Iron-Utilization System in Vibrio vulnificus M2799.

Vibrio vulnificus is a Gram-negative pathogenic bacterium that causes serious infections in humans and requires iron for growth. A clinical isolate, V. vulnificus M2799, secretes a catecholate siderophore, vulnibactin, that captures ferric ions from the environment. In the ferric-utilization system in V. vulnificus M2799, an isochorismate synthase (ICS) and an outer membrane receptor, VuuA, are required under low-iron conditions, but alternative proteins FatB and VuuB can function as a periplasmic-binding protein and a ferric-chelate reductase, respectively. The vulnibactin-export system is assembled from TolCV1 and several RND proteins, including VV1_1681. In heme acquisition, HupA and HvtA serve as specific outer membrane receptors and HupB is a sole periplasmic-binding protein, unlike FatB in the ferric-vulnibactin utilization system. We propose that ferric-siderophore periplasmic-binding proteins and ferric-chelate reductases are potential targets for drug discovery in infectious diseases.

Molecular Mechanism for Conformational Dynamics of Ras·GTP Elucidated from In-Situ Structural Transition in Crystal.

Ras•GTP adopts two interconverting conformational states, state 1 and state 2, corresponding to inactive and active forms, respectively. However, analysis of the mechanism for state transition was hampered by the lack of the structural information on wild-type Ras state 1 despite its fundamental nature conserved in the Ras superfamily. Here we solve two new crystal structures of wild-type H-Ras, corresponding to state 1 and state 2. The state 2 structure seems to represent an intermediate of state transition and, intriguingly, the state 1 crystal is successfully derived from this state 2 crystal by regulating the surrounding humidity. Structural comparison enables us to infer the molecular mechanism for state transition, during which a wide range of hydrogen-bonding networks across Switch I, Switch II and the α3-helix interdependently undergo gross rearrangements, where fluctuation of Tyr32, translocation of Gln61, loss of the functional water molecules and positional shift of GTP play major roles. The NMR-based hydrogen/deuterium exchange experiments also support this transition mechanism. Moreover, the unveiled structural features together with the results of the biochemical study provide a new insight into the physiological role of state 1 as a stable pool of Ras•GTP in the GDP/GTP cycle of Ras.

Expression, purification, crystallization and X-ray crystallographic analysis of the periplasmic binding protein VatD from Vibrio vulnificus M2799.

Vibrio vulnificus is a halophilic marine microorganism which causes gastroenteritis and primary septicaemia in humans. An important factor that determines the survival of V. vulnificus in the human body is its ability to acquire iron. VatD is a periplasmic siderophore-binding protein from V. vulnificus M2799. The current study reports the expression, purification and crystallization of VatD. Crystals of both apo VatD and a VatD-desferrioxamine B-Fe(3+) (VatD-FOB) complex were obtained. The crystal of apo VatD belonged to space group P6422, while the crystal of the VatD-FOB complex belonged to space group P21. The difference in the two crystal forms could be caused by the binding of FOB to VatD.

Structural basis for the inhibitor recognition of human Lyn kinase domain.

Human Lyn tyrosine kinase is expressed in hematopoietic tissues and plays crucial roles in the signal transduction of hematopoietic immune system. Its excess activity is involved in several tumors. The crystal structure has revealed that the potent inhibitor staurosporine binds to human Lyn kinase domain at the ATP-binding site. The remarkable structural features of the staurosporine-binding region will offer valuable structural insights for the structure-based design of novel Lyn-selective inhibitors.

Protein purification and preliminary crystallographic analysis of human Lyn tyrosine kinase.

Lyn is a member of the Src family of non-receptor protein kinase. As well as all members of the Src family, Lyn is thought to participate in signal transduction from cell surface receptors. The crystal structure of Lyn would have a better understanding of Lyn function in various cells. For the purpose of crystallization, C-terminal catalytic segment of human Lyn kinase conjugating hexahistidine purification tag (His-tag) was expressed in Sf21 insect cells. After first step purification utilizing His-tag, an anion-exchange chromatogram yielded four major peaks which had distinguishable phosphorylation manner as judged by Western blot analysis, Native-PAGE analysis and kinase activity measurements. The fractioned samples were separately examined for crystallization screening using a commercial available screening kit. The mono-phosphorylated protein was crystallized with a small rod-shaped and needle clusters. The higher phosphorylated samples corresponding to the other three fractions on the anion-exchange chromatogram were aggregated or precipitated under the above conditions. A crystal of the mono-phosphorylated sample was diffracted to 3.2A with synchrotron source at Photon Factory and a complete X-ray diffraction data set was collected. The coarse structure was solved by a molecular replacement method and further structural refinement is currently underway.