Structural insights into ubiquinone biosynthesis in membranes

Abstract

Biosynthesis of ubiquinones requires the intramembrane UbiA enzyme, an archetypal member of a superfamily of prenyltransferases that generates lipophilic aromatic compounds. Mutations in eukaryotic superfamily members have been linked to cardiovascular degeneration and Parkinson's disease. To understand how quinones are produced within membranes, we report the crystal structures of an archaeal UbiA in its apo and substrate-bound states at 3.3 and 3.6 angstrom resolution, respectively. The structures reveal nine transmembrane helices and an extramembrane cap domain that surround a large central cavity containing the active site. To facilitate the catalysis inside membranes, UbiA has an unusual active site that opens laterally to the lipid bilayer. Our studies illuminate general mechanisms for substrate recognition and catalysis in the UbiA superfamily and rationalize disease-related mutations in humans.

Figure 1. Scheme of UbiA catalysis and structure of ApUbiA

(A) UbiA catalysis. Cleavage of the pyrophosphate group from the IPP substrate generates a carbocation intermediate, which reacts at the meta-position of the PHB substrate to complete the condensation reaction. (B) Apo-structure of ApUbiA viewed from the cytoplasmic side. The cap domain is shown in pink and the TMs are shown in different blue colors. (C) A side view of the same structure. Conserved residues in the central cavity are shown in orange. (D) Cartoon representation of the structure, which has a unique lateral portal that opens to membrane. The large central cavity contains a polar pocket (pink) for pyrophosphate binding, a hydrophobic wall (grey) for the binding of isoprenyl chains, and a small basic pocket (blue) for PHB binding (see fig. S5).

Figure 2. Substrate binding of ApUbiA

(A) The ApUbiA structure in complex with the substrates. The B-factor sharpened experimental map (in purple) is contoured at 1σ. Due to the limited resolution, the interactions (dashed lines) between individual atoms are putative. (B) Conformational changes induced by GSPP binding. Top: the experimental maps (1σ) of the apo (green) and GSPP-bound (purple) structures suggest the change of interactions. Bottom: a cartoon shows that an interaction network is established upon GSPP binding. (C) Comparison ofthe loop region near Arg63 and Arg67 (same maps as in B). The green arrows indicate a few disordered regions in the apo-structure. (D) Trypsin digestion of the ApUbiA protein in presence of GSPP, PHB, and Mg. The primary digestion site is at Arg63 and a minor digestion site (asterisk) is in a linker region of the N-terminal His-tag.

Figure 3. Binding and activity assays of UbiA enzymes

(A) ITC measurements of the GSPP (left) and PHB (right) binding to ApUbiA in presence of Mg. (B) Prenyltransferase activities of EcUbiA mutants. Conserved residues at the central cavity are mutated (n.d.: not detected; error bars: s.e.m. of duplicated assays). Corresponding residues in the ApUbiA structure (Fig. 2A) are labeled in red. R137 is a positive control that is not at the active site in our structure, yet was predicted important in an in silico model (19).

Figure 4. Structural comparison of ApUbiA and a soluble trans-prenyltransferase

The FPPS (1RQI (20)) structure is a top DALI (29) hit of the ApUbiA structure. (A) The superimposed structures of ApUbiA (blue) and FPPS (purple). (B) Electrostatic representation of the binding pockets in UbiA (left) and FPPS (right). The two pockets are generated from superimposed structures and shown in the same orientation.