Mitochondrial ADCK3 employs an atypical protein kinase-like fold to enable coenzyme Q biosynthesis

Abstract

The ancient UbiB protein kinase-like family is involved in isoprenoid lipid biosynthesis and is implicated in human diseases, but demonstration of UbiB kinase activity has remained elusive for unknown reasons. Here, we quantitatively define UbiB-specific sequence motifs and reveal their positions within the crystal structure of a UbiB protein, ADCK3. We find that multiple UbiB-specific features are poised to inhibit protein kinase activity, including an N-terminal domain that occupies the typical substrate binding pocket and a unique A-rich loop that limits ATP binding by establishing an unusual selectivity for ADP. A single alanine-to-glycine mutation of this loop flips this coenzyme selectivity and enables autophosphorylation but inhibits coenzyme Q biosynthesis in vivo, demonstrating functional relevance for this unique feature. Our work provides mechanistic insight into UbiB enzyme activity and establishes a molecular foundation for further investigation of how UbiB family proteins affect diseases and diverse biological pathways.

Figure 1. Unique sequence features of the UbiB family and ADCK3

(A) Domain structures of UbiB family proteins (human, yeast, and E. coli) and PKA (human). Brown triangles represent observed N-termini of mature Coq8p and mature ADCK3 (see D–G). (B) Alignment of a predicted α-helix in the N-terminal extension of UbiB family proteins as listed in (A). (C) Signature motifs identified by a statistical analysis of the UbiB family (foreground) compared to other ePK-like kinase (ELK) sequences (background) with associated sequence logos. Histogram bar height (on an approximately logarithmic scale) represents the selective constraint imposed on unique foreground residue (a measure of “uniqueness”). See also Figure S1A. (D) Confocal microscopy of HEK293 cells transfected with ADCK3-FLAG and MLS-GFP (mitochondrial marker). Nuclear DNA is visualized by Hoechst stain. (E) Anti-FLAG immunoblot of ADCK3-FLAG immunopreciptated from HEK293 cells (3 biological replicates). (F) N-terminal sequence (FxQDQ) of a Coomassie-stained band of mature, IP’d ADCK3-FLAG at ~55 kDa as determined by Edman degradation (‘x’ indicates an unclear residue), and a parallel lane analyzed by anti-FLAG immunoblot. (G) Domain structures of precursor, mature, and crystallized ADCK3. The location of the observed mitochondrial ADCK3-FLAG N-terminus (FHQDQ) in the full-length protein is indicated, along with the predicted molecular weights of all three proteins. See also Figure S2. (H) Cartoon models of precursor, mature and crystallized ADCK3. The NΔ254 model is based on our crystal structure (see Figure 2).

Figure 2. X-ray crystal structure of ADCK3^NΔ254

(A) Overall structure of ADCK3^NΔ254 with domains colored as in Figure 1A and the KxGQ motif residues represented with black spheres. (B) Surface representation of ADCK3^NΔ254 with domains colored as in (A). (C) Topology map of ADCK3^NΔ254 colored as in (A). (D) Overall structure of PKA (PDB: 1ATP) (Zheng et al., 1993) with domains colored as in Figure 1A and bound ATP represented with black sticks. (E) Surface representation of PKA with a peptide substrate analog represented as sticks and domains colored as in (D). (F) Topology map of PKA colored as in (D). See also Figure S2.

