Katti, S. S. | bioRxiv Science

bioRxiv2022

Mechanisms by Which Small Molecule Inhibitors Arrest Sec14 Phosphatidylinositol Transfer Protein Activity

Phosphatidylinositol transfer proteins (PITPs) promote phosphoinositide signaling by enhancing phosphatidylinositol (PtdIns) 4-OH kinase activities in producing signaling pools of PtdIns-4-phosphate. As such, PITPs are key regulators of lipid signaling in eukaryotic cells. While the PITP phospholipid exchange cycle is the engine that stimulates PtdIns 4-OH kinase activity, the protein and lipid dynamics associated with this critical process are not understood. Herein, we use an integrative structural approach that takes advantage of small molecule inhibitors (SMIs) directed against the major yeast PITP (Sec14) to gain new insights into the mechanics of the Sec14 phospholipid exchange cycle from the perspective of protein, phospholipid and SMI dynamics. Moreover, as Sec14 has emerged as an attractive target for next-generation antifungal drugs, the structures of Sec14 bound to SMIs of four different chemotypes reported in this study provide critical information required for structure-based design of next-generation lead compounds that target Sec14 PITPs of virulent fungi.

biophysics↗

bioRxiv2021

Structural anatomy of C1 domain interactions with DAG and other agonists

Diacylglycerol (DAG) is a versatile lipid whose 1,2-sn-stereoisomer serves both as second messenger in signal transduction pathways that control vital cellular processes, and as metabolic precursor for downstream signaling lipids such as phosphatidic acid1,2. DAG effector proteins compete for available lipid using conserved homology 1 (C1) domains as DAG-sensing modules. Yet, how C1 domains recognize and capture DAG in the complex environment of a biological membrane has remained unresolved for the 40 years since the discovery of Protein Kinase C (PKC)3 as the first member of the DAG effector cohort. Herein, we report the first high-resolution crystal structures of a C1 domain (C1B from PKC{delta}) complexed to DAG and to each of four potent PKC agonists that produce different biological readouts and that command intense therapeutic interest. This structural information details the mechanisms of stereospecific recognition of DAG by the C1 domains, the functional properties of the lipid-binding site, and the identities of the key residues required for the recognition and capture of DAG and exogenous agonists. Moreover, the structures of the five C1 domain complexes provide the high-resolution guides for the design of agents that modulate the activities of DAG effector proteins.

biophysics↗