Hideyuki Miyatake

Crystal structures of bacterial lipoprotein localization factors, LolA and LolB

Lipoproteins having a lipid‐modified cysteine at the N‐terminus are localized on either the inner or the outer membrane of Escherichia coli depending on the residue at position 2. Five Lol proteins involved in the sorting and membrane localization of lipoprotein are highly conserved in Gram‐negative bacteria. We determined the crystal structures of a periplasmic chaperone, LolA, and an outer membrane lipoprotein receptor, LolB. Despite their dissimilar amino acid sequences, the structures of LolA and LolB are strikingly similar to each other. Both have a hydrophobic cavity consisting of an unclosed β barrel and an α‐helical lid. The cavity represents a possible binding site for the lipid moiety of lipoproteins. Detailed structural differences between the two proteins provide significant insights into the molecular mechanisms underlying the energy‐independent transfer of lipoproteins from LolA to LolB and from LolB to the outer membrane. Furthermore, the structures of both LolA and LolB determined from different crystal forms revealed the distinct structural dynamics regarding the association and dissociation of lipoproteins. The results are discussed in the context of the current model for the lipoprotein transfer from the inner to the outer membrane through a hydrophilic environment.

Crystal structure of rat biliverdin reductase.

Biliverdin reductase (BVR) is a soluble cytoplasmic enzyme that catalyzes the conversion of biliverdin to bilirubin using NADH or NADPH as electron donor. Bilirubin is a significant biological antioxidant, but it is also neurotoxic and the cause of kernicterus. In this study, we have determined the crystal structure of rat BVR at 1.4 Å resolution. The structure contains two domains: an N-terminal domain characteristic of a dinucleotide binding fold (Rossmann fold) and a C-terminal domain that is predominantly an antiparallel six-stranded β-sheet. Based on this structure, we propose modes of binding for NAD(P)H and biliverdin, and a possible mechanism for the enzyme.

Redox Properties and Coordination Structure of the Heme in the CO-sensing Transcriptional Activator CooA*

The CO-sensing transcriptional activator CooA contains a six-coordinate protoheme as a CO sensor. Cys75 and His77 are assigned to the fifth ligand of the ferric and ferrous hemes, respectively. In this study, we carried out alanine-scanning mutagenesis and EXAFS analyses to determine the coordination structure of the heme in CooA. Pro2 is thought to be the sixth ligand of the ferric and ferrous hemes in CooA, which is consistent with the crystal structure of ferrous CooA (Lanzilotta, W. N., Schuller, D. J., Thorsteinsson, M. V., Kerby, R. L., Roberts, G. P., and Poulos, T. L. (2000) Nat. Struct. Biol. 7, 876–880). CooA exhibited anomalous redox chemistry, i.e. hysteresis was observed in electrochemical redox titrations in which the observed reduction and oxidation midpoint potentials were −320 mV and −260 mV, respectively. The redox-controlled ligand exchange of the heme between Cys75 and His77 is thought to cause the difference between the reduction and oxidation midpoint potentials.

The RIKEN structural biology beamline II (BL44B2) at the SPring-8

A SPring-8 bending magnet beamline, the RIKEN structural biology beamline II (BL44B2), is dedicated to monochromatic/Laue macromolecular crystallography and X-ray absorption fine structure studies. The design and the performance of the beamline are presented. # 2001 Elsevier Science B.V. All rights reserved.

Structure and Characterization of Amidase from Rhodococcus sp. N-771: Insight into the Molecular Mechanism of Substrate Recognition

