CryoEM reveals oligomeric isomers of a multienzyme complex and assembly mechanics

Abstract

Propionyl-CoA carboxylase (PCC) is a multienzyme complex consisting of up to six α-subunits and six β-subunits. Belonging to a metabolic pathway converging on the citric acid cycle, it is present in most forms of life and irregularities in its assembly lead to serious illness in humans, known as propionic acidemia. Here, we report the cryogenic electron microscopy (cryoEM) structures and assembly of different oligomeric isomers of endogenous PCC from the parasitic protozoan Leishmania tarentolae (LtPCC). These structures and their statistical distribution reveal the mechanics of PCC assembly and disassembly at equilibrium. We show that, in solution, endogenous LtPCC β-subunits form stable homohexamers, to which different numbers of α-subunits attach. Sorting LtPCC particles into seven classes (i.e., oligomeric formulae α₀β₆, α₁β₆, α₂β₆, α₃β₆, α₄β₆, α₅β₆, α₆β₆) enables formulation of a model for PCC assembly. Our results suggest how multimerization regulates PCC enzymatic activity and showcase the utility of cryoEM in revealing the statistical mechanics of reaction pathways.

Keywords: Conformation; Propionyl-CoA; Rate constant; Statistical mechanics; Thermodynamics.

Fig. 1

Structure of the LtPCC α₆β₆ dodecamer and the α-β binding site. (A) Atomic model of LtPCC α₆β₆ dodecamer, shown as ribbons and colored by domains. (B) A bird’s eye view of the atomic model in (A). Dashed lines indicate the boundaries between neighboring subunits. (C) Domain organization of LtPCC α- and β-subunits; the color scheme is used throughout the manuscript. (D) Representative cryoEM densities superimposed with the atomic model of LtPCC shown as ribbons and sticks. (E) Atomic model of an α-subunit and a β-subunit of LtPCC colored by domains and shown as ribbons, with active sites labeled. Residues at the α-β binding site are boxed with dotted lines in (F) and labeled in zoomed-in views (G-I). Residues that form hydrogen bonds are labeled in blue. BTI stands for biotin. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

LtPCC conformations differ only in occupancies of the α-β binding sites. Cartoon representations of α₆β₆ (A) α₅β₆ (B) and α₄β₆ (C) cryoEM densities, with α-subunits in orange and β-subunits in green (top row). Two views of the cryoEM densities for α₆β₆ (A) α₅β₆ (B) and α₄β₆ (C) colored by domain as in Fig. 1C (middle rows), superimposed with their respective atomic models represented as ribbons (bottom row). In (A), the BC domain cryoEM density was displayed at a threshold of 0.0142, BT domain at 0.0193, BCCP domain at 0.0181, BT-BCCP linker at 0.0163, C-CT domain at 0.0187, and N-CT domain at 0.0244. In (B) and (C) the cryoEM density of all domains were displayed at a threshold of 0.0105 and 0.0121, respectively. Dust was hidden with “Hide Dust” in ChimeraX. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Sorting method to determine LtPCC oligomeric formula distributions. (A) Cartoon representation of the sorting method, with α-subunits in orange and β-subunits in green. (B) Frequency graph of LtPCC oligomeric formulae calculated from the sorting method. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Reaction mechanics of LtPCC. In the center is the graph of LtPCC reaction rate versus reaction product, showing exponential decay of reaction rate with increasing numbers of α-subunits in the reaction product. Surrounding the graph is the reaction diagram of LPCC assembly/disassembly, with oligomeric isomers of each oligomeric formula grouped together. α-subunits are colored in orange and β-subunits are colored in green. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)