The target of rapamycin (TOR) is a large (281 kDa) conserved Ser/Thr protein kinase that functions as a central controller of cell growth. TOR assembles into two distinct multiprotein complexes: TORC1 and TORC2. A defining feature of TORC1 is the interaction of TOR with KOG1 (Raptor in mammals) and its sensitivity to a rapamycin-FKBP12 complex. Here, we have reconstructed in three dimensions the 25 A resolution structures of endogenous budding yeast TOR1 and a TOR-KOG1 complex, using electron microscopy. TOR features distinctive N-terminal HEAT repeats that form a curved tubular-shaped domain that associates with the C-terminal WD40 repeat domain of KOG1. The N terminus of KOG1 is in proximity to the TOR kinase domain, likely functioning to bring substrates into the vicinity of the catalytic region. A model is proposed for the molecular architecture of the TOR-KOG1 complex explaining its sensitivity to rapamycin.
In this work, we analyzed at high resolution the sugar-binding mode of the recombinant N-terminal ricin-B domain of the hemolytic protein LSLa (LSL(150)) from the mushroom Laetiporus sulphureus and also provide functional in vitro evidence suggesting that, together with its putative receptor-binding role, this module may also increase the solubility of its membrane pore-forming partner. We first demonstrate that recombinant LSL(150) behaves as an autonomous folding unit and an active lectin. We have determined its crystal structure at 1.47 Å resolution and also that of the [LSL(150):(lactose)β, γ)] binary complex at 1.67 Å resolution. This complex reveals two lactose molecules bound to the β and γ sites of LSL(150), respectively. Isothermal titration calorimetry indicates that LSL(150) binds two lactoses in solution with highly different affinities. Also, we test the working hypothesis that LSL(150) exhibits in vivo properties typical of solubility tags. With this aim, we have fused an engineered version of LSL(150) (LSL(t)) to the N-terminal end of various recombinant proteins. All the designed LSL(150)-tagged fusion proteins were successfully produced at high yield, and furthermore, the target proteins were purified by a straightforward affinity procedure on agarose-based matrices due to the excellent properties of LSL(150) as an affinity tag. An optimized protocol for target protein purification was devised, which involved removal of the LSL(150) tag through in-column cleavage of the fusion proteins with His(6)-tagged TEV endoprotease. These results permitted to set up a novel, lectin-based system for production and purification of recombinant proteins in E. coli cells with attractive biotechnological applications.
The BzdR transcriptional regulator that controls the P N promoter responsible for the anaerobic catabolism of benzoate in Azoarcus sp. CIB constitutes the prototype of a new subfamily of transcriptional regulators. Here, we provide some insights about the functional-structural relationships of the BzdR protein. Analytical ultracentrifugation studies revealed that BzdR is homodimeric in solution. An electron microscopy three-dimensional reconstruction of the BzdR dimer has been obtained, and the predicted structures of the respective N-and C-terminal domains of each BzdR monomer could be fitted into such a reconstruction. Gel retardation and ultracentrifugation experiments have shown that the binding of BzdR to its cognate promoter is cooperative. Different biochemical approaches revealed that the effector molecule benzoyl-CoA induces conformational changes in BzdR without affecting its oligomeric state. The BzdRdependent inhibition of the P N promoter and its activation in the presence of benzoyl-CoA have been established by in vitro transcription assays. The monomeric BzdR4 and BzdR5 mutant regulators revealed that dimerization of BzdR is essential for DNA binding. Remarkably, a BzdR⌬L protein lacking the linker region connecting the N-and C-terminal domains of BzdR is also dimeric and behaves as a super-repressor of the P N promoter. These data suggest that the linker region of BzdR is not essential for protein dimerization, but rather it is required to transfer the conformational changes induced by the benzoyl-CoA to the DNA binding domain leading to the release of the repressor. A model of action of the BzdR regulator has been proposed.
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