Figure 3. ADCK3 adopts an atypical PKL active site that binds nucleotides

(A) Structure of the A-rich loop and the QKE triad of ADCK3^NΔ254 colored as in Figure 2. (B) Fold changes in apparent K_d^Mg-ATP and K_d^Mg-ADP for ADCK3^NΔ250 mutants compared to wild type as assessed by differential scanning fluorimetry (DSF) (mean ± s.d., n = 3). (C) Superposition of the nucleotide binding pockets of ADCK3^NΔ254 (darker colors and black text) and PKA (PDB: 2QCS) (Kim et al., 2007) (lighter colors and gray text) colored as in Figure 2. The nucleotide (AMPPNP) (green) and cations (black spheres) are from the PKA structure. Red arrows highlight the unusual conformation of D488^ADCK3. (D) Simulated annealing composite omit maps (2mF_o–DF_c) contoured at 1.4σ (gray mesh) of the QKE triad and the serine of the AAAS motif. (E) Simulated annealing composite omit map (2mF_o–DF_c) of D488 and R611 contoured at 1.8σ (gray mesh). (F) ΔT_m of ADCK3^NΔ250 due to addition of various ligands and cations (mean ± s.d., n = 3 independent ΔT_m determinations). (G) Average differences (over 5 time points) in deuterium exchange of ADCK3^NΔ250 due to the presence of ATPγS or ADP mapped onto the structure of ADCK3^NΔ254. (H) Ribbon maps of ADCK3 showing deuterium exchange levels in three separate conditions (Basal, Mn²⁺ only; +ADP, MnADP; +ATPγS, MnATPγS) at five separate time points. Conditions with MnADP and MnATPγS are shown as changes in deuteration compared to the basal levels for each time point. Incubation times with D₂O are shown at the bottom right. See also Figure S3.

Figure 4. A single A-to-G mutation of the A-rich loop flips nucleotide selectivity and enables ADCK3 autophosphorylation

(A) Comparison of the G-rich loop of PKA (PDB: 2QCS) and the A-rich loop of ADCK3^NΔ254 (dark gray). AMPPNP (green) is from the PKA structure. The overall structural superposition is the same as in Figure 3C. (B) Nucleotide selectivity of ADCK3^NΔ250 A339G compared to wild type (WT). Apparent K_d values were assessed by DSF (mean ± s.d., n = 3; 3 independent K_d measurements were made for each of 3 different protein preparations of WT and A339G). (C and I) K_d^Mg-ATP and K_d^Mg-ADP for ADCK3^NΔ250 variants as assessed by DSF (mean ± s.d., n = 3 independent K_d determinations). (D, E, F, H, J) SDS-PAGE analysis of in vitro [γ-³²P]ATP autophosphorylation reactions with ADCK3^NΔ250 variants. MgATP was used for all reactions except those noted in (H). (E) Time course of ADCK3^NΔ250 A339G autophosphorylation. (F) Dependence of ADCK3^NΔ250 A339G autophosphorylation on ATP concentration. (G) Relative quantification of radioactivity from ³²P-ADCK3^NΔ250 A339G bands in (F). See also Figure S4.

Figure 5. UbiB-specific features of Coq8p are required for yeast growth and CoQ biosynthesis

(A and C) Serial dilutions of yeast transformed with the indicated Coq8p variants grown on agar plates with glucose or glycerol. Homologous ADCK3 residues are indicated to the left of the Coq8p residues. Red ‘X’ symbols indicate no growth on glycerol, yellow dash symbols indicate moderate or low growth on glycerol, and green check mark symbols indicate wild type-like growth on glycerol. (B) Fold changes in CoQ₆ abundance of yeast with Coq8p point mutations compared to wild type Coq8p as determined by LC-MS (mean with 95% c.i., n = 3 biological replicates). See also Figure S5.

Figure 6. Pathogenic ADCK3 mutations disrupt protein stability

(A) Residues mutated in patients with cerebellar ataxia mapped onto the ADCK3^NΔ254 structure as spheres. Domains are colored as in Figure 2. (B) Predicted structural effects of pathogenic ADCK3 mutations. (C) Fold changes in ΔT_m of ADCK3^NΔ250 mutants compared to wild type as assessed by DSF (mean ± s.d., n = 3 independent ΔT_m determinations). (D) Fold changes in apparent K_d^MgADP for ADCK3^NΔ250 mutants compared to wild type as assessed by DSF (mean ± s.d., n = 3 independent K_{d, app} determinations). *p-value < 0.05, **p-value < 0.01.