic inflammation from adjuvant arthritis (AA). We used transgenic rats that express OXT and the monomeric red fluorescent protein 1 (mRFP1) fusion gene to visualise both the magnocellular and parvocellular OXTergic pathways. OXT‐mRFP1 fluorescence intensity was significantly increased in the PVN, SON, dorsal horn of the spinal cord and posterior pituitary in AA rats. The levels of OXT‐mRFP1 mRNA were significantly increased in the PVN and SON of AA rats. These results suggested that OXT was up‐regulated in both hypothalamic magnocellular neurosecretory cells and parvocellular cells by chronic inflammation, and also that OXT in the PVN‐spinal pathway may be involved in sensory modulation. OXT‐mRFP1 transgenic rats are a very useful model for visualising the OXTergic pathways from vesicles in a single cell to terminals in in vitro preparations.

The gene expression of the hypothalamic feeding-regulating peptides in cisplatin-induced anorexic rats.

Cisplatin has been widely used; however, various disadvantageous side effects afflict patients. Rikkunshito (RKT), a traditional Japanese herbal medicine, has been widely prescribed in Japan to improve anorexia; but the mechanisms are unknown. Here we studied whether RKT could improve anorexia induced by cisplatin and changes in feeding-regulating peptides in the hypothalamus in rats. Adult male rats were divided into 4 groups: water+saline (WS), water+cisplatin (WC), RKT+saline (RS), and RKT+cisplatin (RC) groups. Water or RKT (1g/kg) was intragastrically administered for 4 days, from day -1 to day 2, and saline or cisplatin (6mg/kg) was intraperitoneally (i.p.) administered at day 0. After i.p. administration, cumulative food intake, water intake, urine volume and body weight were measured. The rats were then decapitated, followed by removal of the brain, and feeding-regulating peptides in the hypothalamus were measured by in situ hybridization histochemistry. In the three-day measurements, there were no significant changes in cumulative water intake and urine volume. The body weight and cumulative food intake in WC significantly decreased compared to WS, whereas these were not observed in RC. Pro-opiomelanocortin (POMC) and cocaine and amphetamine-regulated transcript (CART) in the arcuate nucleus (ARC) in WC significantly increased, and neuropeptide Y (NPY) in the ARC decreased compared to WS, whereas those in RS and RC were comparable to WS. These results suggest that RKT may have therapeutic potential for anorexia induced by cisplatin.

Crystallization and preliminary X-ray analysis of hyperthermophilic L-threonine dehydrogenase from the archaeon Pyrococcus horikoshii.

Recombinant L-threonine dehydrogenase from the hyperthermophilic archaeon Pyrococcus horikoshii was prepared using an Escherichia coli expression system. The hyperthermostable L-threonine dehydrogenase consists of 348 amino acids with a molecular weight of 37.7 kDa. The enzyme was crystallized by the hanging-drop vapour-diffusion method at 277 K and preliminary X-ray crystallographic analysis was carried out. Diffraction data were collected to 2.20 A resolution under cryogenic conditions. P. horikoshii L-threonine dehydrogenase crystals belong to space group I4(1)22, with unit-cell parameters a = b = 143.84, c = 304.13 A. The presence of three subunits of the enzyme per asymmetric unit was estimated to give a Matthews coefficient (VM) of 3.5 A3 Da(-1) and a solvent content of 64.7%(v/v).