Highlights d Integrated structural model of full-length monomeric nuclear receptor LRH-1 d Model accurately predicts a novel inter-domain charge clamp d DNA binding and transcriptional activation domains dock via conserved helices d Binding of a known transcriptional co-regulator alters LRH-1 inter-domain dynamics

Nuclear receptors are ligand-activated transcription factors whose diverse biological functions are classically regulated by cholesterol-based small molecules. Over the past few decades, a growing body of evidence has demonstrated that phospholipids and other similar amphipathic molecules can also specifically bind and functionally regulate the activity of certain nuclear receptors, suggesting a critical role for these non-cholesterol-based molecules in transcriptional regulation. Phosphatidylcholines, phosphoinositides and sphingolipids are a few of the many phospholipid like molecules shown to quite specifically regulate nuclear receptors in mouse models, cell lines and in vitro. More recent evidence has also shown that certain nuclear receptors can “present” a bound phospholipid headgroup to key lipid signaling enzymes, which can then modify the phospholipid headgroup with very unique kinetic properties. Here, we review the broad array of phospholipid / nuclear receptor interactions, from the perspective of the chemical nature of the phospholipid, and the cellular abundance of the phospholipid. We also view the data in the light of well established paradigms for phospholipid mediated transcriptional regulation, as well as newer models of how phospholipids might effect transcription in the acute regulation of complex nuclear signaling pathways. Thus, this review provides novel insight into the new, non-membrane associated roles nuclear phospholipids play in regulating complex nuclear events, centered on the nuclear receptor superfamily of transcription factors.

Inositol polyphosphate multikinase (IPMK) is a member of the IPK-superfamily of kinases, catalyzing phosphorylation of several soluble inositols and the signaling phospholipid PI(4,5)P2 (PIP2). IPMK also has critical non-catalytic roles in p53, mTOR/Raptor, TRAF6 and AMPK signaling mediated partly by two disordered domains. Although IPMK non-catalytic functions are well established, it is less clear if the disordered domains are important for IPMK kinase activity or ATP binding. Here, kinetic and structural analyses of an engineered human IPMK lacking all disordered domains (ΔIPMK) are presented. Although the KM for PIP2 is identical between ΔIPMK and wild type, ΔIPMK has a 1.8-fold increase in kcat for PIP2, indicating the native IPMK disordered domains decrease IPMK activity in vitro. The 2.5 Å crystal structure of ΔIPMK is reported, confirming the conserved ATP-grasp fold. A comparison with other IPK-superfamily structures revealed a putative “ATP-clamp” in the disordered N-terminus, we predicted would stabilize ATP binding. Consistent with this observation, removal of the ATP clamp sequence increases the KM for ATP 4.9-fold, indicating the N-terminus enhances ATP binding to IPMK. Together, these structural and kinetic studies suggest in addition to mediating protein-protein interactions, the disordered domains of IPMK impart modulatory capacity to IPMK kinase activity through multiple kinetic mechanisms.

Inositol polyphosphate multikinase (IPMK) is a member of the IPK-superfamily of kinases, catalyzing phosphorylation of several soluble inositols and the signaling phospholipid PI(4,5)P 2 (PIP 2 ). IPMK also has critical non-catalytic roles in p53, mTOR/Raptor, TRAF6 and AMPK signaling mediated partly by two disordered domains. Although IPMK non-catalytic functions are well established, it is less clear if the disordered domains are important for IPMK kinase activity or ATP binding. Here, kinetic and structural analyses of an engineered human IPMK lacking all disordered domains (∆IPMK) are presented. Although the K M for PIP 2 is identical between ∆IPMK and wild type, ∆IPMK has a 1.8-fold increase in k cat for PIP 2 , indicating the native IPMK disordered domains decrease IPMK activity in vitro. The 2.5 Å crystal structure of ∆IPMK is reported, confirming the conserved ATP-grasp fold. A comparison with other IPKsuperfamily structures revealed a putative "ATP-clamp" in the disordered N-terminus, we predicted would stabilize ATP binding. Consistent with this observation, removal of the ATP clamp sequence increases the K M for ATP 4.9-fold, indicating the N-terminus enhances ATP binding to IPMK. Together, these structural and kinetic studies suggest in addition to mediating protein-protein interactions, the disordered domains of IPMK impart modulatory capacity to IPMK kinase activity through multiple kinetic mechanisms. ________________________________________ As an essential gene in mammalian development with ubiquitous expression in all eukaryotes 1 , IPMK has important functions at the nexus of many important signaling, metabolic and regulatory pathways, exerting influence through both kinase-dependent and independent roles 2 . The kinase activity of IPMK is a well-characterized node connecting inositol polyphosphate to lipid phosphoinositide signaling, and is critical for AKT activation 3 , Wnt signaling 4 , PLC-mediated IP 3 /Calcium signaling 1,5-7 , nuclear receptor transcriptional activation 8 , mRNA export 9 , and many other roles 2,10 . However, IPMK also has enzyme-independent roles, which utilize protein-protein interactions to regulate several crucial signaling proteins. IPMK acts in an enzyme-independent manner interacting with the p53 tumor suppressor, acting as a transcriptional co-activator for p53 11,12 . IPMK participates in mTOR signaling by serving as a classic adaptor protein, connecting mTOR and Raptor in mammalian cells 13 . IPMK has been

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