An invariant C-terminal tryptophan in McdB mediates its interaction and positioning function with carboxysomes

Abstract

Bacterial microcompartments (BMCs) are widespread, protein-based organelles that regulate metabolism. The model for studying BMCs is the carboxysome, which facilitates carbon-fixation in several autotrophic bacteria. Carboxysomes can be distinguished as type or {beta}, which are structurally and phyletically distinct. We recently characterized the Maintenance of Carboxysome Distribution (Mcd) systems responsible for spatially regulating - and {beta}-carboxysomes, consisting of the proteins McdA and McdB. McdA is an ATPase that drives carboxysome positioning, and McdB is the adaptor protein that directly interacts with carboxysomes to provide cargo specificity. The molecular features of McdB proteins that specify their interactions with carboxysomes, and whether these are similar between - and {beta}-carboxysomes, remain unknown. Here, we identify C-terminal motifs containing an invariant tryptophan necessary for - and {beta}-McdBs to associate with - and {beta}-carboxysomes, respectively. Substituting this tryptophan with other aromatic residues reveals corresponding gradients of carboxysome colocalization and positioning by McdB in vivo. Intriguingly, these gradients also correlate with the ability of McdB to form condensates in vitro. The results reveal a shared mechanism underlying McdB adaptor protein binding to carboxysomes, and potentially other BMCs. Our findings also implicate condensate formation as playing a key role in this association. SIGNIFICANCE STATEMENTO_LIMaintenance of carboxysome distribution protein B (McdB) is necessary for positioning a widespread class of protein-based organelles in bacteria that regulate metabolism. Without McdB, these organelles aggregate and lose functionality. How McdB interacts with and positions these organelles is unknown. C_LIO_LIWe determine that an invariant tryptophan is necessary for McdB to interact with and position its organelle. A similar mechanism occurs in two diverse bacterial cell types, both relying on the invariant tryptophan. C_LIO_LIThis class of bacterial organelle includes compartments involved in bacterial pathogenesis and carbon fixation. Our results therefore advance our understanding and applications of these organelles. C_LI