Structural Insight into the Mechanisms of Transport across the Salmonella enterica Pdu Microcompartment Shell

Abstract

Bacterial microcompartments are a functionally diverse group of proteinaceous organelles that confine specific reaction pathways in the cell within a thin protein-based shell. The propanediol utilizing (Pdu) microcompartment contains the reactions for metabolizing 1,2-propanediol in certain enteric bacteria, including Salmonella. The Pdu shell is assembled from a few thousand protein subunits of several different types. Here we report the crystal structures of two key shell proteins, PduA and PduT. The crystal structures offer insights into the mechanisms of Pdu microcompartment assembly and molecular transport across the shell. PduA forms a symmetric homohexamer whose central pore appears tailored for facilitating transport of the 1,2-propanediol substrate. PduT is a novel, tandem domain shell protein that assembles as a pseudohexameric homotrimer. Its structure reveals an unexpected site for binding an [Fe-S] cluster at the center of the PduT pore. The location of a metal redox cofactor in the pore of a shell protein suggests a novel mechanism for either transferring redox equivalents across the shell or for regenerating luminal [Fe-S] clusters.Bacterial microcompartment organelles are enclosed proteinaceous structures that encapsulate the sequential reaction steps for particular metabolic pathways (for recent reviews, see Refs. 1-3). Previous studies have elucidated several classes of microcompartment organelles, categorized according to their encapsulated enzymes. The prototypical member of these microcompartments is the carboxysome, which exists in autotrophic cyanobacteria and some chemoautotrophic bacteria. The carboxysome catalyzes inorganic carbon fixation from ribulose-1,5-bisphosphate and HCO 3 Ϫ via two sequentially acting enzymes, carbonic anhydrase and RuBisCO (for review, see Ref. 4). Microcompartment organelles that catalyze other metabolic reactions exist in heterotrophic bacteria, particularly the enteric facultative anaerobes. Functionally diverse microcompartments share a similar protein-based shell. Despite encapsulating distinct sets of enzymes in their interiors, the shells are all constructed by the assembly of proteins belonging to the same family of homologous BMC (bacterial microcompartment) proteins (Refs. 5 and 6; for review, see Ref.3).The enteric proteobacterium Salmonella enterica typhimurium forms a propanediol-utilizing (Pdu) 2 microcompartment for the initial steps in metabolizing the substrate 1,2-propanediol (5, 7-9). The Pdu microcompartments are heterogeneous in size, between 120 -160 nm across, and appear polyhedral in shape with irregular facets (Fig. 1) (9). The architecture of the Pdu shell is likely similar to the carboxysome based on the similarity of the shell proteins in the two systems (5, 9, 10), although carboxysome microcompartments have been shown to form more regular icosahedral structures (11-13). The genes for the formation of the Pdu microcompartment are encoded within the ϳ19-kbp pdu operon, which codes for 21 genes whose products are all related to 1,2-pr...

The effects of time, temperature, and pH on the stability of PDU bacterial microcompartments

“…While these may still be intact MCPs, another potential explanation is that the MCPs and the reporter protein formed aggregates. In agreement with this latter hypothesis, a time course of cells expressing PduP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] -GFP in the absence of MCP induction shows diffuse fluorescence for several days, and also shows potential aggregation after eight days, as seen by the emergence of fluorescent puncta (Supporting Information 2, Fig. S1).…”

“…[7][8][9][10] The structures of several of these shell proteins have been solved, providing insight on how the subunits assemble to form the polyhedral shell. [11][12][13] The major constituents of the Pdu MCP shell form homohexamers and self-assemble to create the facets of the MCP [ Fig. 1(C)].…”

Diverse Bacterial Microcompartment Organelles

“…The first crystal structures of BMC domain proteins revealed that they pack tightly together into extended two-dimensional layers with only a few small openings (63). The most notable openings were central pores that were proposed to function in selective metabolite transport (63,68). Subsequent crystallography revealed that the pores of different BMC domain proteins diverge in their properties, consistent with a role in the selective transport of various metabolites (11,12,61).…”

“…For the PduA protein, which is a component of an MCP used for 1,2-PD degradation, its central pore is lined with numerous H-bond donors and acceptors. This property is proposed to allow preferential movement of 1,2-PD compared to propionaldehyde, which must be held inside the MCP to prevent cellular toxicity (68). In addition, modeling suggests that the PduJ and PduK shell proteins have central pores similar to those of PduA and might also transport 1,2-PD.…”

“…Later structures identified shell proteins whose monomers have two fused BMC domains. The double-BMC-domain proteins form trimers composed of six BMC domains in a pseudohexameric arrangement that is similar overall to that of the hexameric single-BMC-domain proteins (65)(66)(67)(68). These trimers likely arose by gene duplication, and this variation may allow protein conformational changes by eliminating the strict 6-fold symmetry of the BMC domain hexamer (11).…”