Inhibition and Crystal Structure of the Human DHTKD1-Thiamin Diphosphate Complex

Abstract

DHTKD1 is the E1 component of the 2-oxoadipic acid dehydrogenase complex (OADHc), which functions in the L-lysine degradation pathway. Mutations in DHTKD1 have been associated with 2-aminoadipic and 2-oxoadipic aciduria, Charcot-Marie-Tooth disease type 2Q (CMT2Q) and eosinophilic esophagitis (EoE). A crystal structure and inhibitors of DHTKD1 could improve the understanding of these clinically distinct disorders, but are currently not available. Here we report the identification of adipoylphosphonic acid and tenatoprazole as DHTKD1 inhibitors using targeted and high throughput screening, respectively. We furthermore elucidate the DHTKD1 crystal structure with thiamin diphosphate bound at 2.1 Å. The protein assembles as a dimer with residues from both monomers contributing to cofactor binding. We also report the impact of ten DHTKD1 missense mutations on the encoded proteins by enzyme kinetics, thermal stability and structural modeling. Some DHTKD1 variants displayed impaired folding (S777P and S862I), whereas other substitutions rendered the enzyme inactive (L234G, R715C and R455Q) or affected the thermal stability and catalytic efficiency (V360A and P773L). Three variants (R163Q, Q305H and G729R) surprisingly showed wild type like properties. Our work provides a structural basis for further understanding of the function of DHTKD1 and a starting point for selective small molecule inhibitors of the enzyme, which could help tease apart the role of this enzyme in several human pathologies.

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

“…There are, however, X-ray structures available for the N-terminally truncated E. coli E1o Δ77 (39% identity, missing the N-terminal 77 amino acids) [ 66 ], the N-terminally truncated Mycobacterium smegmatis α-ketoglutarate decarbo-xylase ( Ms KGD Δ115 , 41% identities, missing the N-terminal 115 amino acids) [ 67 ], the Ms KGD Δ360 catalytic domain, which showed overall structural similarity to the E. coli E1o [ 68 ], and the Ms KGD Δ360 catalytic domain in complex with phosphonate analogues of OG [ 69 ]. X-ray structures were recently reported for the N-terminally truncated human E1a (residues 45-919, missing the N-terminal 44 residues) at 1.9 Å [ 35 ] and for human E1a (residues 25-919, missing the N-terminal 24 residues) at 2.25 Å resolution (38.5% identities between E1o and E1a) [ 36 ]. Single-particle cryo-EM reconstruction of the E2o inner core domain at 4.7 Å global resolution [ 35 ] has been reported showing 24 E2o catalytic domains assembled as eight trimers and positioned at each of the eight vertices of the cubic core with octahedral symmetry ( Figure 2 B).…”

“…Next, it was shown that the E1a has about 40-fold preference in catalytic efficiency with OA versus OG [ 43 ], suggesting that the E1a active center has evolved to accommodate the slightly longer OA compared to OG (one additional CH 2 group) substrate. These are important determinants for the rational design of E1a inhibitors that could be useful for diseases such as GA1 [ 36 ].…”

“…The recently solved X-ray structures of the human E1a at 1.9 Å (PDB code 6sy1) [ 35 ] and at 2.25 Å resolution (PDB code 6U3J) [ 36 ] revealed its structural homology to Ms KGD ( Ms KGD Δ115 ; PDB code 2XT6; 38% identity) and to E. coli E1o (PDB code 2jgd; 38% sequence identity) [ 66 ]. The E1a forms a tight homodimer of over 200 kDa, common to all ThDP-dependent enzymes, burying a more than 5000 Å 2 area of monomeric accessible surface at the dimer interface ( Figure 3 A) [ 36 ]. Each E1a subunit is structurally composed of four distinct domains displaying the common fold of ThDP-dependent enzymes ( Figure 3 , top) [ 35 ].…”

“…The ThDP cofactor is bound between the two subunits and is supported by highly conserved hydrogen bonds and hydrophobic interactions, including Asp333 and Asn366 from the known ThDP-binding fold ( Figure 3 B) [ 87 , 88 ]. From the reported structural studies several amino acid residues in the E1a active center have been suggested to interact with OA, residues which are not conserved with those involved in OG binding in E1o’s: more polar residues including Tyr190 (Phe506 in Ms KGD; Phe227 in E. coli E1o), Tyr370 (Phe682 in Ms KGD; Phe394 in E. coli E1o), a less bulky Ser263 (Tyr578 in Ms KGD; Tyr297 in E. coli E1o), and a conserved Asp707 (Asp1019 in Ms KGD; Asp688 in E. coli E1o) [ 35 , 36 ]. To gain further structural insight into the E1a active center, models have been built of its active center with covalent reaction intermediates derived from OA based on the reported X-ray structures of human E1a [ 35 , 36 ] and Ms KGD [ 68 ], as well as the mechanism of OGDHc with OA presented in Scheme 2 .…”

“…Of special interest is recognition of the formation of a hybrid complex between OGDHc and OADHc. In the second half of this review, we discuss the structural insight into the architecture of the human E1a active site during catalysis, of which two independent X-ray structures have been reported recently [ 35 , 36 ]. Taking into consideration both in vitro and in vivo evidence for the interaction between E1a and E2o from two distinct metabolic pathways, we next present the models constructed for the E1o-E2o and E1a-E2o assembly to advance our understanding of protein-protein interactions in human OGDHc and OADHc in the absence of X-ray crystallographic or cryo-EM-based atomic structure of intact OGDHc or OADHc.…”