Systematic simulation of the interactions of pleckstrin homology domains with membranes.

Pleckstrin homology (PH) domains can recruit proteins to membranes by recognition of phosphatidylinositol phosphate (PIP) lipids. Several family members are linked to diseases including cancer. We report the systematic simulation of the interactions of 100 mammalian PH domains with PIP-containing membranes. The observed PIP interaction hotspots recapitulate crystallographic binding sites and reveal a number of insights: (i) The ß1 and ß2 strands and their connecting loop constitute the primary PIP interaction site but are typically supplemented by interactions at the ß3-ß4 and ß5-ß6 loops; (ii) we reveal exceptional cases such as the Exoc8 PH domain; (iii) PH domains adopt different membrane-bound orientations and induce clustering of anionic lipids; and (iv) beyond family-level insights, our dataset sheds new light on individual PH domains, e.g., by providing molecular detail of secondary PIP binding sites. This work provides a global view of PH domain/membrane association involving multivalent association with anionic lipids.

Sizing up DNA nanostructure assembly with native mass spectrometry and ion mobility.

Recent interest in biological and synthetic DNA nanostructures has highlighted the need for methods to comprehensively characterize intermediates and end products of multimeric DNA assembly. Here we use native mass spectrometry in combination with ion mobility to determine the mass, charge state and collision cross section of noncovalent DNA assemblies, and thereby elucidate their structural composition, oligomeric state, overall size and shape. We showcase the approach with a prototypical six-subunit DNA nanostructure to reveal how its assembly is governed by the ionic strength of the buffer, as well as how the mass and mobility of heterogeneous species can be well resolved by careful tuning of instrumental parameters. We find that the assembly of the hexameric, barrel-shaped complex is guided by positive cooperativity, while previously undetected higher-order 12- and 18-mer assemblies are assigned to defined larger-diameter geometric structures. Guided by our insight, ion mobility-mass spectrometry is poised to make significant contributions to understanding the formation and structural diversity of natural and synthetic oligonucleotide assemblies relevant in science and technology.

Characterization of the membrane interactions of phospholipase Cγ reveals key features of the active enzyme.

PLCγ enzymes are autoinhibited in resting cells and form key components of intracellular signaling that are also linked to disease development. Insights into physiological and aberrant activation of PLCγ require understanding of an active, membrane-bound form, which can hydrolyze inositol-lipid substrates. Here, we demonstrate that PLCγ1 cannot bind membranes unless the autoinhibition is disrupted. Through extensive molecular dynamics simulations and experimental evidence, we characterize membrane binding by the catalytic core domains and reveal previously unknown sites of lipid interaction. The identified sites act in synergy, overlap with autoinhibitory interfaces, and are shown to be critical for the phospholipase activity in cells. This work provides direct evidence that PLCγ1 is inhibited through obstruction of its membrane-binding surfaces by the regulatory region and that activation must shift PLCγ1 to a conformation competent for membrane binding. Knowledge of the critical sites of membrane interaction extends the mechanistic framework for activation, dysregulation, and therapeutic intervention.