Engineering the Self-assembly of Bacterial Microcompartment Shell Proteins via Charged Mutations

Abstract

AbstractProtein self-assembly is a fundamental biological process of great importance for the design and synthesis of biomaterials. Developing the ability to precisely manipulate protein assembly would greatly expand both our understanding of the process and our biotechnological capabilities. Within bacteria, proteins that self-organize to form bacterial microcompartments (MCPs) offer an excellent model system for studying protein self-assembly and advancing biomaterial design capabilities. MCPs consist of irregular polyhedral shells that encase an enzyme core, functioning as enzymatic nanoreactors. In isolation, the abundant shell proteins of the 1,2-propanediol utilization (Pdu) MCP, PduA and PduJ, have a high propensity to self-assemble into tubular structures, analogous in form to carbon nanotubes. Here, we design and characterize tubular structures formed by hexameric PduA and PduJ proteins. We demonstrate that altering hexamer charge offers a systematic strategy for modulating the higherorder assembly of PduA and PduJ across multiple contexts by integrating molecular dynamics simulations with heterologous overexpression, cell-free, and in vivo Salmonella enterica serovar Typhimurium LT2 experiments. First, using molecular simulation, we find that tube chirality and radius play critical roles in determining structural stability and flexibility. Next, overexpression and cell-free experiments show that increasing the overall negative charge of assembling subunits consistently promotes self-assembly into tubular structures. We find that this holds true in the native MCP system, as these same mutations promote the formation of tubular MCP structures in S. enterica LT2. Our results collectively reveal that both electrostatic interactions and fields generated by charges on proteins can be leveraged to control protein-based nanostructures.