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abstracts
Iron is an essential element for the growth and survival of nearly all living organisms. However, it is difficult for most organisms to get enough iron from the environment, because of the extremely low solubility of ferric ion. One of the strategies for iron acquisition is to use the ATP-binding cassette (ABC) transport system. In Gram-negative bacteria, a typical iron uptake ABC transporter consists of a ferric ion-binding protein (Fbp) located in periplasm (FbpA), two transmembrane proteins that form a pathway for ferric ions (FbpB), and two peripheral ATP-binding proteins located at the cytoplasm side of the inner membrane (FbpC). TtFbpA is a ferric ion-binding protein of a putative iron uptake ABC transporter from Thermus thermophilus HB8. Here we report the crystal structures of the apo-form and ferric ion-bound form of TtFbpA at 1.8-Å and 1.7-Å resolutions, respectively [1]. The crystal structure of the ferric ion-bound TtFbpA shows that a ferric ion binds to a specific site of TtFbpA to form a six-coordinated complex by three tyrosine residues, two bicarbonates and a water molecule, revealing a novel mode of coordination to a ferric ion. Another crystal structure of ferric ion-bound TtFbpA reported earlier showed the bound ferric ion is five-coordinated by three tyrosine residues and a carbonate bound in the bidentate manner [2]. The different modes of the coordination would probably result from the different pHs used for crystallization: pH 5.5 (six-coordinated) vs. pH 7.5 (five-coordinated). The Gram negative bacterium T. thermophilus HB8 can live in a wide pH range of 3.4-9.6. We propose that TtFbpA, a periplasmic protein of T. thermophilus HB8, can act as a ferric ion-binding protein over the wide pH range by taking at least two different coordination manners to a ferric ion depending on pH. This is the first example of a periplasmic ferric iron-binding protein that can coordinate a ferric ion via multiple types of coordination complex formation.
abstracts
The PhoP-PhoR two-component system plays a key role in regulating virulence of Mycobacterium tuberculosis. The response regulator PhoP is a transcription regulator, and it regulates expression of more than 100 genes. PhoP belongs to the OmpR/PhoB subfamily of response regulators. Despite extensive research in recent years, the molecular mechanism of DNA sequence recognition by this large subfamily of response regulators is not fully understood, especially the role of the N-terminal regulatory domain on DNA binding of the effector domain. Here we present a crystal structure of the full-length PhoP in complex with a direct-repeat DNA sequence. PhoP binds to DNA as a dimer. The two effector domains bind in tandem, each interacting with a half site of the direct repeat DNA. The DNA recognition helix inserts into the major groove, reading the sequence of a 7-bp motif. The wing residues interact with the downstream sequence, with a conserved arginine side chain inserting into the minor groove. Surprisingly, the regulatory domain also forms a tandem arrangement, instead of the anticipated symmetric dimer. The regulatory domain of the upstream protomer interacts with both domains of the downstream protomer. The structural elements of the alpha4-beta5-alpha5 face, which often found to be involved in dimer interface of the regulatory domain, play important roles in the interactions between the protomers. The crystal structure explains why PhoP recognizes direct repeats of two 7-bp motifs with a strict spacing of 4 bp and the highly cooperative binding of the two monomers. Detailed analysis of the structure along with analysis of the DNA sequence requirements and ITC measurements of protein-DNA binding interactions will be presented.
