Functional Attributes and Self-Assembly Behavior of a Shell Protein the 1,2 Propanediol Utilization Prokaryotic Metabolosome

Abstract

Complex subcellular organization enable cells to synergize multiple metabolic reactions simultaneously. Many species of bacteria have the ability to compartmentalize enzymatic cascade within a nano-sized protein compartments called bacterial microcompartments. These microcompartments have evolved to perform catabolic or anabolic reactions and some of them aid bacteria to survive under energy dearth environment. Within my research, I have focused on deciphering the assembly and function of the proteins that make up the architecture of a bacterial microcompartment involved in 1,2-propanediol metabolism (1,2- propanediol utilization microcompartment) in Salmonella enterica LT2. The component proteins of these microcompartments are encoded by genes belonging to a single operon. The operon encodes for two types of proteins, the shell proteins that form the outer cover of the compartment and the enzymes that are encapsulated within and work in cascade. My study shows that a major shell protein called PduBB’, exhibits high thermal stability and preserves the catalytic activity and native conformation of an encapsulated diol dehydratase enzyme called PduCDE. The individual components of the shell protein (PduB and PduB’) in isolation fail to influence the activity of PduCDE and do not show any protective role. The combination of PduB and PduB’ provides solubility and stability to the shell protein PduBB’, improving its association with the enzyme PduCDE. The longer component PduB has extra 37 amino acids N-terminal extension that has a short helical segment followed by a disordered region. Similarly, the middle subunit (subunit D) of PduCDE also has a helical portion followed by disordered region. A combination of biochemical, spectroscopic and computational modeling studies suggests that the N-terminal extensions of PduB and PduD might be involved in mediating shell protein-enzyme interaction. We propose that the disordered regions provide flexibility to the N-terminal extensions of shell protein and enzyme, facilitating their association mediated by the helical segments. PduBB’ displays an interesting self-assembly behavior and under crowded environment and appropriate ionic strength, it undergoes liquid-liquid phase separation. While PduB' has a high self-associating property displaying liquid-solid transition, PduB has the lowest tendency to undergo phase separation. This implies that the combination of two proteins in PduBB’ results in a wellbalanced self-assembly behavior. The co-phase separation of shell protein PduBB’ and enzyme PduCDE enhances the catalytic efficiency of the enzyme, highlighting the significance of shell-enzyme association and phase separation in improving the catalytic ix performance of the enzyme. My study has two major implications. First, it suggests that formation of PduMCP is likely to be mediated by a combination of protein-protein interaction and phase separation, where disordered N-terminal regions provide flexible association between shell protein and enzyme. Second, the chaperone like behavior of a major shell protein of contributes towards higher thermal stability and catalytic activity of an encapsulated native enzyme.