In this study, we have structurally characterized the amidase of a nitrile-degrading bacterium, Rhodococcus sp. N-771 (RhAmidase). RhAmidase belongs to amidase signature (AS) family, a group of amidase families, and is responsible for the degradation of amides produced from nitriles by nitrile hydratase. Recombinant RhAmidase exists as a dimer of about 107 kDa. RhAmidase can hydrolyze acetamide, propionamide, acrylamide and benzamide with kcat/Km values of 1.14+/-0.23 mM(-1)s(-1), 4.54+/-0.09 mM(-1)s(-1), 0.087+/-0.02 mM(-1)s(-1) and 153.5+/-7.1 mM(-1)s(-1), respectively. The crystal structures of RhAmidase and its inactive mutant complex with benzamide (S195A/benzamide) were determined at resolutions of 2.17 A and 2.32 A, respectively. RhAmidase has three domains: an N-terminal alpha-helical domain, a small domain and a large domain. The N-terminal alpha-helical domain is not found in other AS family enzymes. This domain is involved in the formation of the dimer structure and, together with the small domain, forms a narrow substrate-binding tunnel. The large domain showed high structural similarities to those of other AS family enzymes. The Ser-cis Ser-Lys catalytic triad is located in the large domain. But the substrate-binding pocket of RhAmidase is relatively narrow, due to the presence of the helix alpha13 in the small domain. The hydrophobic residues from the small domain are involved in recognizing the substrate. The small domain likely participates in substrate recognition and is related to the difference of substrate specificities among the AS family amidases.

Iron coordination structures of oxygen sensor FixL characterized by Fe K-edge extended x-ray absorption fine structure and resonance raman spectroscopy.

FixL is a heme-based O2 sensor protein involved in a two-component system of a symbiotic bacterium. In the present study, the iron coordination structure in the heme domain of Rhizobium meliloti FixLT (RmFixLT, a soluble truncated FixL) was examined using Fe K-edge extended x-ray absorption fine structure (EXAFS) and resonance Raman spectroscopic techniques. In the EXAFS analyses, the interatomic distances and angles of the Fe-ligand bond and the iron displacement from the heme plane were obtained for RmFixLT in the Fe2+, Fe2+O2, Fe2+CO, Fe3+, Fe3+F−, and Fe3+CN− states. An apparent correlation was found between the heme-nitrogen (proximal His-194) distance in the heme domain and the phosphorylation activity of the histidine kinase domain. Comparison of the Fe-CO coordination geometry between RmFixLT and RmFixLH (heme domain of RmFixL), based on the EXAFS and Raman results, has suggested that the kinase domain directly or indirectly influences steric interaction between the iron-bound ligand and the heme pocket. Referring to the crystal structure of the heme domain ofBradyrhizobium japonicum FixL (Gong, W., Hao, B., Mansy, S. S., Gonzalez, G., Gilles-Gonzalez, M. A., and Chan, M. K. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 15177–15182), we discussed details of the iron coordination structure of RmFixLT and RmFixLH in relation to an intramolecular signal transduction mechanism in its O2 sensing.

Crystal Structure of Human Importin-α1 (Rch1), Revealing a Potential Autoinhibition Mode Involving Homodimerization

In this study, we determined the crystal structure of N-terminal importin-β-binding domain (IBB)-truncated human importin-α1 (ΔIBB-h-importin-α1) at 2.63 Å resolution. The crystal structure of ΔIBB-h-importin-α1 reveals a novel closed homodimer. The homodimer exists in an autoinhibited state in which both the major and minor nuclear localization signal (NLS) binding sites are completely buried in the homodimerization interface, an arrangement that restricts NLS binding. Analytical ultracentrifugation studies revealed that ΔIBB-h-importin-α1 is in equilibrium between monomers and dimers and that NLS peptides shifted the equilibrium toward the monomer side. This finding suggests that the NLS binding sites are also involved in the dimer interface in solution. These results show that when the IBB domain dissociates from the internal NLS binding sites, e.g., by binding to importin-β, homodimerization possibly occurs as an autoinhibition state.

