Exploring bacterial organelle interactomes: a model of the protein-protein interaction network in the Pdu microcompartment

Abstract

Bacterial microcompartments (MCPs) are protein-bound organelles that carry out diverse metabolic pathways in a wide range of bacteria. These supramolecular assemblies consist of a thin outer protein shell, reminiscent of a viral capsid, which encapsulates sequentially acting enzymes. The most complex MCP elucidated so far is the propanediol utilizing (Pdu) microcompartment. It contains the reactions for degrading 1,2-propanediol. While several experimental studies on the Pdu system have provided hints about its organization, a clear picture of how all the individual components interact has not emerged yet. Here we use co-evolution-based methods, involving pairwise comparisons of protein phylogenetic trees, to predict the protein-protein interaction (PPI) network governing the assembly of the Pdu MCP. We propose a model of the Pdu interactome, from which selected PPIs are further inspected via computational docking simulations. We find that shell protein PduA is able to serve as a "universal hub" for targeting an array of enzymes presenting special N-terminal extensions, namely PduC, D, E, L and P. The varied N-terminal peptides are predicted to bind in the same cleft on the presumptive luminal face of the PduA hexamer. We also propose that PduV, a protein of unknown function with remote homology to the Ras-like GTPase superfamily, is likely to localize outside the MCP, interacting with the protruding β-barrel of the hexameric PduU shell protein. Preliminary experiments involving a bacterial two-hybrid assay are presented that corroborate the existence of a PduU-PduV interaction. This first systematic computational study aimed at characterizing the interactome of a bacterial microcompartment provides fresh insight into the organization of the Pdu MCP.

Figure 1. An idealized model of the Pdu MCP shell and its encapsulated pathway.

The MCP shell is assembled from a few thousand copies of proteins belonging to the BMC (bacterial microcompartment) protein family. Several distinct paralogs from the BMC family are present within a single shell. BMC proteins self-assemble into cyclical hexamers (in blue). Also present in fewer copies are proteins from a distinct family, referred to as BMVs, which are pentameric proteins (in yellow) forming the vertices of the polyhedral structure. The polyhedron is shown here idealized as an icosahedron, while the Pdu MCP is typically less regular in shape. Sequentially acting enzymes (in black) carrying out the Pdu pathway are enclosed by the shell (A). The Pdu pathway degrades 1,2-propanediol to propionaldehyde via a B₁₂-dependent catalytic mechanism, the aldehyde being subsequently converted to 1-propanol or propionyl-phosphate (B).

Figure 2. Description of the procedure for defining pairwise coevolution descriptors.

Calculation of coevolution descriptors relies on the comparison of phylogenetic trees. For each given pair of Pdu gene products, three descriptors are extracted from a topological comparison of their respective phylogenetic trees (blue and green) and the Tree of Life (ToL, pink), while four other descriptors are calculated by comparing the distance matrices that underlie these trees. These seven descriptors are further combined into a vector for subsequent analysis by the RF classifier.

Figure 3. A model of the Pdu interactome.

The Pdu PPI network, inferred from predictions made by the RF classifier in its analysis of coevolution descriptors. Individual Pdu gene products are represented as nodes. Enzymes are shown in light blue, while shell proteins are shown in gray; the shell proteins include several BMC type proteins and a single protein (PduN) from the BMV family presumed to be pentameric vertex proteins. Edges connecting two nodes correspond to predicted PPIs. The numerous PPIs emerging from the PduA node are highlighted in pink. It is not possible to fully convey the likely spatial relationships of all the proteins and enzymes (some of whose locations remain uncertain), but nodes for the shell proteins have been placed at the periphery of the layout to convey their outer locations.

Figure 4. Models of N-terminal peptide extensions from different enzymes docked onto a PduA hexamer. All the models were aligned and overlaid using the PduA structure as guide.

(A) Six N-terminal peptides are docked on the concave (presumptively luminal) face of the PduA hexamer. Four of the five identified earlier as probable targeting sequences (PduC, PduD, PduE, PduP) were folded into α-helices by the flexible docking procedure (see text and Methods) and were docked in the same cleft on the PduA surface. The tail of PduL adopted a less regular conformation during the simulation. The tail from PduQ, which was not predicted to act as a targeting sequence and thereby serves as a control, exhibits an apparently spurious binding mode. To convey depth, the surface of PduA is shaded according to diffusion accessibility [106]. (B) The five targeting peptides, when docked onto the other (flat) face of the PduA shell protein, were found scattered across the surface in arrangements exhibiting poorer interaction interfaces. (C) Binding statistics are reported for all the docking simulations. In all cases, both the predicted energy score (in Rosetta Energy Units) and the buried surface at the interface yielded better values when peptides were docked onto PduA’s concave side. Because the shell protein hexamer is 6-fold symmetric, in all cases the solutions were rotated by multiples of 60° around the axis of symmetry to allow internal consistency.

Figure 5. N-terminal extension sequences and atomic details of the PduD peptide docked onto a PduA hexamer.

(A) Sequences of the five N-terminal extensions proposed to be acting as targeting peptides. An arginine is recurrently found near the center of the peptide (red). (B) The hydrophobic surface (in beige) of the PduD N-terminal tail peptide is predicted to interact with the C-terminal tail of PduA. A central arginine (in red), which is found in all of the N-terminal peptides predicted to dock in the cleft, is consistently oriented to make interactions with a glutamate in the BMC domain.

Figure 6. Model of PduV docked onto a PduU hexamer.

(A) Docking calculations predict that the N-terminal region of PduV binds the PduU β-barrel that protrudes from the conserved BMC domain. Binding statistics for the PduU-PduV docking and three control simulations are reported in a separate table (B). Those latter, which included the docking of PduU to a non-cognate GTPase homolog of PduV (labeled PduU-ERA), a truncated version of PduU lacking the beta-barrel docking to PduV (labeled PduU Δ17-PduV), and PduV docking to PduA instead of PduU (labeled PduA-PduV), all had substantially worse binding statistics than the PduU-PduV model.