A Model for Allosteric Control of Pore Opening by Substrate Binding in the Eutl Microcompartment Shell Protein

Abstract

The ethanolamine utilization microcompartment (eut MCP) is a giant protein assembly that acts as a metabolic organelle in enteropathogenic bacteria, allowing them to proliferate in the human gut. The eut MCP consists of a polyhedral shell, reminiscent of a viral capsid, which encapsulates several sequentially acting metabolic enzymes that convert ethanolamine to ethanol and acetyl phosphate. The tightly-packed molecular shell surrounding the eut MCP is believed to act as a semi-permeable barrier, allowing the passage of substrates, products, and larger cofactor molecules, while minimizing the efflux of a toxic acetaldehyde intermediate. Previous structural studies of the eut MCP demonstrated that a conformational change of the EutL shell protein opens a 10-15A pore through the shell. That observation led to a model for how the protein shell might interconvert between high and low permeability conformations, but the mechanism controlling the pore opening has remained unclear. Here we present structural and biophysical studies directed toward understanding how the conformational switch is regulated in EutL. The X-ray crystal structure of EutL bound to ethanolamine provides evidence that binding of this small metabolite stabilizes the closed-pore conformation by sterically blocking rearrangement to the open conformation. Specific binding of ethanolamine to EutL was verified by isothermal titration calorimetry (ITC). Thermodynamic parameters derived from ITC experiments were rationalized through analysis of molecular contacts revealed by X-ray crystallography and molecular dynamics simulations. We show that ethanolamine binding is specific; i.e. EutL does not bind to other small molecules associated with the metabolic reactions carried out in the eut MCP. Our results suggest a model for EutL function in which the presence of ethanolamine decreases the porosity of the MCP shell by modulating the interconversion between open and closed pore conformations.