No abstract

The GntR superfamily of dimeric transcription factors, with more than 6200 members encoded in bacterial genomes, are characterized by N-terminal winged-helix DNA-binding domains and diverse C-terminal regulatory domains which provide a basis for the classification of the constituent families. The largest of these families, FadR, contains nearly 3000 proteins with all-alpha-helical regulatory domains classified into two related Pfam families: FadR_C and FCD. Only two crystal structures of FadR-family members, those of Escherichia coli FadR protein and LldR from Corynebacterium glutamicum, have been described to date in the literature. Here, the crystal structure of TM0439, a GntR regulator with an FCD domain found in the Thermotoga maritima genome, is described. The FCD domain is similar to that of the LldR regulator and contains a buried metal-binding site. Using atomic absorption spectroscopy and Trp fluorescence, it is shown that the recombinant protein contains bound Ni(2+) ions but that it is able to bind Zn(2+) with K(d) < 70 nM. It is concluded that Zn(2+) is the likely physiological metal and that it may perform either structural or regulatory roles or both. Finally, the TM0439 structure is compared with two other FadR-family structures recently deposited by structural genomics consortia. The results call for a revision in the classification of the FadR family of transcription factors.

Dynactin is a protein complex required for the in vivo function of cytoplasmic dynein, a microtubule (MT)-based motor. Dynactin binds both dynein and MTs via its p150 Glued subunit, but little is known about the 'pointedend complex' that includes the protein subunits Arp11, p62 and the p27/p25 heterodimer. Here, we show that the p27/p25 heterodimer undergoes mitotic phosphorylation by cyclin-dependent kinase 1 (Cdk1) at a single site, p27 Thr186, to generate an anchoring site for polo-like kinase 1 (Plk1) at kinetochores. Removal of p27/p25 from dynactin results in reduced levels of Plk1 and its phosphorylated substrates at kinetochores in prometaphase, which correlates with aberrant kinetochore-MT interactions, improper chromosome alignment and abbreviated mitosis. To investigate the structural implications of p27 phosphorylation, we determined the structure of human p27. This revealed an unusual left-handed b-helix domain, with the phosphorylation site located within a disordered, C-terminal segment. We conclude that dynactin plays a previously undescribed regulatory role in the spindle assembly checkpoint by recruiting Plk1 to kinetochores and facilitating phosphorylation of important downstream targets.

The NudC family consists of four conserved proteins with representatives in all eukaryotes. The archetypal nudC gene from Aspergillus nidulans is a member the nud gene family, involved in maintenance of nuclear migration. This family also includes nudF whose human orthologue, Lis1, codes for a protein essential for brain cortex development. Three paralogues of NudC are known in vertebrates, NudC, NudC-like (NudCL) and NudC-like 2 (NudCL2). The fourth distantly related member of the family, CML66, contains a NudC-like domain. The three principal NudC proteins have no catalytic activity, but appear to play as yet poorly defined roles in proliferating and dividing cells. We present crystallographic and NMR studies of the human NudC protein, and discuss the results in the context of structures recently deposited by Structural Genomics centers, i.e. NudCL and mouse NudCL2. All proteins share the same core CS-domain characteristic for proteins acting either as co-chaperones of Hsp90, or as independent small heat shock proteins. However, while NudC and NudCL dimerize via an N-terminally located coiled-coil, the smaller NudCL2 lacks this motif and instead dimerizes as a result of unique domain swapping. We show that NudC and NudCL, but not NudCL2, inhibit aggregation of several target proteins, consistent with an Hsp90-independent heat shock protein function. Importantly, and in contrast to several previous reports, none of the three proteins are able to form binary complexes with Lis1. The availability of structural information will be of help in further studies of cellular functions of the NudC family.

For almost half a century, the structure of the full-length Bacillus thuringiensis (Bt) insecticidal protein Cry1Ac has eluded researchers, since Bt-derived crystals were first characterized in 1965. Having finally solved this structure we report intriguing details of the lattice-based interactions between the toxic core of the protein and the protoxin domains. The structure provides concrete evidence for the function of the protoxin as an enhancer of native crystal packing and stability.

Activatable magnetic resonance imaging (MRI) contrast agents that can be selectively stimulated at a tumor region are urgently demanded to realize the efficient and accurate diagnosis of cancers. Here, extremely small iron oxide nanoparticles (ESIONPs) modified with citric acid (ESIONPs-CA) are encapsulated in disulfide-cross-linked poly(carboxybetaine methacrylate) (poly(CBMA)) nanogels, and a cyclo[Arg-Gly-Asp-D-Tyr-Lys] (c(RGD)) ligand is further introduced to obtain ESIONP-packaged poly(CBMA) nanogels equipped with tumortargeted c(RGD) (ICNs-RGD). On the basis of the transformation of the clustered ESIONPs into dispersed ones induced by the reducing glutathione (GSH), ICNs-RGD can complete the conversion from a T 2 contrast agent to a T 1 one, realizing the selective activation of the T 1 contrasting effect. The GSH-dependent MRI signal conversion of ICNs-RGD is feasible in the tumor cell and tissue. Moreover, ICNs-RGD exhibits obvious targeting specificity and favorable biocompatibility. In the MRI experiments of tumor-bearing mice, benefiting from the stimuli-responsiveness toward GSH and targeting specificity, the T 1 contrasting effect of tumor tissues can be selectively enhanced after the intravenous injection of ICNs-RGD. Therefore, tumor-targeted ICNs-RGD with a switchable MRI signal derived from the activation of GSH is a potential contrast agent for the efficient and precise tumor diagnosis in the clinic.

Background: The Dbl-family of guanine nucleotide exchange factors (GEFs) activate the cytosolic GTPases of the Rho family by enhancing the rate of exchange of GTP for GDP on the cognate GTPase. This catalytic activity resides in the DH (Dbl-homology) domain, but typically GEFs are multidomain proteins containing other modules. It is believed that GEFs are autoinhibited in the cytosol due to supramodular architecture, and become activated in diverse signaling pathways through conformational change and exposure of the DH domain, as the protein is translocated to the membrane. A small family of RhoA-specific GEFs, containing the RGSL (regulators of G-protein signaling-like) domain, act as effectors of select GPCRs via Gα 12/13 , although the molecular mechanism by which this pathway operates is not known. These GEFs include p115, LARG and PDZRhoGEF (PRG).

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