New methods to prepare iodinated derivatives by vaporizing iodine labelling (VIL) and hydrogen peroxide VIL (HYPER-VIL)

New techniques are presented for the preparation of iodine derivatives, involving vapour diffusion of iodine. Firstly, in the vaporizing iodine labelling (VIL) technique, a small amount of KI/I(2) solution is enclosed in a crystallization well, with the result that gaseous I(2) molecules diffuse into the crystallization droplets without exerting substantial changes in ionic strength in the target crystals. Once they have diffused into the droplet, the I(2) molecules sometimes iodinate accessible tyrosines at ortho positions. Secondly, when iodination is insufficient, the hydrogen peroxide VIL (HYPER-VIL) technique can be further applied to increase the iodination ratio by the addition of a small droplet of hydrogen peroxide (H(2)O(2)) to the crystallization well; the gaseous H(2)O(2) also diffuses into the crystallization droplet to emphasize the iodination. These techniques are most effective for phase determination when coupled with softer X-rays, such as those from Cu Kalpha or Cr Kalpha radiation. The effectiveness of these techniques was assessed using five different crystals. Four of the crystals were successfully iodinated, providing sufficient phasing power for structure determination.

Dynamic light-scattering and preliminary crystallographic studies of the sensor domain of the haem-based oxygen sensor FixL from Rhizobium meliloti.

FixL is a transmitter protein in a two-component system which acts as an oxygen sensor when the symbiotic Rhizobia resides in root nodules of host plants. The oxygen-sensor domain of Rhizobium meliloti FixL (RmFixLH) was purified by His-tag affinity and isoelectronic focusing chromatographies, without the use of gel-filtration chromatography. Dynamic light-scattering measurements of RmFixLH thus obtained revealed it to be monodispersive and present as a homodimer in solution. A single crystal of RmFixLH in the met (Fe3+) form was grown in 100 mM acetic acid/NaOH buffer at pH 4.6 in the presence of 200 mM ammonium acetate, using 40%(w/v) PEG 4000 as a precipitant. A crystal of the ferrous CO form of RmFixLH was also prepared by reduction of the met crystal with Na2S2O4 in an atmosphere of CO. The crystals (0.2 x 0.05 x 0.01 mm) belong to the monoclinic system (C2) with unit-cell parameters a = 60.94, b = 37.44, c = 54.14 A, beta = 115.29 degrees and diffract X-rays to 1.7 A resolution at station BL44B2 of SPring-8, Japan. Bijvoet difference Patterson maps show a clear peak corresponding to the haem iron in RmFixLH.

Thermodynamic Characterization of the Interaction between Prefoldin and Group II Chaperonin

Prefoldin (PFD) is a hexameric chaperone that captures a protein substrate and transfers it to a group II chaperonin (CPN) to complete protein folding. We have studied the interaction between PFD and CPN using those from a hyperthermophilic archaeon, Thermococcus strain KS-1 (T. KS-1). In this study, we determined the crystal structure of the T. KS-1 PFDbeta2 subunit and characterized the interactions between T. KS-1 CPNs (CPNalpha and CPNbeta) and T. KS-1 PFDs (PFDalpha1-beta1 and PFDalpha2-beta2). As predicted from its amino acid sequence, the PFDbeta2 subunit conforms to a structure similar to those of the PFDbeta1 subunit and the Pyrococcus horikoshii OT3 PFDbeta subunit, with the exception of the tip of its coiled-coil domain, which is thought to be the CPN interaction site. The interactions between T. KS-1 CPNs and PFDs (CPNalpha and PFDalpha1-beta1; CPNalpha and PFDalpha2-beta2; CPNbeta and PFDalpha1-beta1; and CPNbeta and PFDalpha2-beta2) were analyzed using the Biacore T100 system at various temperatures ranging from 20 to 45 degrees C. The affinities between PFDs and CPNs increased with an increase in temperature. The thermodynamic parameters calculated from association constants showed that the interaction between PFD and CPN is entropy driven. Among the four combinations of PFD-CPN interactions, the entropy difference in binding between CPNbeta and PFDalpha2-beta2 was the largest, and affinity significantly increased at higher temperatures. Considering that expression of PFDalpha2-beta2 and CPNbeta subunit is induced upon heat shock, our results suggest that PFDalpha1-beta1 is a general PFD for T. KS-1 CPNs, whereas PFDalpha2-beta2 is specific for CPNbeta.