Hiro Nakamura

Structure of PAS-linked histidine kinase and the response regulator complex

We determined the structure of the complex of the sensory histidine kinase (HK) and its cognate response regulator (RR) in the two-component signal transduction system of Thermotoga maritima. This was accomplished by fitting the high-resolution structures of the isolated HK domains and the RR onto the electron density map (3.8 A resolution) of the HK/RR complex crystal. Based on the structural information, we evaluated the roles of both interdomain and intermolecular interactions in the signal transduction of the cytosolic PAS-linked HK and RR system, in particular the O(2)-sensor FixL/FixJ system. The PAS-sensor domain of HK interacts with the catalytic domain of the same polypeptide chain by creating an interdomain beta sheet. The interaction site between HK and RR, which was confirmed by NMR, is suitable for the intermolecular transfer reaction of the phosphoryl group, indicating that the observed interaction is important for the phosphatase activity of HK that dephosphorylates phospho-RR.

Separation of phosphoprotein isotypes having the same number of phosphate groups using phosphate-affinity SDS-PAGE.

Herein, we demonstrate the separation of phosphoprotein isotypes having the same number of phosphate groups using phosphate‐affinity SDS‐PAGE. The phosphate‐affinity site is a polyacrylamide‐bound Phos‐tag that enables the mobility shift detection of phosphoproteins from their nonphosphorylated counterparts. As the first practical example of the separation, we characterized the monophosphorylated Tau isotypes by each of three tyrosine kinases, c‐Abl, MET, and Fyn. Each monophosphoisotype phosphorylated at the Tyr‐394, Tyr‐197, or Tyr‐18 was detected as three distinct migration bands. As a further application, we extended this technique to the mobility shift analysis of His and Asp phosphoisotypes in the Sinorhizobium meliloti FixL/FixJ two‐component system. FixL is autophosphorylated at the His‐285 with ATP, and the phosphate group is transferred to the Asp‐54 of FixJ and subsequently removed by the FixL phosphatase activity. Using this method, we first performed simultaneous detection of the phosphorylated and nonphosphorylated isotypes of FixL and FixJ generated in their phosphotransfer reaction in vitro. As a result, a monophosphoisotype of FixL containing the phosphorylated His residue was confirmed. As for FixJ, on the other hand, two monophosphoisotypes were detected as two distinct migration bands. One is a well‐known isotype phosphorylated at the Asp‐54. The other is a novel isotype phosphorylated at the His‐84.

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.

O2-specific regulation of the ferrous heme-based sensor kinase FixL from Sinorhizobium meliloti and its aberrant inactivation in the ferric form

FixL, a rhizobial heme-based O2-sensing histidine kinase, catalyzes autophosphorylation in the deoxy form at low O2 tension, while the kinase activity is inhibited in the case of the O2-bound form. The present study unambiguously shows that the binding of CO and NO does not significantly inhibit the kinase activity of dithiothreitol (DTT)-reduced ferrous FixL from Sinorhizobium meliloti, which is inconsistent with the spin state mechanism previously reported. Kinase inactivation is caused by aberrant disulfide (S-S) bond formation at Cys301 in the ferric homodimer, which explains these contradictory observations. The addition of DTT cleaved the S-S bond, leading to restoration of kinase activity in the ferric form as well as heme reduction, but, sodium hydrosulfite treatment produced the kinase-inactive deoxy form without S-S cleavage. On the basis of these experimental results, it can be concluded that ferrous FixL discriminates O2 from CO and NO, and signals the O2-bound state by downregulating the phosphoryl transfer reaction.

The uncoupling of oxygen sensing, phosphorylation signalling and transcriptional activation in oxygen sensor FixL and FixJ mutants

The rhizobial FixL/FixJ system, a member of the superfamily of bacterial two‐component signal transducing systems, regulates the expression of nitrogen fixation‐related genes by sensing environmental oxygen tension. Oxygen‐free (deoxy) FixL is autophosphorylated at an invariant histidine residue with ATP, and the phosphoryl group is transferred to FixJ, leading to an enhancement in transcriptional activity at low oxygen tensions, but the histidine kinase activity of the oxygen‐bound (oxy) form is inhibited. To investigate the mechanism of oxygen sensing, we established a FixL/FixJ‐mediated PfixK‐lacZ reporter system in Escherichia coli, and isolated FixL and FixJ mutations conferring an upregulation of lacZ gene expression on the reporter cells even under aerobic conditions. FixL mutant proteins, which contain single amino acid changes near the autophosphorylation site, showed elevated levels of autophosphorylation and a concomitant phosphoryl transfer to FixJ in the presence of oxygen, although their oxygen‐binding affinities were unimpaired. These mutational analyses suggest that the autophosphorylation domain plays a crucial role in regulatory coupling between oxygen binding and kinase activity. FixJ mutants in helix α1 and strand β5 of the N‐terminal half exhibited the formation of a stable acyl phosphate bond. In contrast, those in helices α4 and α5 constitutively bound to the fixK promoter in a monomeric form, suggesting that the α4 and α5 helices may be involved in the post‐phosphorylation/dimerization signal transfer to liberate the DNA‐binding activity of the C‐terminal domain, not only serving as a dimerization interface.

