Structures and comparison of endogenous 2-oxoglutarate and pyruvate dehydrogenase complexes from bovine kidney

Abstract

The α-keto acid dehydrogenase complex family catalyzes the essential oxidative decarboxylation of α-keto acids to yield acyl-CoA and NADH. Despite performing the same overarching reaction, members of the family have different component structures and structural organization between each other and across phylogenetic species. While native structures of α-keto acid dehydrogenase complexes from bacteria and fungi became available recently, the atomic structure and organization of their mammalian counterparts in their native states remain unknown. Here, we report the cryo electron microscopy (cryoEM) structures of the endogenous cubic 2-oxoglutarate dehydrogenase complex (OGDC) and icosahedral pyruvate dehydrogenase complex (PDC) cores from bovine kidney determined at 3.5 Å and 3.8 Å resolution, respectively. The structures of multiple protein were reconstructed from a single lysate sample, allowing direct structural comparison without the concerns of differences arising from sample preparation and structure determination. Although native and recombinant E2 core scaffold structures are similar, native structures are decorated with their peripheral E1 and E3 subunits. Asymmetric sub-particle reconstructions support heterogeneity in the arrangements of these peripheral subunits. Additionally, despite sharing a similar monomeric fold, OGDC and PDC E2 cores have distinct interdomain and intertrimer interactions, which suggests a means of modulating self-assembly to mitigate heterologous binding between mismatched E2 species. The lipoyl moiety lies near a mobile gatekeeper within the interdomain active site of OGDC E2 and PDC E2. Analysis of the two-fold related intertrimer interface identified secondary structural differences and chemical interactions between icosahedral and cubic geometries of the core. Taken together, our study provides direct structural comparison of OGDC and PDC from the same source and offers new insights into determinants of interdomain interactions and of architecture diversity among α-keto acid dehydrogenase complexes.

MRPS36 provides a missing link in the eukaryotic 2-oxoglutarate dehydrogenase complex for recruitment of E3 to the E2 core

“…In the eukaryotic E2o sequences, the motif in the corresponding stretch is clearly distinct with a conservation of proline and alanine residues that form an unstructured loop as predicted by AlphaFold2 (SI Figure 2B). The logos were generated using the MEME Suite (45) eukaryotic OGDHC to facilitate the binding of functional assemblies of E3 (dimeric) to E2o (trimeric) (27,28,54). The final structural model highlights how dimeric E3 may be recruited to the trimeric E2o core via MRPS36 (Figure 4B).…”

“…Several recently published structural models of eukaryotic OGDHC revealed an octahedral core containing 24 E2o subunits (27)(28)(29). However, these models do not provide comprehensive details about the exact orientation of E1o and E3 with respect to the core, and fully overlooked the presence and role of MRPS36 (27)(28)(29).…”

“…Several recently published structural models of eukaryotic OGDHC revealed an octahedral core containing 24 E2o subunits (27)(28)(29). However, these models do not provide comprehensive details about the exact orientation of E1o and E3 with respect to the core, and fully overlooked the presence and role of MRPS36 (27)(28)(29). To understand how all subunits of the eukaryotic OGDHC are assembled into a complex, we set out to use AlphaFold2-Multimer (51) together with our XL-MS data to model the orientations of E1o, E3, and MRPS36 with respect to the E2o core, and to predict potential interfaces.…”

“…Its presence was suggested to be crucial for the efficient recruitment of E3 to the E2o core (32), but due to the lack of experimental evidence its function as an adaptor between E3 and the remainder of the OGDHC remains elusive. Also, in very recent high-resolution structural models of eukaryotic OGDHC, MRPS36 is not observed, or at least not reported on (27,28). Here, we explore the overall composition and architecture of mammalian OGDHC by combining complexome profiling (CP) (33,34) and cross-linking mass-spectrometry (XL-MS) with heart mitochondria from both mice and bovine origin.…”

“…While this catalytic mechanism is well understood, current structural insights into OGDHC lack detailed information about the complex architecture (19,(24)(25)(26) in a close-to-native environment. Recent cryo-electron microscopic (cryo-EM) reconstructions of mammalian OGDHC revealed an octahedral core of 24 E2o subunits in an 8x3 assembly with E1o and E3 suggested to bind at the edges and faces, respectively (27,28). Nonetheless, likely due to the highly conformational flexibility and possible heterogeneity of the complex (29), these structures are not sufficient to understand how the E1o and E3 components are organized with respect to the core.…”