Molecular Sensing and Manipulation of Protein Oligomerization in Membrane Nanotubes with Bolaamphiphilic Foldamers.

Adaptive and reversible self-assembly of supramolecular protein structures is a fundamental characteristic of dynamic living matter. However, the quantitative detection and assessment of the emergence of mesoscale protein complexes from small and dynamic oligomeric precursors remains highly challenging. Here, we present a novel approach utilizing a short membrane nanotube (sNT) pulled from a planar membrane reservoir as nanotemplates for molecular reconstruction, manipulation, and sensing of protein oligomerization and self-assembly at the mesoscale. The sNT reports changes in membrane shape and rigidity caused by membrane-bound proteins as variations of the ionic conductivity of the sNT lumen. To confine oligomerization to the sNT, we have designed and synthesized rigid oligoamide foldamer tapes (ROFTs). Charged ROFTs incorporate into the planar and sNT membranes, mediate protein binding to the membranes, and, driven by the luminal electric field, shuttle the bound proteins between the sNT and planar membranes. Using Annexin-V (AnV) as a prototype, we show that the sNT detects AnV oligomers shuttled into the nanotube by ROFTs. Accumulation of AnV on the sNT induces its self-assembly into a curved lattice, restricting the sNT geometry and inhibiting the material uptake from the reservoir during the sNT extension, leading to the sNT fission. By comparing the spontaneous and ROFT-mediated entry of AnV into the sNT, we reveal how intricate membrane curvature sensing by small AnV oligomers controls the lattice self-assembly. These results establish sNT-ROFT as a powerful tool for molecular reconstruction and functional analyses of protein oligomerization and self-assembly, with broad application to various membrane processes.

Reconstitution and real-time quantification of membrane remodeling by single proteins and protein complexes.

Cellular membrane processes, from signal transduction to membrane fusion and fission, depend on acute membrane deformations produced by small and short-lived protein complexes working in conditions far from equilibrium. Real-time monitoring and quantitative assessment of such deformations are challenging; hence, mechanistic analyses of the protein action are commonly based on ensemble averaging, which masks important mechanistic details of the action. In this protocol, we describe how to reconstruct and quantify membrane remodeling by individual proteins and small protein complexes in vitro, using an ultra-short (80- to 400-nm) lipid nanotube (usNT) template. We use the luminal conductance of the usNT as the real-time reporter of the protein interaction(s) with the usNT. We explain how to make and calibrate the usNT template to achieve subnanometer precision in the geometrical assessment of the molecular footprints on the nanotube membrane. We next demonstrate how membrane deformations driven by purified proteins implicated in cellular membrane remodeling can be analyzed at a single-molecule level. The preparation of one usNT takes ~1 h, and the shortest procedure yielding the basic geometrical parameters of a small protein complex takes 10 h.

Structural Snapshots and Loop Dynamics along the Catalytic Cycle of Glycosyltransferase GpgS.

Glycosyltransferases (GTs) play a central role in nature. They catalyze the transfer of a sugar moiety to a broad range of acceptor substrates. GTs are highly selective enzymes, allowing the recognition of subtle structural differences in the sequences and stereochemistry of their sugar and acceptor substrates. We report here a series of structural snapshots of the reaction center of the retaining glucosyl-3-phosphoglycerate synthase (GpgS). During this sequence of events, we visualize how the enzyme guides the substrates into the reaction center where the glycosyl transfer reaction takes place, and unveil the mechanism of product release, involving multiple conformational changes not only in the substrates/products but also in the enzyme. The structural data are further complemented by metadynamics free-energy calculations, revealing how the equilibrium of loop conformations is modulated along these itineraries. The information reported here represent an important contribution for the understanding of GT enzymes at the molecular level.

Structural basis for selective recognition of acyl chains by the membrane-associated acyltransferase PatA.

The biosynthesis of phospholipids and glycolipids are critical pathways for virtually all cell membranes. PatA is an essential membrane associated acyltransferase involved in the biosynthesis of mycobacterial phosphatidyl-myo-inositol mannosides (PIMs). The enzyme transfers a palmitoyl moiety from palmitoyl-CoA to the 6-position of the mannose ring linked to 2-position of inositol in PIM1/PIM2. We report here the crystal structures of PatA from Mycobacterium smegmatis in the presence of its naturally occurring acyl donor palmitate and a nonhydrolyzable palmitoyl-CoA analog. The structures reveal an α/ß architecture, with the acyl chain deeply buried into a hydrophobic pocket that runs perpendicular to a long groove where the active site is located. Enzyme catalysis is mediated by an unprecedented charge relay system, which markedly diverges from the canonical HX4D motif. Our studies establish the mechanistic basis of substrate/membrane recognition and catalysis for an important family of acyltransferases, providing exciting possibilities for inhibitor design.