Crystal structure of the alpha(6)beta(6) holoenzyme of propionyl-coenzyme A carboxylase

Abstract

Propionyl-coenzyme A carboxylase (PCC), a mitochondrial biotin-dependent enzyme, is essential for the catabolism of the amino acids Thr, Val, Ile and Met, cholesterol and fatty acids with an odd number of carbon atoms. Deficiencies in PCC activity in humans are linked to the disease propionic acidaemia, an autosomal recessive disorder that can be fatal in infants. The holoenzyme of PCC is an alpha(6)beta(6) dodecamer, with a molecular mass of 750 kDa. The alpha-subunit contains the biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) domains, whereas the beta-subunit supplies the carboxyltransferase (CT) activity. Here we report the crystal structure at 3.2-A resolution of a bacterial PCC alpha(6)beta(6) holoenzyme as well as cryo-electron microscopy (cryo-EM) reconstruction at 15-A resolution demonstrating a similar structure for human PCC. The structure defines the overall architecture of PCC and reveals unexpectedly that the alpha-subunits are arranged as monomers in the holoenzyme, decorating a central beta(6) hexamer. A hitherto unrecognized domain in the alpha-subunit, formed by residues between the BC and BCCP domains, is crucial for interactions with the beta-subunit. We have named it the BT domain. The structure reveals for the first time the relative positions of the BC and CT active sites in the holoenzyme. They are separated by approximately 55 A, indicating that the entire BCCP domain must translocate during catalysis. The BCCP domain is located in the active site of the beta-subunit in the current structure, providing insight for its involvement in the CT reaction. The structural information establishes a molecular basis for understanding the large collection of disease-causing mutations in PCC and is relevant for the holoenzymes of other biotin-dependent carboxylases, including 3-methylcrotonyl-CoA carboxylase (MCC) and eukaryotic acetyl-CoA carboxylase (ACC).

Figure 1. Structure of the PCC holoenzyme

(a). Schematic drawing of the structure of the RpPCCα-RdPCCβ chimera, viewed down the three-fold symmetry axis. Domains in the α and β subunits in the top half of the structure are given different colors, and those in the first α and β subunits are labeled. The α and β subunits in the bottom half are colored in magenta and green, respectively. The red arrow indicates the viewing direction of panel b. (b). Structure of the RpPCCα-RdPCCβ chimera, viewed down the two-fold symmetry axis. The red rectangle indicates the region shown in detail in Fig. 2a. (c). Cryo-EM reconstruction of HsPCC at 15 Å resolution, viewed in the same orientation as panel a. The atomic model of the chimera was fit into the cryo-EM envelope. (d). The cryo-EM reconstruction viewed in the same orientation as panel b. The arrows indicate a change in the BCCP position that is needed to fit the cryo-EM map. All the structure figures were produced with PyMOL (www.pymol.org), and the cryo-EM figures were produced with Chimera .

Figure 2. Interactions between the α and β subunits in the PCC holoenzyme

(a). Schematic drawing of the interface between the α and β subunits in the RpPCCα-RdPCCβ chimera. (b). Detailed interactions between the hook in the BT domain of the α subunit and the β subunits. The C-terminal helix (α8) of an adjacent β subunit (labeled β2) is also shown. (c). Detailed interactions between helix αW in the BT domain and the β subunit. The view is related to that of panel a through a 90° rotation around the vertical axis. See Supplementary Fig. 8 for stereo versions of panels b and c.

Figure 3. The active sites of the PCC holoenzyme

(a). Schematic drawing of the relative positioning of the BC and CT active sites in the holoenzyme. One α subunit and a β₂ dimer (β1 from one layer and β4 from the other layer) are shown, and the viewing direction is the same as Fig. 1b. The two active sites are indicated with the stars, separated by 55 Å distance. The bound positions of ADP in complex with E. coli BC and that of CoA in complex with the 12S subunit of transcarboxylase are also shown. (b). Detailed interactions between BCCP-biotin and the C domain of a β subunit. Hydrogen-bonding interactions are indicated with the dashed lines in red. The N1′ atom of biotin is labeled as 1′, hydrogen-bonded to the main-chain carbonyl of Phe397. (c). Molecular surface of the CT active site, showing a deep canyon where both substrates are bound. (d). Schematic drawing of the CT active site. See Supplementary Fig. 14 for stereo versions of panels b and d.

Figure 4. Locations of disease-causing mutations in the PCC holoenzyme

Schematic drawing of the structure of one α subunit and one β₂ subunit dimer of PCC, in the same view as Fig. 3a. The locations of the missense mutations associated with PA are indicated with the spheres, colored by the domains. The BC and CT active sites are indicated with the stars.