Structural basis for the geometry-driven localization of a small protein

Abstract

In bacteria, certain shape-sensing proteins localize to differently curved membranes. During sporulation in Bacillus subtilis, the only convex (positively curved) surface in the cell is the forespore, an approximately spherical internal organelle. Previously, we demonstrated that SpoVM localizes to the forespore by preferentially adsorbing onto slightly convex membranes. Here, we used NMR and molecular dynamics simulations of SpoVM and a localization mutant (SpoVM P9A ) to reveal that SpoVM's atypical amphipathic α-helix inserts deeply into the membrane and interacts extensively with acyl chains to sense packing differences in differently curved membranes. Based on binding to spherical supported lipid bilayers and Monte Carlo simulations, we hypothesize that SpoVM's membrane insertion, along with potential cooperative interactions with other SpoVM molecules in the lipid bilayer, drives its preferential localization onto slightly convex membranes. Such a mechanism, which is distinct from that used by high curvature-sensing proteins, may be widely conserved for the localization of proteins onto the surface of cellular organelles.ArfGAP1 | SpoIVA | DivIVA | curvature sensing | lipid packing

A Quality-Control Mechanism Removes Unfit Cells from a Population of Sporulating Bacteria

“…Assembly of the coat begins with the localization of a small 26-amino acid protein, SpoVM (Levin et al, 1993), which recognizes the outer forespore membrane by preferentially adsorbing onto the forespore’s positively curved membrane surface (Gill et al, 2015; Ramamurthi et al, 2009). SpoVM recruits the structural protein SpoIVA (Price and Losick, 1999; Ramamurthi et al, 2006; Roels et al, 1992; Wu et al, 2015) which polymerizes irreversibly in an ATP-dependent manner around the forespore surface (Castaing et al, 2013; Ramamurthi and Losick, 2008).…”

The Unsolved Problem of How Cells Sense Micron-Scale Curvature

“…Although the wild-type SpoVM N terminus was found to be highly flexible, the SpoVM P9A N terminus was more rigid. It was proposed that the flexible N terminus is important for recruiting additional SpoVM molecules and other forespore proteins to positively curved membranes in a cooperative manner, consistent with the obtained Hill coefficients from saturation binding curves [29]. Cooperativity may emerge from oligomerization and thus it will be important to determine if SpoVM can oligomerize (perhaps mediated by its flexible N-terminus), and if oligomerization is critical for membrane curvature sensing.…”

“…To determine whether SpoVM can distinguish between different curvatures, recombinant SpoVM was incubated with lipid bilayers supported on spherical silica beads of defined curvatures [21, 29]. Saturation binding curves for SpoVM adsorption onto 2 µm and 8 µm diameter surfaces revealed that at low concentrations, SpoVM preferentially binds the 2 µm beads.…”

“…Analysis of the primary sequence followed by solution state NMR of SpoVM revealed that an amphipathic helix extends from amino acid residues 11 to 23, flanked by flexible N and C termini [21, 29]. Amphipathic helices harbor hydrophobic and polar regions on opposite faces of the helix and are a recurring theme of membrane curvature sensors.…”

“…This reveals open sites for amphipathic helix insertion into the membrane [43]. On the micron-scale, defects in lipid packing are likely to manifest differently, and may be at the level of the fatty acyl chain rather than with the phospholipid head group [29]. This could explain why SpoVM penetrates the bilayer and likely localizes well below the phospholipid head group.…”