Phase Separation of Shell Protein and Enzyme: An Insight into the Biogenesis of a Prokaryotic Metabolosome

Bacterial microcompartments are substrate specific metabolic modules that are conditionally expressed in certain bacterial species. These all protein structures have size in the range of 100-150 nm and are formed by the self-assembly of thousands of protein subunits, all encoded by genes belonging to a single operon. The operon contains genes that encode for both enzymes and shell proteins. The shell proteins self-assemble to form the outer coat of the compartment and enzymes are encapsulated within. A perplexing question in MCP biology is to understand the mechanism which governs the formation of these small yet complex assemblages of proteins. In this work we use 1,2-propanediol utilization microcompartments (PduMCP) as a paradigm to identify the factors that drive the self-assembly of MCP proteins. We find that a major shell protein PduBB tend to self-assemble under macromolecular crowded environment and suitable ionic strength. Microscopic visualization and biophysical studies reveal phase separation to be the principle mechanism behind the self-association of shell protein in the presence of salts and macromolecular crowding. The shell protein PduBB interacts with the enzyme diol-dehydratase PduCDE and co-assemble into phase separated liquid droplets. The co-assembly of PduCDE and PduBB results in the enhancement of catalytic activity of the enzyme. A combination of spectroscopic and biochemical techniques shows the relevance of divalent cation Mg2+ in providing stability to intact PduMCP in vivo. Together our results suggest a combination of protein-protein interactions and phase separation guiding the self-assembly of Pdu shell protein and enzyme in solution phase. Significance StatementPresent work shows how surrounding environment modulates the self-assembly behavior of a major shell protein of 1,2-propanediol utilization microcompartment (PduMCP). Appropriate ionic strength and macromolecular crowding bring about liquid-liquid phase separation of the shell protein. Under crowded environment Mg2+ displayed unique property to drive the formation of shell protein liquid condensates. The co-phase separation of a native enzyme along with the shell protein enables it to outperform the enzyme in isolation. This adds on to the existing concept of phase separation being the underlying principle behind the genesis of lipid free organelles in prokaryotes. Further, our results indicate that the divalent metal ion Mg2+ plays an intricate role in the outfitting of the structure-function integrity of PduMCPs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/476392v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@1b95271org.highwire.dtl.DTLVardef@1d3a5b5org.highwire.dtl.DTLVardef@2432eaorg.highwire.dtl.DTLVardef@8aad07_HPS_FORMAT_FIGEXP M_FIG C_FIG