Toward an Understanding of the Structural and Mechanistic Aspects of Protein-Protein Interactions in 2-Oxoacid Dehydrogenase Complexes

Abstract

The 2-oxoglutarate dehydrogenase complex (OGDHc) is a key enzyme in the tricarboxylic acid (TCA) cycle and represents one of the major regulators of mitochondrial metabolism through NADH and reactive oxygen species levels. The OGDHc impacts cell metabolic and cell signaling pathways through the coupling of 2-oxoglutarate metabolism to gene transcription related to tumor cell proliferation and aging. DHTKD1 is a gene encoding 2-oxoadipate dehydrogenase (E1a), which functions in the L-lysine degradation pathway. The potentially damaging variants in DHTKD1 have been associated to the (neuro) pathogenesis of several diseases. Evidence was obtained for the formation of a hybrid complex between the OGDHc and E1a, suggesting a potential cross talk between the two metabolic pathways and raising fundamental questions about their assembly. Here we reviewed the recent findings and advances in understanding of protein-protein interactions in OGDHc and 2-oxoadipate dehydrogenase complex (OADHc), an understanding that will create a scaffold to help design approaches to mitigate the effects of diseases associated with dysfunction of the TCA cycle or lysine degradation. A combination of biochemical, biophysical and structural approaches such as chemical cross-linking MS and cryo-EM appears particularly promising to provide vital information for the assembly of 2-oxoacid dehydrogenase complexes, their function and regulation.

Keywords: enzyme catalysis; glucose metabolism; hydrogen exchange mass spectrometry; molecular modeling; neurodegeneration; protein assembly; protein cross-linking; protein-protein interaction.

Scheme 1

Role of the OGDHc in the TCA cycle and of the E1a in the L-lysine degradation pathway in health and diseases. AASS, 2-aminoadipate-6-semialdehyde synthase; AASDH, 2-amino-adipate-6-semialdehyde dehydrogenase; AADAT, 2-aminoadipate transaminase; GCDH, glutaryl-CoA dehydrogenase; E1o-like, E2o, E3-components of the OGDHc; E1a, 2-oxoadipate dehydrogenase; AMOXAD, α-aminoadipic and α-ketoadipic aciduria; CMT2Q, Charcot–Marie-Tooth disease type 2Q; EoE, eosinophilic esophagitis; GA1, glutaric aciduria 1.

Figure 1

(A) Mechanism of the OGDHc with 2-oxoglutarate (in black) and 2-oxoadipate (in blue) as substrates. (B) X-band EPR spectra of the ThDP-enamine radical species generated on E1o OG and OA. The radical species generated on E1o from OA is ~3 times lower in concentration compared to that from OG (0.9 μM with ~0.2% occupancy of E1o active centers). (C) Time dependence of reductive acylation of the lipoyllysyl groups on LDo by E1o from OG or OA could be analyzed by FT-MS. The fraction of reductively acylated LDo versus total LDo (acylated plus un-acylated LDo) when plotted versus time, allowed evaluation of the rate of acyl transfer from E1o to E2o (k₄ in (A)). (D) Mechanism of succinyl transfer from S8-succinyldihydrolipoyllysyl-E2o to CoASH with suggested role of His375 and Asp374. Pathway A: direct attack by the conjugate base thiolate anion of CoASH assuming a low pK_a for the CoASH, or by the thiol form itself. Pathway B: initial proton transfer from CoASH to His375 forming the conjugate base CoAS^-, which is the attacking agent. Both pathways then proceed by an oxyanionic tetrahedral intermediate [41].

Scheme 2

Mechanism of the OADHc (comprising multiple copies of the E1a, E2o and E3 components) showing the formation of the enamine intermediate on E1a from 2-oxoadipate and reductive glutarylation reaction between E1o and E2o according to a concerted mechanism [39].