CuB promotes both binding and reduction of dioxygen at the heme-copper binuclear center in the Escherichia coli bo-type ubiquinol oxidase☆

A CuB-deficient mutant of the Escherichia coli bo-type ubiquinol oxidase exhibits a very low oxidase activity that is consistent with a decreased dioxygen binding rate. During the turnover, a photolabile reaction intermediate persists for a few hundred milliseconds, due to much slower heme o-to-ligand electron transfer. Thus, the lack of CuB seems to have endowed the mutant enzyme with myoglobin-like properties, thereby stabilizing the CO-bound form, too. Accordingly we conclude that CuB plays a pivotal role in preferential trapping and efficient reduction of dioxygen at the heme-copper binuclear center.A CuB‐deficient mutant of the Escherichia coli bo‐type ubiquinol oxidase exhibits a very low oxidase activity that is consistent with a decreased dioxygen binding rate. During the turnover, a photolabile reaction intermediate persists for a few hundred milliseconds, due to much slower heme o‐to‐ligand electron transfer. Thus, the lack of CuB seems to have endowed the mutant enzyme with myoglobin‐like properties, thereby stabilizing the CO‐bound form, too. Accordingly we conclude that CuB plays a pivotal role in preferential trapping and efficient reduction of dioxygen at the heme‐copper binuclear center.

Heme-dependent autophosphorylation of a heme sensor kinase, ChrS, from Corynebacterium diphtheriae reconstituted in proteoliposomes

Corynebacterium diphteriae employs the response regulator, ChrA, and the sensor kinase, ChrS, of a two‐component signal transduction system to utilize host heme iron. Although ChrS is predicted to encode a heme sensor, the sensing mechanism remains to be characterized. In this report, ChrS expressed in Eshcherichia coli membranes was solubilized and purified using decylmaltoside. ChrS protein incorporated into proteoliposomes catalyzed heme‐dependent autophosphorylation by ATP. Other metalloporphyrins and iron did not stimulate kinase activity. The UV–Vis spectrum of hemin in the ChrS–proteoliposomes indicated that heme directly interacts with ChrS. This is the first functional reconstitution of a bacterial heme‐sensing protein.

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.

Chimeric sensory kinases containing O2 sensor domain of FixL and histidine kinase domain from thermophile

To explore the functional mechanism of inter-domain interaction in a sensor histidine kinase, five chimeric sensory kinases were constructed. In each of these chimeric proteins (CskA254, CskA264, CskA274, CskA284, and CskA294), the sensor domain of heme-based O(2) sensor FixL, obtained from Sinorhizobium meliloti, was fused with the histidine kinase domain from a hyperthermophile, Thermotoga maritima, each at a systematically different position. The UV-visible (UV-vis), resonance Raman (RR), and circular dichroism (CD) spectral characteristics of the CskAs indicated that the secondary and heme environmental structures of all five CskAs examined are identical to those of FixL. In spite of these structural similarities, all CskAs did not exhibit O(2)-dependent regulation of autophosphorylation activity. Furthermore, their functional properties were much different from those of FixL: The O(2) binding affinity and the autophosphorylation activity for CskA254, CskA264, and CskA274 were similar to those of the truncated sensor and histidine kinase domain, whereas CskA284 and CskA294 display extremely low O(2) affinity and low autophosphorylation activity, as compared with each truncated domain. These observations indicated that the interdomain interaction was presented in those CskAs, and that interaction could be related to the O(2)-dependent regulatory interaction of FixL. In the present study, we demonstrated that the interaction in the physiological sensor histidine kinase would be strictly and finely controlled to mediate the signal ligation-dependent autophosphorylation activity in its histidine kinase domain.

Crystal structure of bacterial haem importer complex in the inward-facing conformation.

Pathogenic bacteria remove iron from the haem of host tissues and use it as a catalytic center of many enzymes. Haem uptake by pathogenic bacteria is facilitated by the membrane-integrated haem importer, which belongs to the type II ATP-binding cassette (ABC) transporter. Here we present crystal structures of Burkholderia cenocepacia haem importer BhuUV complexed with the periplasmic haem-binding protein BhuT and in the absence of BhuT. The transmembrane helices of these structures show an inward-facing conformation, in which the cytoplasmic gate of the haem translocation pathway is completely open. Since this conformation is found in both the haem- and nucleotide-free form, the structure of BhuUV-T provides the post-translocation state and the missing piece in the transport cycle of the type II importer. Structural comparison with the outward-facing conformation reported for the haem importer ortholog HmuUV from Yersenia pestis gives mechanistic insights into conformational transitions and haem secretion during the haem transport cycle.