Loquacious-PD facilitates Drosophila Dicer-2 cleavage through interactions with the helicase domain and dsRNA.

Loquacious-PD (Loqs-PD) is required for biogenesis of many endogenous siRNAs in Drosophila In vitro, Loqs-PD enhances the rate of dsRNA cleavage by Dicer-2 and also enables processing of substrates normally refractory to cleavage. Using purified components, and Loqs-PD truncations, we provide a mechanistic basis for Loqs-PD functions. Our studies indicate that the 22 amino acids at the C terminus of Loqs-PD, including an FDF-like motif, directly interact with the Hel2 subdomain of Dicer-2's helicase domain. This interaction is RNA-independent, but we find that modulation of Dicer-2 cleavage also requires dsRNA binding by Loqs-PD. Furthermore, while the first dsRNA-binding motif of Loqs-PD is dispensable for enhancing cleavage of optimal substrates, it is essential for enhancing cleavage of suboptimal substrates. Finally, our studies define a previously unrecognized Dicer interaction interface and suggest that Loqs-PD is well positioned to recruit substrates into the helicase domain of Dicer-2.

Drosophila dicer-2 cleavage is mediated by helicase- and dsRNA termini-dependent states that are modulated by Loquacious-PD.

In previous studies we observed that the helicase domain of Drosophila Dicer-2 (dmDcr-2) governs substrate recognition and cleavage efficiency, and that dsRNA termini are key to this discrimination. We now provide a mechanistic basis for these observations. We show that discrimination of termini occurs during initial binding. Without ATP, dmDcr-2 binds 3' overhanging, but not blunt, termini. By contrast, with ATP, dmDcr-2 binds both types of termini, with highest-affinity binding observed with blunt dsRNA. In the presence of ATP, binding, cleavage, and ATP hydrolysis are optimal with BLT termini compared to 3'ovr termini. Limited proteolysis experiments suggest the optimal reactivity of BLT dsRNA is mediated by a conformational change that is dependent on ATP and the helicase domain. We find that dmDcr-2's partner protein, Loquacious-PD, alters termini dependence, enabling dmDcr-2 to cleave substrates normally refractory to cleavage, such as dsRNA with blocked, structured, or frayed ends.

A sterol-binding protein integrates endosomal lipid metabolism with TOR signaling and nitrogen sensing.

Kes1, and other oxysterol-binding protein superfamily members, are involved in membrane and lipid trafficking through trans-Golgi network (TGN) and endosomal systems. We demonstrate that Kes1 represents a sterol-regulated antagonist of TGN/endosomal phosphatidylinositol-4-phosphate signaling. This regulation modulates TOR activation by amino acids and dampens gene expression driven by Gcn4, the primary transcriptional activator of the general amino acid control regulon. Kes1-mediated repression of Gcn4 transcription factor activity is characterized by nonproductive Gcn4 binding to its target sequences, involves TGN/endosome-derived sphingolipid signaling, and requires activity of the cyclin-dependent kinase 8 (CDK8) module of the enigmatic "large Mediator" complex. These data describe a pathway by which Kes1 integrates lipid metabolism with TORC1 signaling and nitrogen sensing.

Resurrection of a functional phosphatidylinositol transfer protein from a pseudo-Sec14 scaffold by directed evolution.

Sec14-superfamily proteins integrate the lipid metabolome with phosphoinositide synthesis and signaling via primed presentation of phosphatidylinositol (PtdIns) to PtdIns kinases. Sec14 action as a PtdIns-presentation scaffold requires heterotypic exchange of phosphatidylcholine (PtdCho) for PtdIns, or vice versa, in a poorly understood progression of regulated conformational transitions. We identify mutations that confer Sec14-like activities to a functionally inert pseudo-Sec14 (Sfh1), which seemingly conserves all of the structural requirements for Sec14 function. Unexpectedly, the "activation" phenotype results from alteration of residues conserved between Sfh1 and Sec14. Using biochemical and biophysical, structural, and computational approaches, we find the activation mechanism reconfigures atomic interactions between amino acid side chains and internal water in an unusual hydrophilic microenvironment within the hydrophobic Sfh1 ligand-binding cavity. These altered dynamics reconstitute a functional "gating module" that propagates conformational energy from within the hydrophobic pocket to the helical unit that gates pocket access. The net effect is enhanced rates of phospholipid-cycling into and out of the Sfh1* hydrophobic pocket. Taken together, the directed evolution approach reveals an unexpectedly flexible functional engineering of a Sec14-like PtdIns transfer protein-an engineering invisible to standard bioinformatic, crystallographic, and rational mutagenesis approaches.