Figure 2

(A) Domain structure of the human E2o showing the lipoyl domain (LDo) and the E2o core or catalytic domain (CDo) connected by a linker region. (B) Electron micrograph reconstruction of the human E2o core domain structure at 4.7 Å resolution [35]. Figure reproduced with permission of the International Union of Crystallography). The trimer building blocks (the individual subunits are shown in blue, green and orange) are assembled into the 24-meric core via four-fold (left), two-fold (middle) and three-fold symmetry axes, respectively. Insert shows the first residue 219 (red spheres) from each subunit of the 24-mer E2o core which is exposed to the surface of the E2o core. (C) On human E2o, the region comprising residues from the core domain (in bold) and E2o linker region (¹⁴⁴AEAGAGKGLRSEHREKMNRMRQRIAQRLKE¹⁷⁴) displayed a significant decrease in the level of the deuterium uptake during the first 3 min upon E1o binding, suggesting a unique subunit-binding mode in human OGDHc assembly, where the E2o core domain also participates in the interaction with E1o. (D) Sequence alignment of the human E2o linker-core region involved in interaction with E1o with some known E1-binding domains in E2 components identified conserved residues, indicating that E2o shares some but not all sequence features of other subunit-binding domains involved in E1o binding. The abbreviations are denoted: human (h), Escherichia coli (Ec), Bacillus stearothermophilus (Bst), Pseudomonas putida (Pp), and Azotobacter vinelandii (Av), 2-oxoglutarate dehydrogenase complex (o), pyruvate dehydrogenase complex (p), and branched chain 2-oxoacid dehydrogenase complex (b). Alignment of multiple sequences was carried out using the Clustal Omega program with default settings [70].

Figure 3

Structure of the human E1a component. (A) Overall fold of the E1a (α₂ homodimer) as follows from X-ray structures of the human E1a [35,36]. One monomer is shown in green and the other in cyan and the ThDP cofactor in two active cites is shown in yellow. (B) Active site of E1a with ThDP bound which is stabilized by highly conserved hydrogen bonds and hydrophobic interactions (A,B) reprinted in part with permission from [36]. (C) Modeling of the E1a active site with pre-decarboxylation intermediate generated from the nucleophilic attack by the ylide carbanion of ThDP on C2 atom of 2-oxoadipate. (D) Modeling of the E1a active site with the post-decarboxylation intermediate formed after the release of CO₂. (E) Schematic representation of acyl transferase reaction between the E1a and E2o components. (F) Modeling of the E1a active site with lipoyllysyl-E2o showing two sulfur atoms of the lipoyllysyl-E2o interacting with His⁴³⁵ and His⁷⁰⁸ in the active center of E1a. Modeling of the E1a active site in panels (C–F) were built by using the X-ray structure of E1a (PDB: 6sy1) [35] and MsKGD (PDB: 3zht) [68] and the PyMOL visualization system (v 2.2.1).

Figure 4

Deuterium uptake of the human E1a peptic peptides on interaction with E2o. (A) Difference plot showing the changes in deuterium incorporation of peptic peptides of E1a (ΔΔD, y axis) (deuterons exchanged in peptic peptides of E1a by itself minus deuterons exchanged in the peptic peptides of E1a on interaction with E2o). (B) The E1a peptic peptides protected from HDX on interaction with E2o are color-coded according to the deuterium uptake difference at the first 30s and at 90 min time points [85].

Figure 5

Inter-component cross-links within the human E1o-E2o and E1a-E2o sub-complex identified by CL-MS by using disuccinimidyl dibutyric urea (DSBU, Cα-Cα distance ~27 Å to be bridged) as a cross-linker. The bar plots with cross-link network were generated with xiView visualization tool for xiNET [94].

Figure 6

Modeling of the human E1o-E2o and E1a-E2o sub-complex structures. (A) Overview of the modeling workflow using intra-component and inter-component cross-links as residue distance constraints. (B) Mapping of the compatible inter-component cross-links into E1a-E2o sub-complex structure created by protein-protein docking [100]. (C) Mapping of the compatible inter-component cross-links into the E1o-E2o sub-complex structure. The Lys⁸² and Lys⁸⁷ are from different subunits of the E2o trimer [94,104].

Figure 7

Modeling of the human E1o-E2o and of E1a-E2o sub-complex structures. (A) The E1o (α₂) and the E1a (α₂) homodimers are colored in yellow and in purple, respectively. The E2o core formed by tightly associated trimers is shown in violet. The E2o lipoyl domains are shown in dark blue. The N-terminal residues of E1o (residues 27–40) and E1a (residues 24–47) are shown in red. The E1o and E1a active centers located between the two subunits are shown in orange. (B) View of the E1o-E2o and E1a-E2o sub-complex structures at a different angle. Three regions of the E2o with α-helix secondary structure comprising residues 191–208 from the linker region (in blue), and residues 273–289 (in cyan) and 370–386 (in purple) both from the E2o core are indicated showing their different orientation versus E1o (left) and E1a (right) [94,104].