Bacterial microcompartment shells of diverse functional types possess pentameric vertex proteins

Abstract

Bacterial microcompartments (MCPs) are large proteinaceous structures comprised of a roughly icosahedral shell and a series of encapsulated enzymes. MCPs carrying out three different metabolic functions have been characterized in some detail, while gene expression and bioinformatics studies have implicated other types, including one believed to perform glycyl radical-based metabolism of 1,2-propanediol (Grp). Here we report the crystal structure of a protein (GrpN), which is presumed to be part of the shell of a Grp-type MCP in Rhodospirillum rubrum F11. GrpN is homologous to a family of proteins (EutN/PduN/CcmL/CsoS4) whose members have been implicated in forming the vertices of MCP shells. Consistent with that notion, the crystal structure of GrpN revealed a pentameric assembly. That observation revived an outstanding question about the oligomeric state of this protein family: pentameric forms (for CcmL and CsoS4A) and a hexameric form (for EutN) had both been observed in previous crystal structures. To clarify these confounding observations, we revisited the case of EutN. We developed a molecular biology-based method for accurately determining the number of subunits in homo-oligomeric proteins, and found unequivocally that EutN is a pentamer in solution. Based on these convergent findings, we propose the name bacterial microcompartment vertex for this special family of MCP shell proteins.

Figure 1

Comparison of the structure of GrpN with other bacterial microcompartment vertex (BMV) proteins. (A) An idealized model of an MCP showing pentameric units at the vertices of the polyhedral shell. (B) Space-filling model of the pentameric structure of GrpN. (C) Superposition of a GrpN monomer with CcmL (top, yellow), CsoS4A (middle, blue), and EutN (bottom, magenta). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 2

Application of the OCAC (oligomeric characterization by the addition of charge) method to the EutN shell protein. (A) Diagram of primary structure of EutN before and after cleavage with TEV protease. The plus symbol “ + ” denotes one additional positive charge. (B) For an n-oligomer, n + 1 possible charge states exist. Shown are the six possible charge-states of a pentamer. (C) EutN OCAC native gel. The engineered EutN(+) oligomer was incubated with TEV protease for 15 min, quenched with iodoacetamide, and then run on a native gel. From left to right, TEV protease concentrations are 0.0, 0.002, 0.01, 0.02, 0.1, and 0.5 mg/mL. The presence of six distinct bands shows that EutN is a pentamer. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]